development of enkephalin mrna interference in the rat brain · shenk and comparing it to...
TRANSCRIPT
Development of Enkephalin mRNA Interference
in the Rat Brain
Mémoire
Daniel Szaroz
Maîtrise en Neurobiologie
Maître ès sciences (M.Sc.)
Québec, Canada
© Daniel Szaroz, 2014
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Résumé
Les enképhalines (ENK), étant neuromodulateurs, ont un rôle prépondérant dans plusieurs
circuits neuronaux tels que ceux de la récompense, la peur et l‟anxiété. Dans cette étude,
nous avons ciblé les ENK et atténué leur expression dans le noyau accumbens et
l‟amygdale centrale par le biais d‟injections de vecteurs lentiviraux exprimant un shRNA
spécifique à l‟ENK. Les injections des lentivirus exprimant un shENK ont été comparées à
des hémisphères intacts et à des injections du même vecteur exprimant un shRNA témoin,
pour révéler des diminutions de l'ARNm des ENK de 62 %. Ces quantifications ont été
validées in vivo par la comparaison du signal radioactif des sondes pour l‟ARNm des ENK
dans les régions infectées par le virus, ces régions ayant été identifiées par
immunohistochimie. Nous démontrons une spécificité de l‟atténuation de l‟ARNm des
ENK puisqu‟aux sites des injections, il n'y a pas eu de diminution de l‟ARNm de la
GAD65.
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Abstract
Enkephalin (ENK), a prominent endogenous opioid mediator of the behavioural response,
elicits its function in important circuits of the brain such as reward, fear and anxiety. In this
study, we have targeted the downregulation of ENK expression by the delivery of a
lentiviral vector with an expressing shRNA specific to ENK mRNA in ENK rich regions,
such as the nucleus accumbens and central amygdala. By injecting a vector expressing an
shENK and comparing it to non-injected hemispheres, as well as to injections of the same
vector yet expressing a scrambled shRNA, we have observed an average downregulation of
62% ENK mRNA. Quantifications were performed in vivo, by collecting the in situ
hybridization radioprobe signal for ENK mRNA of regions infected by the virus; the latter
visualized immunohistochemically. Our results show a knockdown specificity of ENK
mRNA and tissue integrity, as demonstrated by the lack of GAD65 mRNA disruption.
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Table des matières
Résumé ........................................................................................................................................... iii Abstract ........................................................................................................................................... v Table des matières ........................................................................................................................ vii Liste des tableaux ........................................................................................................................... xi
Liste des figures ........................................................................................................................... xiii Liste des abréviations .................................................................................................................... xv Remerciements ............................................................................................................................. xxi
1 INTRODUCTION ................................................................................................. 1
1.1 Endogenous opioids ............................................................................................................ 2
1.1.1 Analgesic compounds of yore ................................................................................... 2
1.1.2 The endogenous opioid superfamily ......................................................................... 3 1.1.3 Opioid receptor cross-over ........................................................................................ 6 1.1.4 Enkephalin distribution ............................................................................................. 8
1.1.5 A tale of two receptors: distribution and function ................................................... 12 1.1.6 Enkephalins and anxiety .......................................................................................... 20
1.2 Lentiviral mRNA interference .......................................................................................... 21
1.2.1 The advent of gene-silencing ................................................................................... 21 1.2.2 Transient and long-term methods of silencing ........................................................ 23
1.2.3 Advantages of viral induced silencing..................................................................... 23
1.2.4 Viral transductions in silencing ............................................................................... 24 1.2.5 Rationale in relation to ENK-silencing: Using a stable viral construct ................... 26
Research Hypothesis and Objectives ............................................................................................ 30
2 MATERIALS AND METHODS ........................................................................ 33
2.1 Production of an shRNA Lentiviral System ..................................................................... 33
2.1.1 Plasmid amplification .............................................................................................. 34 2.1.2 Vector construction.................................................................................................. 34
2.1.2.1 DNA cloning; a brief summary ............................................................ 37
2.1.2.2 Bacterial transformation; day 1 ............................................................. 37 2.1.2.3 Bacterial inoculation; day 2 .................................................................. 39 2.1.2.4 Plasmid extraction; day 3 ...................................................................... 40
2.1.3 Lentivirus production .............................................................................................. 42
2.2 PC12 cell differentiation ................................................................................................... 45
2.2.1 Cell culture .............................................................................................................. 45 2.2.2 Semi-quantitative RT-PCR ...................................................................................... 48 2.2.3 Total RNA extraction .............................................................................................. 48 2.2.4 Reverse transcription ............................................................................................... 49 2.2.5 Primer dilution ......................................................................................................... 50
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2.2.6 PCR amplification .................................................................................................... 50
2.2.7 Gel organization ....................................................................................................... 51
2.3 In vivo targeting of ENK mRNA with an shRNA lentivector .......................................... 52
2.3.1 Animals .................................................................................................................... 52 2.3.2 Pre-surgical stereotaxic treatments .......................................................................... 54 2.3.3 Stereotaxic anaesthesia ............................................................................................ 54 2.3.4 Stereotaxic surgeries ................................................................................................ 55
2.3.5 Animal euthanasia .................................................................................................... 56 2.3.6 Brain harvesting ....................................................................................................... 57 2.3.7 Immunohistological resolution of lentiviral diffusion and neuronal cell
transduction: a brief summary ............................................................................................. 57
2.3.8 Radioprobe synthesis for ENK and GAD65 mRNA hybridization ......................... 59 2.3.9 Immunohistochemistry for lentiviral localization: combined protocol ................... 60 2.3.10 Solutions required for immunohistochemistry ......................................................... 60
2.3.11 Immunohistochemistry for copGFP ......................................................................... 61 2.3.12 In situ hybridization integration ............................................................................... 62
2.3.13 Pre-hybridization and radioprobe hybridization ...................................................... 62 2.3.14 Post-hybridization .................................................................................................... 63
2.4 Quantification .................................................................................................................... 64
2.4.1 Data analysis ............................................................................................................ 64 2.4.2 Co-localization of copGFP and ENK mRNA for quantification and analysis ........ 65
2.4.3 GAD65 mRNA quantifications in areas injected with an shRNA ENK
expressing vector ................................................................................................................. 67 2.4.4 Statistical analysis .................................................................................................... 68
3 RESULTS ............................................................................................................ 69
3.1 ShRNA expression vectors ................................................................................................ 69
3.1.1 High-titer viral production reproducibility .............................................................. 69
3.2 Cellular culture results analysis ......................................................................................... 69
3.2.1 PC12 differentiation analysis overview ................................................................... 69 3.2.2 Cellular decline & anti-proliferative effects ............................................................ 70 3.2.3 Shape, maturational & morphological concerns for PC12 cells .............................. 71
3.2.4 Agarose gel analysis of opioid mRNA from PC12 cells ......................................... 71 3.2.5 Attempts at experimental troubleshooting ............................................................... 74
3.3 In vivo delivery of ShRNA expression vectors ................................................................. 75
3.3.1 Injection sites revealed by immunohistochemical analysis ..................................... 75
3.3.2 Representative rats and quantifiable coronal sections ............................................. 83
3.4 Quantification of ENK mRNA from ENKergic Neurons ................................................. 84
3.4.1 Injection sites and area quantifications .................................................................... 84 3.4.2 Markedly important reductions in ENK expression mediated by an shENK
expressing vector but not the shSCR expressing vector ...................................................... 86
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3.4.2.1 Optical density measurements in the targeted area injected with the
viral solution ......................................................................................................... 86
3.4.3 Specification for shRNA expression vector in vivo quantification control with
GAD65 ................................................................................................................................ 89 3.4.3.1 GAD65 mRNA quantifications reveals no differences for integrated
density and optical density measurements ............................................................ 91
4 DISCUSSION ..................................................................................................... 93
4.1 Methodological development of lentiviral production...................................................... 94
4.1.1 Optimization of selected steps for a high-titer viral production and in-between
batch reproducibility ............................................................................................................ 94
4.1.2 Preliminary technical notes and considerations....................................................... 95 4.1.3 A functional transfection foreshadows the generation of high-titer particles ......... 95 4.1.4 Purification and concentration methodology ........................................................... 96 4.1.5 Selection of different producer and titration cell lines, enhances the production
of lentiviral particles ............................................................................................................ 98 4.1.6 HeLa cell titration .................................................................................................... 99
4.1.7 A stricter measure of strength provided by the HeLa cell line .............................. 100
4.2 In vitro knockdown system: PC12 rat pheochromocytoma cell line .............................. 102
4.2.1 Adequate and representative in vitro system ......................................................... 102
4.2.1.1 The PC12 in vitro system .................................................................... 102 4.2.1.2 Inducible maturational effects by specific differentiation agents tailor
bidirectional outcomes for PC12 cells ................................................................ 103 4.2.1.3 The overall role on transcription by NaB is tied to cellular arrest ...... 104
4.2.2 Validity of controls and technical considerations.................................................. 107 4.2.3 NaB differentiation did not fully induce the chromaffin phenotype expected ...... 108 4.2.4 Unattained morphological end points show a link to unattained biochemical
endpoints ........................................................................................................................... 109 4.2.5 The effect of NaB for DYN is dose-responsive due to unreversed PC12
phenotype .......................................................................................................................... 110 4.2.6 We suggest that a positive culture microenvironment provides proper nutrient
absorption and is essential for our PC12 cell line‟s biochemical consistency .................. 110
4.2.7 We suggest that integrin ligand-coated culture plates may help to stabilize our
PC12 cell line .................................................................................................................... 113
4.2.8 The availability of growth factors in our cell culture medium may also have
contributed to unexpected cell behaviour .......................................................................... 113
4.2.9 Our results confirm NaB as a potent inhibitor of histone deacetylase .................. 114 4.2.10 Final considerations and final notes ...................................................................... 115
4.3 In vivo Knockdown system: Stereotaxic injections of shRNA vectors .......................... 115
4.3.1 Immunohistochemistry in our protocol: usefulness and limitations ...................... 116 4.3.2 Preliminary notes and knockdown insight provided by immunohistochemistry .. 116 4.3.3 Viral spreading as witnessed by copGFP visualization shows limited diffusion .. 117
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4.3.4 Very efficient transduction and knockdown of ENK mRNA in different
ENKergic populations ........................................................................................................ 118
4.3.5 Percentage knockdown of ENK mRNA and ENKergic populations bias ............. 118 4.3.6 Viral inhibitions of ENKergic innervations ........................................................... 121 4.3.7 Considerations for area selection subject to quantification from IHC staining ..... 122 4.3.8 Injection tracks carry efficient infectious titer of viral solution refluxed from
amygdalar injection sites ................................................................................................... 123
4.3.9 In vivo controls for quantifications ........................................................................ 124 4.3.10 Anatomical hurdles and limitations ....................................................................... 125
Conclusion ................................................................................................................................... 129 Bibliography ................................................................................................................................ 131
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Liste des tableaux
Table 1. Endogenous opioid superfamily and receptors ........................................................ 6 Table 2 - Different features of viral vehicles ....................................................................... 26 Table 3- Summary of major steps conducive to efficient lentiviral particle production ..... 44 Table 4- Selected measurements from ENK expression for regions where an shENK was
found (by copGFP) staining are illustrated here (mean ± standard error). ................... 85 Table 5-Different centrifugation times and parameters for different viral preparations. ..... 98
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Liste des figures
Figure 1. Classic endorphin cell bodies across the rodent brain ........................................... 10 Figure 2. Localization of Enkephalin‟s receptor proteins across the rodent brain. .............. 14 Figure 3. Brief summary of receptor knockouts for mood disorders and addiction. ............ 19 Figure 4. Lentiviral silencing mechanism for targeted mRNA repression. .......................... 22
Figure 5. A vast choice of viral vectors exist today. ............................................................. 24 Figure 6. As inserted in the expression vector PSIH1-H1copGFP, top and bottom strands of
the short-hairpins generators. ........................................................................................ 36 Figure 7. Figurative representation of sodium butyrate (NaB) pheochromocytoma cells
(PC12) differentiation schedule. ................................................................................... 47
Figure 8. Schematic representation of the timeline with requirements for efficient viral
production. .................................................................................................................... 53
Figure 9. Elucidation of the quantification method used in this study for a representative
brain section with an ShENK vector delivered within Nucleus Accumbens (NAc). ... 66
Figure 10. Most representative agarose gel of relative RT-PCR analysis for ENK and DYN
mRNA expression after sodium butyrate cell differentiation. ...................................... 73
Figure 11. Representative brain sections quantified for viral injection of ShSCR and
ShENK into the nucleus accumbens (NAc) and central amygdala (CEA). .................. 77 Figure 12. Injections in the NAc core and shell. The following figure shows the sites
obtained from injections in the nucleus accumbens. .................................................... 79 Figure 13. Injections targeting the basolateral amygdala. .................................................... 82
Figure 14. Optical density measurements of ENK mRNA expression in the area injected
with the viral solution. .................................................................................................. 88
Figure 15. Representative brain sections demonstrating viral delivery of the lentiviral
vector expressing an ShENK in the striatum. ............................................................... 90
Figure 16. The following figure shows optical density measurements for GAD65 mRNA
expression in targeted areas, injected with a vector expressing an shENK. ................. 92 Figure 17. The inhibition of histone deactylases and cellular outcomes. ........................... 106
Figure 18. Microenvironment requirements of the mammalian cell. ................................. 112
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Liste des abréviations
AAV Adeno-Associated virus
ABC Avidin-Biotin Complex
ADV Adenovirus
AMC Amygdalar Capsule
AmpR Ampicillin Anti-biotic Resistance Gene
AMY Amygdala
BamHI Bacillus Amyloliquefaciens type II Restriction Endonuclease
BAME Bovine Adrenal Medullary Endothelial Cells
Beta-END β-endorphin
BLA Basolateral Amygdala
BLAa Anterior Basolateral Amygdala
BLAp Posterior Basolateral Amygdala
BSA Bovine Albumin Serum
bp Base Pairs
BST Bed Nucleus of the Stria Terminalis
CaCl2 Calcium Chloride
CCAC Canadian Council of Animal Care
CDK Cyclin Dependent Kinase
cDNA Complementary DNA
CEA Central Amygdala
CEAc Central Amygdala capsular division
CEAl Central Amygdala lateral division
CEAm Central Amygdala medial division
CHUQ Centre Hospitalier Universitaire de Québec
CMV Cytomegalovirus
CNS Central Nervous System
Col IV Collagen IV
CopGFP Copepod Green Fluorescent Protein
CP Caudate Putamen
CPAC Comité de la Protection des Animaux
CS Conditioned Stimulus
DAB Diaminobenzidine
DAMGO ([D-Ala2-N-Me-Phe4-glycol 5-enkephalin
DEPC Diethylpyrocarbonate
DG-HIP Dentate Gyrus of the Hippocampus
DH5α Doug Hanahan 5, E. Coli competent cell strain
DMEM Dulbecco's Modified Eagle Medium
DNA Deoxyribonucleic Acid
dNTP Deoxynucleotide
DOR δ-opioid Receptor
DS Double Stranded
DYN Dynorphin
EC External Capsule
ECM Extra Cellular Matrix
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E. Coli Escherichia Coli
EcoR1 E.Coli type II Restriction Endonuclease
ECS Extra Cellular Space
EDTA Ethylenediaminetetraacetic Acid
END Endorphin
EndA- Endonuclease 1 in E.coli
ENK Enkephalin
ER Endotoxin Removal
EtBr Ethidium Bromide
EtOH Ethanol
FACS Fluorescence-activated Cell Sorting
FBS Fetal Bovine Serum
FQ Phenylalanine & Glutamine
FN Fibronectin
GABA γ-Aminobutyric Acid Neurotransmitter
GAD65 Glutamic Acid Decarboxylase 65
GAPDH Glyceraldehyde 3-phosphate Dehydrogenase
GFP Green Fluorescent Protein
GP Globus Pallidus
GPCR G-Protein Coupled Receptor
HAT Histone Acetyltransferase
HDAC Histone Deacetylase
HeLa Human “Henrietta Lacks” Cell Line
HEPES Buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HIP Hippocampus
HIV-1 Human Immunodeficiency Virus type 1
HPA Hypothalamic Pituitary Axis
HRP Horse Radish Peroxidase
HS Horse Serum
HSV-1 Herpes-Simplex 1 virus
HTH Hypothalamus
IA Intercalated Nuclei of the Amygdala
ID Integrated Density
IFU Infectious Unit
IgG Immunoglobulin G
IgM Immunoglobulin M
IHC Immunohistochemistry
ISH In Situ Hybridization
KCl Potassium Chloride
KOR κ-opioid Receptor
LB Luria Bertani Medium
LC Locus Coeruleus
Leu5-ENK H-Tyr-Gly-Gly-Phe-Leu-OH
LN Laminin
LPS Lipopolycaccharides
LS Lateral Septum
MAPK Mitogen-Activated Protein Kinase
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MEA Medial Amygdala
MLV Muloney Leukemia Virus
MOPS 3-(N-morpholino) Propanesulphonic Acid
MOR μ-opioid Receptor
mPFC Medial Pre-frontal Cortex
mRNA Messenger RNA
Met5-ENK H-Tyr-Gly-Gly-Phe-Met-OH
MSH Melanocyte-stimulating Hormone
NaB Sodium Butyrate
NaCl Sodium Chloride
NAc Nucleus Accumbens
NAcS Nucleus Accumbens Shell
NAcC Nucleus Accumbens Core
NaOH Sodium Hydroxide
NeoR Neomycin Resistance gene
NGF Nerve Growth Factor
NMDA N-methyl-D-aspartate receptor
NTS Nucleus of Solitary Tract
OD Optical Density
Oprd-/- δ- opioid receptor deficient mice
Oprk-/- κ- opioid receptor deficient mice
Oprm-/- μ- opioid receptor deficient mice
ORL-1 Orphan-Receptor-Like 1
OFQ/N Orphanin FQ/nociceptin
PAG Periaqueductal Grey Matter
PB Parabrachial Nucleus
PBS Phosphate Buffered Saline
PC Parabrachial Complex
PC12 Rat Adrenal Pheochromocytoma Cells
PCR Polymerase Chain Reaction
PDYN Prodynorphin
PENK Proenkephalin
PFA Paraformaldehyde
PLC Phospholipase C
PNOQ Pronociceptin/Orphanin FQ
PNS Peripheral Nervous System
POA Preoptic Area
POMC Pro-opiomelanocortin
PPDKO Preprodynorphin Knock-out
PPEKO Preproenkephalin Knock-out
ppENK Preproenkephalin
PVH Paraventricular Nucleus of the Hypothalamus
PVN Paraventricular Nucleus
PVT Paraventricular Nucleus of the Thalamus
QBT Qiagen Equilibration buffer
QC Qiagen Wash buffer
QN Qiagen Elution buffer
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RB101 C31H38N2O3S3
RecA Recombinase A protein in E.coli
RISC RNA-induced Silencing Complex
RNA Ribonucleic Acid
RNAi Ribonucleic Acid Interference
RNase Ribonuclease
RT-PCR Reverse Transcriptase-PCR
SCFA Short-Chain Fatty Acids
SDS Sodium Dodecyl Sulphate
SEM Standard Error of the Means
shSCR Short-hairpin Scrambled RNA
shENK Short-hairpin Enkephalin
shRNA Short-hairpin RNA
siRNA Small Interfering RNA
SIA Stress-Induced-Analgesia (Antinociception)
S.O.C Super Optimal Broth with Catabolite repression
SSC Standard Saline Citrate
SV40 Simian Virus 40
TE Tris-EDTA Endotoxin-free
TEA Triethanolamine
TM Transmembrane
TU/ml Transducing Units/millilitre
US Unconditioned Stimulus
UV Ultraviolet
VLM Ventrolateral Medulla
VP Ventral Pallidum
VSVg Vesicular Stomatitis Virus G Protein
VTA Ventral Tegmental Area
V/V Volume Percent Concentration
W/V Percent Weight By Volume
V/V Volume Percent Concentration
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Dedicated to my parents…
Through thick and thin you were there
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Remerciements
En premier lieu, j‟aimerais remercier le Dr. Guy Drolet pour m‟avoir accueilli dans son
laboratoire. Je suis très reconnaissant pour l‟encadrement reçu et aussi pour m‟avoir laissé
entrer dans le fabuleux monde des Enképhalines.
J‟aimerais aussi remercier Sylvie Laforest, pour m‟avoir patiemment appris les nombreuses
techniques de laboratoire et d‟avoir partagé avec moi son savoir pratique et ces recettes,
tant expérimentales… que culinaires! La cohorte « Lentivirus » de Jean-François Poulin,
qui a fait et testé le plasmid shENK, à Caroline Jeudi, merci! À Aurore « Frenchie », ton
humour et ta bonne présence dans le lab ont égayé tant de nombreuses journées. À Camille
B-Vincent, ton intégrité et ta droiture tant scientifique que personnelle, ont été des plus
admirables. Merci pour les beaux moments passés à faire des expériences et les tests d‟
« elevated-plus-maze » et de « social interaction », et les Friends! À Antoine H-Blanchet,
merci pour ton humeur positive et contagieuse. Au Dr. Rouillard, pour avoir fait parti de
mon comité depuis mon entrée à la maîtrise; pour ta bonne humeur, les diverses jazettes et
les plusieurs réponses à mes questions.
À Serge, tu as s‟en doute été l‟une des personnes les plus importantes pendant toutes mes
études supp. Merci, pour ta clairvoyance, et les dimanches superbowl! To my home away
from home, Elsa, thank you for your home cooked meals and the numerous weekends spent
around the house with Miche and the girls. The memories will forever be a part of me. To
Don, where do I begin?! Thank you for your sound counsel, and your friendship these past
few years. I was and am, very grateful to have you in my life.
To all the wonderful people who make Quebec City a really cool place, thank you. Whether
it was bootcamping, volunteering, teaching, learning, chilling, whichever it was, those were
fun times outside the lab; Chris B, Ez, Caleb S, Albert Y, Justin W, Iris K, Adam P, Tom F,
James O, Elizabeth D, Melissa N, Jon R, Glenn K, Walter A, Eddy R, Eva M, Timber Milz,
Abi L, Eli Laynar M, Coco, David N, Alice R & JP R, Jeremy F & Manu M, Matt V, Steph
P, Morsen M, Norbert, Rebecca L, Stacey H, Alex C, Josh C, Cassie D, Joshua L, Chuli &
JP, you all have a very special spot in my heart. À Jacks C-A et Mimi L pour tant de bons
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moments!! A special thanks to Laurence P and Andrew E for being so laid back and for the
support during the writing phase!
Für Elyse… For all those times you stood by me, for your companionship amidst long lab
days, support and love as much during the experiment phase, as during the writing of this
thesis.
To my hometown folks, thank you for always being there, whenever I made it back to
town. To Scott Irwin, definitely the best of friends anyone could have had all along. Thank
you for your continual friendship, Mini-Ire, snowboarding, the visits to the city and for
being there since the start of grad school. To my kid brother, Sam, thank you for the many
visits, for keeping me sane during the writing process and for your entertaining ways!
To Pops, for the constant pep talk all along. Can‟t wait to spend more time on the golf
course!
Saving the best for last, to Delia and Jeff, for everything imaginable. Much of this work
wouldn‟t have been possible without the two of you. You guys have supported me in so
many ways, sharing my moments. Jeff, special thanks for Illustrator 6 and the techy part of
this thesis. You guys were there at every step of the way, through thick and thin, a gift that
can‟t ever be replaced…
“I fear these things, but vaguely, for my brain buzzes in the merciful wash of endorphins
that preclude any thought from occupying it too long.”
—Louise Erdrich, May 1993—
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In Memoriam
Le 4 octobre 2011, dans le contexte du cours « Projet de Maitrise », le Dr. Rouillard, Dr.
Drolet et le Dr. Mouginot ont siégé en tant que membres de jury à la présentation des
résultats obtenus durant le parcours de la maitrise, pour agir comme comité d‟encadrement.
Les suggestions et les commentaires émis par chaque membre du jury au cours de la
présentation, furent hautement appréciés. Entre autres, plusieurs de ses suggestions furent
notés et ont aidé à la rédaction de ce mémoire. Quoique la participation et la position de
chaque membre du jury, était reliée aux résultats qui furent présentés alors, Didier en a
profité pour tirer un lien au mémoire. Dans la production des lentivirus, 2 lignées
cellulaires ont étés utilisés : HeLa et 293TN. Suite à la présentation, Didier s‟est alors
exprimé en disant, « il faudra le mentionner, pourquoi ça, dans le mémoire ». Il avait ainsi
souhaité qu‟une description soit émise au sujet du raisonnement derrière l‟utilisation de ces
deux types cellulaires. C‟est donc en hommage à Didier que les sections 4.1.6 et 4.1.7 ont
étés écrites. Didier aimait beaucoup la science et s‟intéressait beaucoup aux nombreux
projets à travers l‟axe. Pour ceux qui l‟ont connu, Didier pouvait discuter tant de science
que d‟autres choses et pouvait tisser des liens avec n‟importe qui sur l‟échelon académique.
À la douce mémoire du Dr. Didier Mouginot, un neuroscientifique qui a tant marqué la
science et qui ne sera jamais oublié.
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1
CHAPTER 1
1 Introduction
Of the many scientific systems yet adopted by popular culture, none have better been
connotated with well-being than the endogenous opioids. These peptides, more generally
known as endorphins, have proved their belonging in the layperson‟s lexicon mainly to
describe sensations of pleasantness, numbness and soothing effects. Though often
jargonized in cultural media with a broad and general understanding, some reported
perceptions have now been given scientific credibility, such as “runner‟s high” (Partin,
1983; Morgan, 1985). This positive psychophysical effect is merely one example of the
actions mediated by a natural and central release of endorphins, which in this case, is
provoked by strenuous exercise (Boecker et al., 2008). Historical evidence has also
contributed to their popularity by acclaiming endorphins as key mediators in pain
management and antinociception, showing similarities to the analgesic effects of
morphine (Pert and Yaksh, 1974; Hughes, 1975; Hughes et al., 1975; Loh et al., 1976;
Millan, 2002; Galdino et al., 2010). By binding specific opioid receptors located centrally
for review, see (Mansour et al., 1995a) and peripherally (Stein et al., 1993; Schäfer et al.,
1994), the analgesic action of endogenous opioids has been well-characterized and has
even unveiled prominent avenues of clinical development (Stein et al., 2003). Decades of
research have lengthened the list of ever-growing naturally-produced opioids. This
pronounced ligand diversity and complexity unravels a plethora of possibilities, which
contrary to expectations, culminates on 3 major opioid receptors.
In spite of all these rising possibilities in the brain, endorphins were proven capable of
affecting other moods and behaviours. Therefore, at the crossroads between pain research
and an acute stress response, a phenomenon now well classified as “Stress-Induced-
Analgesia (or Antinociception)” aka SIA, showed the prevalence of endogenous opioids
in promoting pain relief (Butler and Finn, 2009; Vachon-Presseau et al., 2013). Center to
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this prodigious intersection, such studies proved that pain reduction inversely correlated
with a rise in endogenous opioids, provided that the stress remained acute (Madden et al.,
1977; Willer et al., 1981; Terman et al., 1984). This intersection in the most literal sense,
was not only hallmarked in principle of purpose by brain pathways but also streamlined
investigations towards the role of endogenous peptides in the broad stress response. Since
then, numerous elaborate roles have been accredited for particular members of this opioid
class in many more physiological paradigms of stress and resulting anxiety behaviours.
Our mission statement lies at the heart of stress-related endogenous opioid interactions.
The focus of this work has been to target the absolution of the Enkephalin (ENK)
peptide (a “classical” member of the endogenous opioids) in select centers of the brain
capable of mediating a stress-response. We have hypothesised that by attenuating the
expression of ENK, we would pave the way for its lack of function in a plausible stress-
related paradigm. Therefore, by using a specific and targeted form of RNA interference,
the expression of ENK could be first attenuated. We have therefore developed a consistent
and reproducible method of attenuation of the ENK protein and targeted the nucleus
accumbens and the amygdala, 2 very relevant ENK-producing regions. An appreciation
for ENK will solicit an understanding of its vast and broad implication, profusely in stress
biology and also in the neuroanatomical caveats of its partial depletion.
1.1 Endogenous opioids
1.1.1 Analgesic compounds of yore
Endogenous opioids as we now know them, have been discovered and classified
based on the preliminary discovery of exogenous opioids found in opium, extracted from
Poppys (papaver somniferum). As a very old analgesic and sedative, the properties of
opium have been reported throughout history dating as far back as the classical antiquity
for extensive review, see (Duarte, 2005). In 1806, Friedrich Sertürner first isolated a
crystalline substance he initially called “Principium Somniferum” but later renamed as we
know it today, „morphine‟. Thirty years later, work with opium by Robinet and
Thibouméry led to the isolation of 2 other alkaloids found in smaller percentages, Codeine
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(0.7-5%) and Thebaine (0.1-2.5%) (Robinette, 2008). However, morphine remained the
most important opium-derived substance to date useful in the synthesis of other semi-
synthetic analogs, acting as a comparative standard for other drugs and actively used for
research purposes for review, see (Brownstein, 1993).
Evidence for opioid receptors sites was validated by 3 distinct teams who assessed
competitive binding for these receptors by potent morphine-derived agonists and
antagonists such as naloxone, agreeing on a functional specificity (Pert and Snyder, 1973;
Simon et al., 1973; Terenius, 1973). Ironically, these studies employed exogenous ligands
showing adequate affinity to unknown receptors, only hinting at the possibility of an
endogenous system of ligands. Such a hypothesis was adopted by one of the groups who
unveiled opioid receptors originally, in their quest at the time to identify an „endogenous
factor‟ (singular) also able to act on the receptor (Terenius and Wahlström, 1975).
Inevitably, within the next 4 years, the 3 major classes of endogenous opioid ligands were
discovered. Today they comprise the generally referred-to “classical endogenous opioids”.
First isolated was ENK from pig brain, for having morphine-like effects (Hughes, 1975),
followed by β-endorphin (beta-END) comprising of the 31 C-terminal amino acids of β-
lipotropin (Li and Chung, 1976) and finally the potent Dynorphin (DYN) in 1979
(Goldstein et al., 1979). These 3 classes of peptides concur with the 1967 originally-
postulated terminology of “opioid”, denoting an analgesic compound with morphine-like
effects or antagonistic-like effects, as was suggested for nalorphine (Martin, 1967).
1.1.2 The endogenous opioid superfamily
Decades of research have increased the superfamily of naturally-produced opioids
which now includes over 25 distinct classical endogenous peptides and over 4 non-classical
or newly-discovered endorphins (Bloom, 1983; Akil et al., 1984; Mansour et al., 1995b;
Weisinger, 1995; Okutsu et al., 2006). The reason for the expansion of the “classical”
endogenous peptides has been mainly in spite of discoveries revealing post-translational
processing derived from the biologically inert precursor proteins for ENK, beta-END and
DYN. In order to be synaptically active, these peptides are derived from a longer pro-
hormone and cleaved by peptidases, which caters for further enzymatic modifications in
4
their process to peptidic maturation (Hook et al., 1994; Yasothornsrikul et al., 2003). Once
cleaved and matured, they become biologically active peptides able to act on either of 3
major membrane-bound rhodopsin-like GPCR protein receptors. These are conventionally
called the μ- (MOR), κ- (KOR), and δ- (DOR) opioid receptors, named after the
compounds that have led to their discovery (morphine, ketocyclazocine respectively) or the
region in which they were discovered (vas deferens for the DOR) (Martin, 1967;
Brownstein, 1993; Stevens, 2009). Each cleavage product instills a cascade of downstream
signalling hereby providing clues about specific functions attributed to each family of
opioids.
All “classical” opioids are coded from 3 separate genes and all stem from the
processing of either-proenkephalin (PENK), pro-opiomelanocortin (POMC), or
prodynorphin (PDYN). ENK as isolated then, consisted of 2 closely related peptides
comprised of 5 core and crucial amino acids; Met5-enkephalin (H-Tyr-Gly-Gly-Phe-Met-
OH) and Leu5-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH). The human PENK now contains
4 exons (as opposed to 3 in the rat) and is proteolytically processed into a total of 7
different peptides; 1 copy of Leu-ENK, 4 copies of Met-ENK, 1 copy of a heptapeptide and
1 copy of an octapeptide (Weisinger, 1995). Although other products of ENK cleavage
have been reported in different species and organs, they are longer peptides that extend
beyond the core of the Valine residue of the ENK octapeptide (Mansour et al., 1995b).
Interestingly, though each propeptide is translated from a different gene, the classical
opioids show a preserved ENK-like peptidic domain at the N-terminus of each derived
product (Akil et al., 1984). Processing of the PDYN precursor shows a certain similarity to
ENK products, in that the residue core of all 7 products is merely an Arginine extension
beyond Leu-ENK. The products of PDYN processing are: Big Dyn (1-32), Dyn A (1-17),
Dyn A (1-8), Leumorphin (1-29), Dyn B (1-13), α-neoendorphin, and β-neoendorphin
(Chen et al., 2007). In a similar way, POMC cleavage products also show sequence
conservation of Met-ENK in the amino acid sequence of β-endorphin (beta-END), the
active opioid producing signalling effects on the MOR. Older studies mistakenly attributed
the source of Met-ENK as derived from beta-END, knowing today, that only an external
endopeptidase can mediate this cleavage and that beta-END is not the source of Met-ENK,
5
for review see (Bloom, 1983). As was initially discovered, (beta-END) is derived from β-
lipotropin, a longer peptide cleaved to also yield β-MSH (Pritchard et al., 2002). Contrary
to belief at the time, β-lipotropin was not the source of Met-ENK either. Taken together,
the classical endogenous peptides are therefore united based on their N-terminal domain
similarity and cross-functional properties. However, new members have also been added to
this superfamily of opioids with properties that challenge traditional concepts of ligand-
receptor selectivity.
The “non-classical” opioids do not contain the trademark N-terminal domain but
can be divided into 2 sub-classes; those derived from the precursor pronociceptin/orphanin
FQ (PNOQ) and those with unknown precursor, the endomorphins. The derived peptides
from PNOQ have been discovered to be Nociceptin and Nocistatin (Meunier et al., 1995;
Okuda-Ashitaka and Ito, 2000). Now known as Nociceptin (originally called orphanin FQ
upon discovery (Reinscheid et al., 1995), it been comprised in the endogenous opioids for
its resemblance to Dyn A and its ability to bind the Orphan-Receptor-Like 1 (ORL-1)
which scored a 52% amino-acid homology to MOR, DOR, KOR (Darlison and Richter,
1999). Though the receptor for Nocistatin is still under investigation, it was suggested that
its role is opposite that of Nociceptin by attenuating hyperalgesia (Okuda-Ashitaka and Ito,
2000). Interestingly, neither Nociceptin nor Nocistatin display an N-terminal sequence
beginning with Tyrosine, which disables their binding to any of the 3 major opioid peptides
(Mogil and Pasternak, 2001). However, since both Endormorphin-1 and Endomorphin-2
contain Tyrosine in their N-terminal at the start of their peptidic sequences, they have been
shown to strictly bind the MOR with more affinity and selectivity than for DOR and KOR
(Zadina et al., 1997). Their small quadrapeptidic structures have been validated as bioactive
and selective for the MOR given by stereochemical achievements of the first Tyrosine
residue and third pharmacophoric residues (Fichna et al., 2007). As the N-terminal domain
confers togetherness to a versatile class of peptides, receptor selectivity diverges this
molecular signature to broad, practical and enhanced possibilities.
6
1.1.3 Opioid receptor cross-over
It is well recognized that none of the 3 classic families of opioids hold a particular
specificity and selectivity for any of the opioid receptors in particular. Rather, Mansour et
al, showed that opioids with the Met-ENK or Leu-ENK core are required to bind the DOR
and the MOR relatively well and that opioids with an Arginine-X extension, in addition to
the conserved N-terminal core beyond the sixth residue, can also bind the KOR (Mansour
et al., 1995b). Though a general consensus has been achieved through competition binding
studies and later confirmed by double knockout studies for ENK and DYN (Mansour et al.,
1995b; Law and Loh, 1999; Clarke et al., 2003) illustrated in (Table 1), there is
nevertheless a major and inherent overlap between these receptors‟ sequence and structure
(Stevens, 2009).
Table 1. Endogenous opioid superfamily and receptors
7
For simplicity, only the first 8 amino acids of each corresponding peptide are shown
in the second column. The third column provides an approximate ranking system for the
affinity of receptors based on competition constants of selective ligands performed in vitro.
All comparisons are approximate to serve as a visual aid. As illustrated, ENK products
derived from PENK primarily bind MOR and DOR whereas DYN products derived from
PDYN bind KOR. Source: Modified from (Mansour et al., 1995b)
Given the central dogma of biochemistry. a sequence similarity engenders a similar
structure which inevitably is linked to function and validates a structure-function
relationship (Lodish, 2008). For opioid receptors, this influences greatly the binding cross-
over of endogenous ligands given by the preserved homology of their binding domains. By
sequence analysis of cloned opioid receptors, these receptors were revealed to have an
incredible amino acid homology of 73%-76% in the 7 transmembrane domains (TM) of
their GPCR structures and showed to diverge most in the N-terminus (9-10%) and
extracellular loops (14-72%) yet overall exerting a resemblance of over 60% (Law and Loh,
1999). Inevitably, given their high structural resemblance, the downstream events they
mediate are also similar. These GPCRs interact with inhibitory G-proteins cascades, which
after opioid binding, mediate the inhibition of adenyl cyclase, activate PLC, regulate ionic
channels and activate the MAPK pathway for transcription purposes (Davis and Pasternak,
2009). Therefore, given specific receptor selectivity and this vast endogenous ligand
diversity, synergistic pairing mediates downstream signalling. However, it is selective
receptor activation that very often leads to dissimilar pharmacological endpoints and
proposes specific physiological processes. These very diverse processes depict specific
ligand-receptor pairing, yielding processes such as pain, reward, addiction, anxiety, more
importantly but also, tolerance, pregnancy, learning, eating, sexual activity, seizures,
locomotion and many more. These have been readily achieved, marked and combined in
the literature for the past 30 years in the annual publication, “Endogenous opiates and
behaviour” (Bodnar, 2012). Nevertheless, the distribution of an opioid such as ENK is of
first primordial importance in specific receptor binding mechanisms and stress-related
neurobiology.
8
1.1.4 Enkephalin distribution
Given the wide spectrum of physiological processes involving endogenous opioids,
it is inevitable that as they function as neurotransmitters in the brain and paracrine
agents/modulators in peripheral tissues, they exhibit a complex distribution. ENK as a
major contributor within this class, shares and mediates these processes given its complex
distribution in the brain, the periphery and even in non-neuronal tissues (Weisinger, 1995).
Shortly after its discovery, early studies using antisera directed against the 2 ENK
pentapeptides originally discovered, mapped the distribution of ENK in all levels of the
brain (telencephalon, diencephalon, mesencephalon and rhombencephalon and spinal cord)
(Elde et al., 1976; Hökfelt et al., 1977; Simantov et al., 1977; Sar et al., 1978; Wamsley et
al., 1980). Although these IHC studies revealed neurons containing the 2 pentapeptides, it
also revealed the presence of Leu-ENK containing peptides such as DYN (Merchenthaler et
al., 1986) and axonal fibers transporting Met-ENK peptides (Hökfelt et al., 1977; Sar et al.,
1978; Uhl et al., 1979; Wamsley et al., 1980). As was mentioned previously, synonymously
beta-END contains the Met-ENK residues in its N-terminus (Khachaturian et al., 1985),
and extensive antibody cross-reactivity for the N-terminal has been observed with other
opioids also (Gramsch et al., 1983). For this reason, the staining pattern observed in those
studies contained more information than desired and to say the least, was sometimes
imprecise (Merchenthaler et al., 1986). However, it was later discovered that the octa-ENK
is specific to ENK alone and that anti-sera directed against it could represent a better
distribution of immunoreactive ENK (Fallon and Leslie, 1986). Later studies, which
resorted to labelling the RNA messenger of ENK by in situ hybridization (ISH), have been
able to localize solely the cell bodies of Enkergic neurons (Harlan et al., 1987; Hurd, 1996).
Either used alone or in combination with IHC, neurons synthesizing ENK or regions
containing ENK by projection, have since been mapped in all parts of the cerebrum with
greater sensitivity and consensus (Watson et al., 1982, 1984; Khachaturian et al., 1983,
1985; Fallon and Leslie, 1986; Le Merrer et al., 2009). Somas expressing the ENK
transcript are now known to be numerically most distributed and most abundant in the
brain, when compared to the other endogenous opioid cell bodies, (see figure 1) (Le Merrer
et al., 2009).
9
Though this comprises the complete central system of ENKergic neurons, only
select regions have been identified to have a role in stress. These have been identified as:
the cingulated and infralimbic cortex of the medial pre-frontal cortex (mPFC), central,
medial and basolateral amygdala (CEA, MEA, BLA respectively), the lateral and cortical
divisions of the CEA (CEAl, CEAc, respectively), lateral septum (LS), cerebral and
piriform cortex, intercalated nuclei of the amygdala (IA), anterior and posterior bed nucleus
of the stria terminalis (BST), the preoptic area (POA), the hypothalamus (HTH), the
paraventricular nucleus of the thalamus and hypothalamus (PVT, PVH respectively), the
parabrachial complex (PC), the nucleus of solitary tract (NTS), locus coeruleus (LC) and
the ventrolateral medulla (VLM) (Drolet et al., 2001; Poulin et al., 2006, 2008). In the
brain, these regions have laid the foundations for the study of ENK yet localizations of
ENK have also been reported in the peripheral nervous system (PNS) and many non-
neuronal systems with practical corollaries.
10
Endogenous opioid somas across the rodent brain
Figure 1. Classic endorphin cell bodies across the rodent brain
The pattern of distribution of cell bodies expressing enkephalin products as derived from proenkephalin,
is depicted in regions with orange circles. The size of the different „circles‟ designates the density of cell
bodies proportional to the amount of prohormone found within each region as determined by studies of
immunochemistry and in situ hybridization. Particular attention is drawn to orange circles which
designate a widespread distribution of cell somas in all levels of the rodent brain capable of expressing
ENK-related peptides. Obtained from: (Le Merrer et al., 2009)
11
To fathom that a neuropeptide involved in the central neurobiology of pain and
stress could also be involved in the paracrine and autocrine regulation (Weisinger, 1995) of
systems outside the central and peripheral nervous systems, uncovers more of ENK‟s
elusive functional mysteries. Much like for studies in the CNS, cloning studies have
provided the necessary tools to localize PENK, depicting a more accurate distribution of
the origin of the peptides in very vast tissues of the rat anatomy. These have been mapped
in: the testes, spermatids, ovaries, oviduct, uterus, heart, bone, skin, fibroblasts, liver, lung,
kidneys, adrenal medulla, intestines, T-cells and macrophages for review, see (Weisinger,
1995). As derived from studies with bovine species, one of these organs in particular, the
adrenal medulla, harbours chromaffin cells that have been a chosen study prototype in the
biochemical and the molecular biosynthesis of PENK and its secreted ENK products for
many neuroendocrine cell types (Kilpatrick et al., 1980; Jones et al., 1982; Ungar and
Phillips, 1983; Viveros and Wilson, 1983). More specifically, studies involving a bovine
adrenal cDNA probe to PENK hybridized various regions of the bovine brain, suggest a
structure conservation of ENK mRNA in the brain and adrenal medulla (Pittius et al.,
1985). That same study also endorsed the adrenal medulla for being much more abundant
in PENK than any single region of the brain, thus demonstrating its suitability as a model
organ. In the rat, varying amounts of PENK in many regions of the brain have also been
detected, but this time with a human pheochromocytoma cDNA probe revealing not only
brain-adrenal conservation of PENK but also inter-species conversation of principle (Tang
et al., 1983). Although implied when mapping gene transcripts, those studies also
confirmed the correlation between the peptides released after post-translational processing
of PENK and the level of mRNA (Tang et al., 1983; Eiden et al., 1984; Pittius et al., 1985).
Further advancements with this model organ have used a cell line (PC12) derived from a
tumorigenic state of the rat adrenal medulla adequate for the study of endogenous opioids.
Ironically, the PC12 cell line had first been used as a suitable neuronal cell line susceptible
to NGF differentiation in the culture dish before studies proved the adrenal medulla to be a
model organ (Greene and Tischler, 1976). However, this tumorigenic cell line does not
synthesize or express ENK-related peptides unlike a normal and homeostatic adrenal
medulla (Byrd et al., 1987; Fujita et al., 1989; Margioris et al., 1992). Fortunately, later
work showed this cell line was inducible by sodium butyrate (NaB) to express PENK and
12
its gene products (Byrd and Alho, 1987; Byrd et al., 1987). The PC12 cell line thus became
a model cell line stemming from a model organ in the study of neuronal differentiation
either through NGF or NaB, and underlined as a novel in vitro cell line model.
Regardless of its distribution, the action of ENK can only be appreciated if it
coincides with an available receptor. In the neurobiology of stress, the location and the type
of receptor immediately follows ligand distribution and requires careful scrutiny if
behavioural correlations are to be extrapolated. Only central receptors will be discussed,
thus limiting the complexity of their interactions to their central role in the neurobiology of
stress.
1.1.5 A tale of two receptors: distribution and function
Consensus for the binding of endogenous peptides to opioid receptors is arguably
dependent on the final peptide processing as opposed to the derived pro-hormone family. In
the case of PENK- and ENK- derived peptides, most ENK peptides show preferential
binding for the DOR over MOR, while some such as Met-ENK are almost equipotent for
the 2 receptors (Lord et al., 1977; Mansour et al., 1995b; Law and Loh, 1999). The longer
ENK hepta-peptide shows high affinity to all 3 opioid receptors (DOR, MOR, KOR) and
the ENK octa-peptide shows high binding affinity for DOR and KOR with slightly less
potent binding for MOR (Mansour et al., 1995b). For this reason, we therefore
acknowledge and refer to the DORs and MORs as being ENK‟s main recipient receptors.
Much like their endogenous ligands, DORs and MORs have a ubiquitous
distribution in all levels of the brain. Efforts to chart the types and distribution of these
receptors have also utilized a similar approach to the peptide-product vs. mRNA-neuronal
origin, used for the mapping of the endogenous ligands. In the case of the receptors,
however, opioid binding sites were first mainly determined by autoradiographic studies
showing the differential binding dispositions of various ligands and second, by radioprobes
for each receptor type through ISH, for review, see (Le Merrer et al., 2009). Just as for the
cell body locations of endogenous peptides, ISH allowed receptor transcript localizations.
As figure 2 shows, these receptors have been mapped by ligand autoradiography in many
13
nuclei showing the coexistence of multiple receptors at once in most regions of the brain
despite certain exceptions.
14
Endogenous opioid receptor protein locations across the rodent brain
Figure 2. Localization of Enkephalin‟s receptor proteins across the rodent brain.
The following figure depicts most opioid receptor protein locations across the rodent brain. Particular
attention is drawn to the MOR and DOR locations, in red and yellow, respectively which correspond
to ENK‟s preferential receptors. Modified from: (Le Merrer et al., 2009). [Amb, nucleus ambiguus; AD, anterodorsal thalamus; AL, anterior lobe, pituitary; AON, anterior olfactory
nucleus; Arc, arcuate nucleus, hypothalamus; BLA, basolateral nucleus, amygdala; BNST, bed nucleus of the
stria terminalis; CeA, central nucleus, amygdala; Cl, claustrum; CL, centrolateral thalamus; CM, centromedial
thalamus; CoA, cortical nucleus, amygdala; CPu, caudate putamen; CrbN, cerebellar nuclei; DMH, dorsomedial
hypothalamus; DMR, dorsal and medial raphe´ ; DTN, dorsal tegmental nucleus; En, endopiriform cortex; Ent,
entorhinal cortex; FrCx, frontal cortex; G, nucleus gelatinosus, thalamus; G/VP, globus pallidus/ventral
pallidum; HbL, lateral habenula; HbM, medial habenula; HPC, hippocampus; IL, intermediate lobe, pituitary;
IP, interpeduncular nucleus; LC, locus coeruleus; LD, laterodorsal thalamus; LG, lateral geniculate, thalamus;
LH, lateral hypothal- amus; LRN, lateral reticular nucleus; MD, mediodorsal thalamus; Me, median eminence;
MEA, median nucleus, amygdala; MG, medial geniculate; MM, medial mammillary nucleus; MV, medial
vestibular nucleus; NAc, nucleus accumbens; NL, neuronal lobe, pituitary; NRGC, nucleus reticularis
gigantocellularis; NTS, nucleus tractus solitarius; OCx, occipital cortex; PAG, periaqueductal gray; PCx,
parietal cortex; Pir, piriform cortex; PN, pontine nucleus; PnR, pontine reticular; PO, posterior thalamus; POA,
preoptic area; PPTg, pedunculopontine nucleus; PrS, presubiculum; PV, paraventricular thalamus; PVN,
paraventricular hypothalamus; RE, reuniens thalamus; RN, red nucleus; RM, raphe´ magnus; SON, supraoptic
nucleus; SN, substancia nigra; SNT, sensory trigeminal nucleus; STN, spinal trigeminal nucleus; TCx, temporal
cortex; Th, thalamus; Tu, olfactory tubercle; Tz, trapezoid nucleus; VL, ventrolateral thalamus; VM,
ventromedial thalamus; VMH, ventromedial hypothalamus; VPL, ventroposterolat- eral thalamus; VTA, ventral
tegmental area; ZI, zona incerta.]
15
Mismatches between the receptor mRNA and the ligand binding preference have
been reported in various areas of the rodent brain, such as in the cerebellum where DOR
mRNA was found with no ligand binding (also shown in figure 2) (Mansour et al., 1995a).
Although these mismatches remain either mysterious radiolabelling abnormalities or
mRNA transport to presynaptic terminals (Le Merrer et al., 2009), endogenous ENK
knockout by PENK has defined local mechanisms of action for ENK within their effector
regions. Simply, studies have observed receptor upregulation upon genetic ligand
knockouts of ENK, DYN and simultaneous knockouts of both reporting a marked
upregulation of DOR and MOR in limbic areas for PPEKO (Brady et al., 1999; Clarke et
al., 2003). By knockout and implicit developmental upregulation of ENK‟s target
endogenous receptors were confirmed, excluding the KOR which showed 60% more
upregulated in PPDKO mice (Clarke et al., 2003). Most importantly however, these studies
conferred a specific tight circuit of tonic activation by ENK for MOR and DOR in limbic
areas which strongly correlates the lack of ENK with the upregulation of each receptor.
This further involves ENK in the regulation of emotion through MORs and DORs in
regions such as; amygdala (AMY), BST, POA, HTH, periaqueductal grey matter (PAG),
globus pallidus (GP), nucleus accumbens (NAc) and caudate putamen (CP). Concurrently,
these centers adhere to the centers defined within the neurobiology of stress and are
comprised together with the septum, dentate gyrus of the hippocampus (dg-HIP), LC, PB,
NTS, vagus doral motor for those regions expressing MORs and the HIP and AMY for
those regions expressing DORs (Drolet et al., 2001). However, regions like the NTS and
thalamus express very little DOR and regions like the anterior/intermediate/neuronal
pituitary and PVN show no receptors nor mRNA for MORs and DORs, at least in basal
conditions (Le Merrer et al., 2009). What is interesting to note, is that though the
expression of these receptors appears absent, receptor density may increase based on the
expression of ENK mRNA during stress, hereby soliciting its receptors. These regions in
particular are highly modulated by circulating stress hormones in the physiology of the
stress response; the HTH, pituitary and the adrenal glands form the well-known
hypothalamic-pituitary axis (HPA) releasing glucocorticoids (Tsigos and Chrousos, 2002;
Smith and Vale, 2006). The PVH is a complex set of nuclei and has been well recognized
to play a key role in the activation of the adrenocortical stress response by acting as the
16
major source of CRF for the HPA axis, bearing the most CRF-expressing neurons
(Swanson et al., 1987; Sawchenko et al., 1993). In paradigms of acute stress, ENK has been
reported to increase in mRNA levels after an intraperitoneal injection of hypertonic saline
in the parvocellular neurons of the PVN (Lightman and Young, 1989), increase in mRNA
levels after ether stress and immobilization stress in the medioparvocellular neurons of the
PVN (Ceccatelli and Orazzo, 1993) and also increase in mRNA levels for a single stress
session of immobilization in the parvocellular neurons of the PVN (Dumont et al., 2000).
Taken together, these studies show an adaptive role for ENK in select nuclei of the PVN
that is activated under acute stress. Interestingly, under a chronic stress paradigm (repeated
immobilization for 10 days) the ventral portion of the PVH showed a marked increase in
ENK-expressing neurons with sustained activity (Dumont et al., 2000). This study shows a
clear manifestation for a role of ENK, in the adaptation to stress. Furthermore, a similar
study also showed an increase in ENK activation under an acute stress session but in the
ventrolateral medulla (VLM) but with nearly opposite results to the first study, for the
levels of ENK mRNA in the VLM under chronic immobilization (Mansi et al., 2000).
These 2 studies individually corroborate a complex role for ENK, induced under
psychogenic stress and that is ultimately region-specific. These regions therefore underlie a
novel circuit for ENK and demonstrate its activation/inhibition in many key regions that,
under basal conditions, don‟t display high densities of receptors. Though ENK can bind
both DORs and MORs, specificities of each and subtle binding affinities draw a functional
map for ENK as a versatile peptide.
A very interesting phenomenon implicit to the functionality of MOR and DOR and
their existential role in regions of the limbic system, can be extracted from such studies
which have assessed the binding affinity of ENK-related peptides through receptor binding
assays and peptidic competition, (see previous table 1) (Mansour et al., 1995b). Simply
put, if such a derived peptidic product such as the ENK hepta-peptide shows close-to-
perfect equipotent binding to all 3 opioid receptors with high affinity, can these receptors
truly antagonize behaviour? This precipitates the pondering behavioural scientist to
reconsider or disparage the individuality of these receptors. However, these binding
preferences were assessed in vitro, comprising an ideal system to analyse these interactions.
17
The studies were thus devoid of in vivo complexities which define the action of ENK-
related peptides such as: synaptic localization of opioid receptors, differing ENK-related
peptides within select regions (by post-translational processing), endopeptidase degradation
of ENK-ligands, and communicating projections between neurons. Therefore, the role of
these receptors needs in vivo validation and their effector functions are solely dependent on
the ENK peptides post-translationally processed and/or projected to specific regions
harbouring the compatible receptors for said behaviours. Luckily, genetic strains of mice
with MOR and DOR knockouts have in vivo answers for the physiological response of
these receptors.
Although MOR and DOR can both bind ENK-related peptides and often coexist
together in the same regions, a study that targeted the genetic ablation of either receptors in
different mice by homologous recombination showed dichotomous behavioural outputs for
each receptor (Filliol et al., 2000). Specifically, mice lacking the gene (oprd -/-) for DOR
and those lacking (oprm -/-) for MOR, showed opposite results when subjected to the
elevated-plus-maze (Pellow et al., 1985); the DOR deficient mice showed less time and
visits to the open arms of the maze compared to wild-types and the reverse was observed
for oprm -/- (anxiolytic-like behaviour). A similar divergent behaviour between oprd -/-
and oprm -/- mutant mice was also observed when the mice were subjected to the light-
dark box paradigms of anxiety (Bourin, 2003); with the DOR deficient mice showing
preference to the dark compartment. Furthermore this group also reported hyperlocomotion
under no stress for DOR ablated mice while in another study, MOR gene disrupted mice
showed a tendency for hypolocomotion (Matthes et al., 1996). The dichotomy in
physiological roles was also illustrated in yet another elegant study with MOR deficient
mice. Nieto et al, showed that if ENK-products are prevented from being degraded by
endopeptidases via the RB101 selective inhibitor, the continual phasic or tonic effect
solicits the DOR extensively in mood regulation, showing anxiolytic- and anti-depressant-
like functions (Nieto et al., 2005). As was shown during the O-maze test for anxiety
(Shepherd et al., 1994), neither the wild-type mice nor the MOR- ablated mice showed any
divergence in behaviour under the influence of RB101, depicting a clear stimulation of
DORs. However when assessed for jump latency on a hot plate test (Eddy and Leimbach,
18
1953) and after injected with RB101, MOR-deficient mice showed a very short jump
latency compared to their wild-type counterparts, confirming a role solely for MOR in
thermal nociception and not for DOR. Thermal sensitivity has also been reported in other
MOR- deficient studies, alluding to a role of MOR in this particular type of nociception
(Matthes et al., 1998; Qiu et al., 2000; Sora et al., 2001). Although, studies with genetic
ablations for ENK‟s receptors DOR and MOR have shown different physiological roles
varying also in emotional regulation, the roles are not so radical as MOR can also be
anxiogenic or anxiolytic, depending on the contextual components of the study (Wilson and
Junor, 2008).
Morphine, the potent agonist for the which the binding to the MOR was first
observed to mediate analgesia (Martin, 1967) long before the MOR was discovered, is still
used today. Although morphine has preferential binding for the MOR and lower affinity for
the KOR and DOR respectively, the ENK peptides nevertheless have higher binding
affinity than morphine to both DOR and MOR across the board (Raynor et al., 1994;
Mansour et al., 1995b). The specific binding of morphine to the MOR and ensuing
analgesia was validated behaviourally in vivo, in a study with mice disrupted for their MOR
gene product (Matthes et al., 1996). When both strains of mice were administered
morphine, MOR knockout mice showed no (spinal) analgesic effects and interestingly also
no rewarding effects of morphine-induced place preference, in contrast to wild-type mice.
By knockout, this study provided fundamental fuel to all studies building on the MOR and
its role in nociception. Interestingly, the MOR has also been postulated to have an
important role in ethanol consumption. MOR knockout mice experience less ethanol
consumption and reduced rewarding properties of ethanol (Roberts et al., 2000; Hall et al.,
2001). Much can be elaborated on the rewarding properties of the MOR for palatable
substances especially in the NAc (Kelley et al., 2003). The MOR agonist, DAMGO ([D-
Ala2-N-Me-Phe4-glycol 5-enkephalin (DAMGO)) amplifies the “liking” reactions to
sucrose and has carved a place for opioids in the reward circuit between the NAc and VP
(Peciña et al., 2006; Smith and Berridge, 2007). The NAc and VP work closely together in
“wanting”, also mediated by opioids. These structures harbor strong projections and are
both rich in ENK (Zahm et al., 1985; Kalivas et al., 1993; Lu et al., 1997). Additional
physiological repercussions of receptor knockouts are shown in figure 3.
19
The physiological repercussions of endogenous opioid receptor knockouts
Figure 3. Brief summary of receptor knockouts for mood disorders and addiction.
The following figure shows the compiled efforts of the multiple receptor knockouts. An
interesting dichotomous behavioural phenotype results in response to anxiety for the DOR
and MOR knockouts. Although the DORs seem to reduce anxiety, enhanced rewarding
properties are better mediated by the MOR than the other 2 receptors. KOR knockouts
however, seem to manage anxiety yet respond better to ethanol and nicotine. Source: (Lutz
and Kieffer, 2013)
20
Studies involving genetic ablation of these receptors nevertheless have limitations
on what can be observed behaviourally. Even if the genetic disruption of one receptor does
not have major repercussions on the expression of the other 2 opioid receptors for review,
see (Kieffer and Gavériaux-Ruff, 2002), compensatory changes may result in utero, since
the mice show normal growth and homeostatic behaviour (Matthes et al., 1996; Filliol et
al., 2000; Kieffer and Gavériaux-Ruff, 2002). A more interesting profile of action for these
receptors can be complemented by pharmacological substrates in agonist/antagonist
binding to MOR and DOR but, more importantly, looking at genetic ablations of the
PENK, draws directly into the physiological role of the endogenous ligand, ENK.
1.1.6 Enkephalins and anxiety
A conclusive yet preliminary link between ENK and stress came with genetic
knockouts of PPE, which historically created prior receptor knockout strains. Published just
21 days prior to the MOR knockout study (Matthes et al., 1996), ENK was postulated to
modulate pain, thus lowering the threshold of pain in PPEKO mice (König et al., 1996).
Beyond pain, however, PPEKO mice showed heightened vigilance leading them to be
instantly intolerant to intruders in the „resident-intruder‟ test and demonstrate anxiety. Most
importantly, mice showed increased anxiety by demonstrating thigmotaxis (Simon et al.,
1994) in the open field test and testing with the elevated-O-maze showed less time in the
center of the maze (König et al., 1996). These 2 independent studies thus converged on the
principle of pain for the ENK-MOR system, despite a poorly selective ligand class and a
moderately promiscuous receptor. However, further genetic knockout studies showed a
greater role for ENK in anxiety-related experimental endpoints (Ragnauth et al., 2001;
Bilkei-Gorzo et al., 2004). In a paradigm of fear conditioning, homozygous PPEKO mice
showed increased freezing before and after auditory stimulation, on the second day of
testing over-shooting wild-type and heterozygous PPEKO mice, revealing an implicit role
of ENK in fear-related behaviour (Ragnauth et al., 2001). These PPEKO mice also showed
increased thigmotaxis in the open field test and showed less activity and time spent in the
light portion of the light-dark box paradigm, demonstrating a concurring link to anxiety.
Although the excessive anxiety behaviours are reproducible in mice with almost all PPEKO
experiments, the effect is nevertheless unspecific. Given that the genetic ablation of ENK is
21
global, all stress centers of the mouse brain are also affected. In turn, this does not allow
tracing anxiety-like behaviour, functionally nor anatomically. In order to understand and
link function, behaviour and anatomy, a more localized method of analysis would be
required, targeting one region at a time.
1.2 Lentiviral mRNA interference
1.2.1 The advent of gene-silencing
The world acclaimed work of Andrew Fire and Craig Mello has revolutionized our
understanding of cellular machinery with the discovery of RNA interference (Fire et al.,
1998). The principle as discovered then, and elucidated in great detail in later years, has
allowed the trivial assessment of the gene function by the deletion of the mRNA transcript
leading to the synthesis of a peptide (Elbashir et al., 2001b, 2001c). By recruiting the Dicer
enzyme along the endogenous silencing pathway of cells, the complementarity of the
mRNA can be readily detected and processed as a double-stranded (DS) duplex. As figure
4 shows, the processing of the post-transcriptional mRNA by RISC (RNA-induced
silencing complex) ultimately leads to the destruction of the mRNA (Elbashir et al., 2001a;
Ichim et al., 2004). Given the wonderful and numerous applications of RNAi, an armada of
methods and vehicles has advanced silencing techniques, offering advantages and option
specific to each silencing of RNA.
22
Lentiviral silencing mechanism
Figure 4. Lentiviral silencing mechanism for targeted mRNA repression.
Of the 3 methods to activate the mechanism behind RNA interference (RNAi) (1), (2), (3),
lentiviral particle transduction allows for the consistent deletion of mRNA. Short duplexes of
double stranded interfering RNAs (siRNAs) (1) are designed in 19-29 nt length to be inserted in
(2) shRNA plasmid DNA expression vectors. After initial transfection into cells, hijacking of
cellular machinery allows production of replication defective shRNA lentiviral particles (3). By
lentiviral vector particles, shRNA enters the cells by being transduced onto cells. The lentiviral
genome is then reverse-transcribed to be integrated in the chromosome of the host cells. Viral
genes and the encoded shRNA are transcribed together with the cell host genes. After
transcription of the shRNA, the stem-loop generated (a) gets cleaved by Dicer (b) to yield a 21nt
sense-antisense strand duplex yielding the similar DS duplex siRNA originally designed. RISC
incorporates the antisense strand (d) and uses it to destroy the mRNA when perfectly
complementary (e-f) or inhibition of translation of the mRNA when complementarity isn‟t as
prominent (not shown). Lack of repression is observed if no sequence homology exists as for
scrambled sequence control, ShSCR. Adapted from: “ RNAi Gene Silencing”, RNAi-directed
mRNA cleavage; Santa Cruz Biotechnology www.scbt.com/gene_silencers.html
23
1.2.2 Transient and long-term methods of silencing
One of the most capital and discriminating reasons to select a method of RNAi is
the temporal aspect of the silencing required. A comparison between double-stranded
duplex small-interfering RNA‟s (siRNA) and short-hairpin RNA (shRNA), illustrates best
the temporal discrimination that can be achieved through RNAi. Given their synthetic and
small nucleotide structure, siRNAs are readily available commercially and are viewed as
„transient‟ effectors of mRNA silencing. The basic principle of RNA interference is that
after transduction into the cytoplasm of a cell, transfection of the siRNA can bind Dicer in
the cytoplasm or bypass, and load onto RISC directly for nuclear import/export for review,
see (Rao et al., 2009b). Although this may seem conducive to a viable knockdown, not only
is the RISC loading process 10 times less efficient than for shRNA (Sano et al., 2008; Rao
et al., 2009b) but also dividing cells do not propagate the RISC guiding strand. Although
some have appeared to challenge this notion in non-dividing systems, such as a neuronal
system through repeated infusions of siRNA, that is certainly not a unanimous case for all
applications or medical disorders (Zhang et al., 2009). Although shRNA can also be made
to induce either transient or stable silencing, in a transient setting, it employs a more
efficient mechanism which activates RISC by virtue of being synthesized in the nucleus
after its transduction into cells (Cullen, 2005; Rao et al., 2009b). Also in comparison to
shRNAs, siRNAs have been shown to be less potent in silencing mRNA, thus generating
„specific‟ off targets for mismatched mRNA (Lin et al., 2005). „Specific‟ off targets, are
generated when an siRNA mismatches with an mRNA transcript of another gene, and „non-
specific‟ off targets are unintended interactions with mRNA, outside of the actual binding
for review, see (Rao et al., 2009a). Though so far, preferential aspects of silencing have
been attributed to shRNAs for basic molecular advantages, the method of delivery can
transform a transient silencing shRNA plasmid into a long-term silencing method.
1.2.3 Advantages of viral induced silencing
Over time, the transfection method of siRNAs and shRNAs has been segregated,
based on viral and non-viral systems of delivery. A viral system of delivery confers the
interesting possibility to be pseudotyped for a specific cell type and hence can have higher
transfection efficiency (Burns et al., 1993; Cronin et al., 2005). However, not all viral
24
vectors can be altered in tropism. On the other hand, the most likely alternative to a viral
vector is a lipid-based vector, which is a cationic-lipid DNA complex (Elbashir et al.,
2002). However, cationic vectors have generally been known to accompany a degree of
toxicity and delivery is not readily accessible for in vivo experiments given the blood-brain-
barrier (Trülzsch and Wood, 2004; Yew and Scheule, 2005; Lv et al., 2006). Seeing that
many siRNAs are delivered in lipid-form (Dykxhoorn et al., 2003), the viral delivery of
shRNAs is an interesting alternative.
1.2.4 Viral transductions in silencing
To date there are many classes of viruses that can be used to carry short RNAs,
especially in the broad field of gene therapy, as shown below. For review, see (Ginn et al.,
2013).
Vectors used in Gene Therapy in 2012
Figure 5. A vast choice of viral vectors exist today.
The following figure shows the viral vectors that were used in a clinical context in 2012.
Most of these are available and have been used in gene silencing mechanisms. Source:
(Ginn et al., 2013).
25
When considering the choice of a viral vector for silencing purposes, many factors
need to be considered. As was mentioned previously, an shRNA can be made into a
transient or a stable silencing machine (Hommel et al., 2003). This is because, given the
proper delivery vehicle, steps specific in insuring that experimental conditions will extract
the unique features of each vector. Though figure 5 shows multiple vectors, there are 5
commonly used vectors for gene-silencing. These are: herpes-simplex 1 viruses (HSV-1);
oncoretroviruses of which (muloney murine leukemia virus (MLV) is part of); adenovirus
(ADV) and adeno-associated virus vector (AAV) for concise review, see (Kay et al., 2001).
As some would argue, the dividing line for these vectors depends on the requirement for
their integration in the genome of the cells or simply remaining in the cytoplasm (Thomas
et al., 2003), see table 2.
One class in particular has multiple features that promote genome integration very
efficiently. Lentiviruses based on HIV-1 are viewed as a more robust virus, not just
amongst the other retroviral vectors but as a class of their own. While viral members of the
retroviral class are very similar to lentiviruses and equally able to activate the RNAi
machinery, their inability to transfect non-dividing cells makes them less qualified for
neuronal work (Blömer et al., 1997). The exact intricate mechanism limiting the tropism of
retroviruses is still unresolved. The going hypothesis is that some types of retroviral
viruses, like the MLV, enter the nucleus of dividing cells at mitosis, whereas lentiviruses
enter growth-arrested cells through the nuclear pores (Durand and Cimarelli, 2011).
However, though a definite explanation cannot be yet provided, an early comparative study
showed that lentiviruses express transgenes long-term, the longest over MLV, AAV and
ADV vectors (Blömer et al., 1997). The robustness of the genus has been further exploited
for the generation of a packaging cell line accompanied by high-throughput TU/ml (Farson
et al., 2001). Such features make lentiviral vectors useful for many applications despite
major concerns for their capacity to infect humans, as they are derived from a virus which
is responsible for a global pandemic. However, seeing that these construction plasmids are
administered in trans from plasmids lacking a packaging signal, and having missing long-
terminal-repeats (preventing self-replication), the replication of lentiviral particles is
unlikely (Tiscornia et al., 2006) but not unheard of (Logan et al., 2004; Wanisch and
Yáñez-Muñoz, 2009).
26
Table 2 - Different features of viral vehicles
Source: (Thomas et al., 2003)
1.2.5 Rationale in relation to ENK-silencing: Using a stable viral construct
The use of viral technology to study ENK provides an insightful way of looking at
the peptide under a different lens. ENK has been inserted in the HSV-1 vector in multiple
studies, either destined for the CNS or the PNS. The overexpression of ENK by an HSV-1,
vector has been shown in multiple studies to reverse pain in the dorsal root and more
recently in the caudal ventrolateral medulla (Goss et al., 2001; Wilson and Yeomans, 2002;
Lee et al., 2006; Martins et al., 2011). Though the ventrolateral medulla is a known pain
center for its descending inhibitions (Tavares and Lima, 2002), the overexpression of ENK
in the lateral most part of the VLM by a HSV-1 vector, induced antinociception at the
spinal cord, 10 days after injection (Martins et al., 2011) thus activating a local MOR
population. By using a similar HSV-1 vector overexpressing ENK virus, injections in the
central nucleus of the amygdala (CEA) showed a similar antinociceptive effect as assessed
by the formalin test (Kang et al., 1998). This study therefore showed a region-specific role
27
for ENK in antinociception by overexpression, which alludes to a similar role for ENK as
was shown by the global ENK knockout studies and exaggerated pain (König et al., 1996;
Bilkei-Gorzo et al., 2004). This same team used the same HSV-1 vector to overexpress
ENK as injected in the CEA and bordering the BLA of the amygdala, to potentiate an
indirect anxiolytic effect (Kang et al., 2000). Indeed, not the mere overexpression of ENK
but rats injected with diazepam and infected with the vector overexpressing ENK, spent
more time in the open arms of the elevated plus maze as compared to vehicle or control
HSV vectors for diazepam injected-rats. This corroborates with results obtained in our lab,
which have shown that ENK mRNA is greatly found in the lateral part of the CEA (CEAl)
(Poulin et al., 2006) where the much needed GABA/benzodiazepine receptors are located
and required for the full anxiolytic effect (Kang et al., 2000). The CEA is a important
nuclei of the amygdala as it integrates not only intra-amygdaloid ENKergic projections
from the CEAc and CEAl subdivisions onto the CEAm but its CEAm is a major source of
amygdaloid output (Petrovich and Swanson, 1997; Jolkkonen and Pitkänen, 1998; Poulin et
al., 2006). Much like what is reminiscent from the many functions of the amygdala as a
whole (Davis, 1992; Swanson and Petrovich, 1998; Davis and Whalen, 2001), the CEA
mediates a series of functions such as; ethanol reinforcement (Hyytiä and Koob, 1995),
positive emotional learning in reward (Murray, 2007), and of course, fear and anxiety
(Davis, 1992; Fox and Sorenson, 1994; Rogan et al., 1997; Kalin et al., 2004). Under a
paradigm of bilateral excitotoxic lesions to the CEA in primates, bilaterally lesioned
monkeys display reduced fear to snakes and reduced freezing upon human intruder
interference, as compared to controls (Kalin et al., 2004). In studies performed with
rodents, lesions to the CEA also reduce HPA activity (Prewitt and Herman, 1994). By
studying the classical fear conditioning with paired conditioned (CS) and unconditioned
stimuli (US)- , see, (Pape and Pare, 2010), an insightful structural circuit results, involving
the CEA as the primary output structure and the BLA as the primary (sensory) input
structure for CS/US stimuli (Sah et al., 2003; Pape and Pare, 2010; Marek and Strobel,
2013). Though the BLA can receive thalamic and cortical inputs for the propagation of a
CS to the CEAl, different neuronal populations may require distinct inputs offering a bi-
directional outcome (Herry et al., 2008). Upon initiation of the fear response, the BLA
innervates the CEAl, which will innervate the CEAm, and which will innervate the
28
brainstem and hypothalamus (Pape and Pare, 2010). Though pharmacological infusions and
manipulations with various non-NMDA antagonists have been shown to block extinction or
fear conditioning (Falls et al., 1992; Kim et al., 1993), the intercalated nuclei of the
amygdala (IA) after a selective lesion, are indispensible for extinction (Likhtik et al., 2008).
As extinction involving IAs is understood, the BLA transmits the CS to the IA neurons
which then send GABAergic projections to the CEA (Likhtik et al., 2008). When looking at
the neurobiology stress, this is of particular intrigue especially, when contemplating a
partial depletion of ENK.
The BLA has many target outputs across the brain (for review, see (Davis, 2002)),
and various lines of evidence ascribe a possible opioid modulation for some of its target
projections. When looking at the BLA it is well known to send glutamatergic projections to
the IA (Royer et al., 1999, 2000; Poulin et al., 2006); a region which produces ENKs (co-
localized with GABA) and is abundant in MORs (Poulin et al., 2006). Furthermore, the
BLAa sends ENKergic projections to the CEA, which is most likely glutamatergic (Poulin
et al., 2006), outside of the tight system of projections witnessed in the amygdala (Pitkänen
et al., 1997). Finally, the BLAp is also known to project to the anterolateral nuclei of BST
(Dong et al., 2001a), more specifically; the rhomboid nucleus, the subcommissural zone,
juxtacapsular nucleus, less so the oval and fusiform nuclei yet considerable inputs to the
medial group of the anterior division (Dong et al., 2001a). The BST is a versatile structure
able to innervate viceromotor neurons in the hypothalamus and brainstem (Dong et al.,
2001b) when it receives input from the CEAl (amongst others) onto its oval and fusiform
nuclei (Petrovich and Swanson, 1997). Also, it is able to project ENK onto the CEAl
(Poulin et al., 2006). Moreover, the anterolateral part of the BST shows strong projections
to the VTA thus being a center able to receive emotional information yet integrating it in
the reward circuit (Jalabert et al., 2009). In a study with injections of the non-selective
opioid antagonist Methylnaloxonium, injected into the BST and NAc Shell, suppression of
heroin intake was observed in dependent rats (Walker et al., 2000). Furthermore, the BST
was shown to mediate stress-induced reinstatement for cocaine, in deprived rats by the
induction of CRF, in the CEA to BST pathway (Erb et al., 2001). Taken together, though
the IA, BST and CEA ultilize ENK in different ways, its artificial removal from a circuit
29
could help determine its role whether in circuits of fear, reward or anxiety. When
contemplating a partial depletion of ENK, the BLA, has an important proportion of
ENKergic cells which could thus probe for its involvement in projected structures given
anxiety/fear paradigms and shed light on the projections modulating behaviours.
30
Research Hypothesis and Objectives
Research Rationale and Hypothesis Many studies have acknowledged the functional role of ENK as essential and
an endogenous modulator within the neurobiology of stress both within acute
and chronic stress paradigms. Results from our lab strongly corroborate these
results with fluctuations in the levels of ENK expressed. As noticed under our
chronic stress paradigms within the NAc and the BLA (Poulin, 2010), a
decrease in ENK mRNA is reported for vulnerable animals compared to
control and resilient groups. We thus hypothesize that by developing RNA
interference for ENK mRNA in these regions, we would generate a realistic yet
efficient downregulation of its expression. In light of this perspective, the
following objectives comprise our concise plan of action.
Research Objectives Our first objective is to be able to develop a high-titer production of lentivirus
particles harbouring an expressing shRNA capable of attenuating specifically
ENK mRNA expression. Given that lentiviruses have been engineered and
developed to express a short-hairpin subject to activate Dicer in RNA
silencing, they can efficiently be used as vectors to activate the gene silencing
mechanisms of neuronal cells (Hommel et al., 2003). By carefully
implementing a constitutive shENK expression cassette, prior to an H1
promoter within an expression vector plasmid imbedded in a third-generation
packaging system, it has enabled us to construct viruses (Dull et al., 1998).
Finally, by improving steps of the production methodology, in order to satisfy a
neuronal in vivo system a proper, reproducible and high-titered virus batch can
be generated (Salmon and Trono, 2006; Tiscornia et al., 2006; Barde et al.,
2010).
Our second objective serves to establish a cell line capable of harbouring and
demonstrating the effects of a lentiviral transduction to attenuate ENK mRNA
expression in vitro. By using the tumorigenic and undifferentiated rat adrenal
31
pheochromocytoma cell line, PC12 as our model cell line, it can first be
differentiated with sodium butyrate (NaB). PC12 cells can be differentiated
with NaB over the course of multiple differentiation sessions as many teams
have shown the upregulation of ENK mRNA and the secretion of ENK
peptides (Byrd and Alho, 1987; Byrd et al., 1987; Mally et al., 2004). Thus, by
differentiating a cell line and inducing the expression of ENK mRNA, these
cells can be transduced with high-titer lentiviral particles to witness an
apprehensive, in vitro knockdown. By first witnessing a knockdown in vitro, as
transcript output can be measured by RT-PCR, the level of knockdown can be
quantified. This in vitro system would serve to evaluate the knockdown in a
simple system, prior to the delivery of lentiviral particles in the rat brain.
Our third objective serves to stereotaxically deliver lentiviral particles
expressing an shRNA specific to ENK mRNA and quantify the downregulation
of the transcript. By targeting ENK rich areas such as the NAc and the
amygdala, lentiviral particles can be delivered to transduct neuronal cells with
high efficiency. ENK knockdown can thus be quantified by first localizing the
integrated viral expression vector by immunohistochemistry (IHC) of
polyclonal antibody directed against the reporter gene expressed by the vector,
copGFP. Then, by classic in situ hybridization techniques, the resulting mRNA
expression of ENK can be radioactively probed for and quantified in vivo.
32
33
CHAPTER 2
2 MATERIALS AND METHODS
2.1 Production of an shRNA Lentiviral System
In order to produce an effective silencing mechanism for the specific and long-term
attenuation of ENK mRNA, multiple technical steps are required to be compatible and to
integrate with one another. In our case we have chosen to proceed with an HIV-1 derived
third generation lentivirus which confers novel silencing features upon delivery to recipient
cells. By first designing an shRNA specific to our target protein, and inserting an
expression cassette into an expression plasmid, the entire plasmid can be easily replicated
in bacteria along with the packaging plasmids. The expression plasmid bears cis- acting
elements which will optimize the production and packaging of the lentiviral genome (Dull
et al., 1998). Three necessary packaging plasmids representing the HIV-1 accessory genes
gag-pol, rev and envelope protein (pMDL, pREV and pVSVg) are administered in trans-,
to ensure the proper packaging of the lentiviral genome leading to the production of viral
particles. After infection, gag-pol proteins from the pMDL plasmid will mediate events
leading to the integration of lentiviral genome into the host cell (Tiscornia et al., 2006).
Preceded by a strong promoter, when integrated, the expression cassette is constitutively
expressed giving rise to a stem-loop structure which will activate the RNAi silencing
mechanisms of the cell.
By selecting a robust cell line capable of producing high-titer particles, an adequate
titer of particles will be useful in attenuating ENK mRNA after transduction to target cells.
Higher-titer particle generation, wide cell tropism and stable integration, make lentiviral
34
particles ideal for both in vivo delivery and cultured cell lines. For both in vitro and in vivo
experimentation, lentiviral particles should be built the same way with the same short-
hairpin template of silencing RNA, directed for ENK mRNA downregulation. Therefore we
have sought to synchronise the steps of our production protocol to achieve desirable and
infectious viral particles.
2.1.1 Plasmid amplification
Given the nature of a third-generation lentiviral expression system, the production
protocol called for 4 plasmids; one expression plasmid with a gene-specific target shRNA
template and 3 „accessory‟ or packaging plasmids, aiding in the formation of lentiviral
particles (replication, transduction, packaging). Together, these 4 plasmids were transfected
into eukaryotic productive cells, such as (293TN), which resulted in the production of a
viable infectious unit (IFU) viral titer. Viable IFU‟s are viral particles with successful
infection-competent qualities for other cells.
The cellular machinery of the 293TN cells had to recognize promoter sequences,
origins of replications, poly-adenylation signals and long terminal repeats in order to have
successfully generated viral particles. A definite threshold amount of each plasmid was
required for each transfection of the producer cells (293TN). Hence, plasmids had to be
amplified in larger quantities respective to the efficient quantitative (μg) ratio for each
plasmid. These recommendations as well as necessary materials for the successful
production of viral particles were inspired from the user manual: “Guide to Packaging and
Transduction of Target Cells Lentivector Expression Systems: User Manual” written by
System Biosciences.
2.1.2 Vector construction
The purpose in constructing an expression vector targeting ENK mRNA was to create
an easily transfectable plasmid able to express a short-hairpin attenuating the expression of
ENK by means of a specific template for ENK mRNA. Preliminary work in our lab aided
in the building of an efficient silencing shRNA sequence. Based on 2 validated shRNA
sequences from Qiagen, the design of our short-hairpin mainly relied on the ability of these
35
2 sequences to target adequately ENK mRNA and successfully attenuate its expression.
The 2 sequences targeted different bases on the ENK mRNA (560-579) and (135-154) of
the preproenkephalin mRNA (NM_017139). Each of these 2 shRNA candidate sequences
was designed to target the ENK mRNA, with the conditional possibility of successfully
activating either of the 2 sites for the RISC and Dicer-processing enzymes. As a fully
functional silencing hairpin, the shRNA template for ENK silencing consists of a total of 58
bases that can fold and self-hybridize and that is joined by a loop to complete the hairpin
(see figure 6 for base pairs). In its linear form, the 58 nucleotides were annealed and ligated
into the pSIH-H1-copGFP expression vector (System Bioscience) via BamHI and EcoRI
restriction sites. As a means to verify the infectivity of the virus, preliminary vector
construction protocols indicated a virulence and knock-down efficiency attributed to the
shRNA ENK template targeting the 560-579 base pairs as manifested in coronal sections of
the striatum (Poulin, 2010). Hence, the first shRNA sequence targeting bases (560-579)
was selected in the construction of a proper expression vector and was used in the
attenuation of the ENK mRNA cytoplasmic prepropeptide, for the present study (fig 6).
A control sequence, named „scrambled sequence‟ built of 58 oligonuclelotides was
also synthesized and sequenced but this time, with no homology to any mRNA within the
total rat RNA content. The control shRNA was annealed within the same expression vector
pSIH-HI-copGFP plasmid (System Biosciences) (see figure 6 for full sequence of the
shSCR). This expression vector was carefully selected for both types of shRNA templates
because of its great advantage to express viral genes essential for viral packaging and no
demeanor towards the shRNA template design.
36
5'- gatcc GGGATAACATCGACATGTA TTCAAGAGA TACATGTCGATGTTATCCC tttttgaat -3’
3’- ctagg CCCTATTGTAGCTGTACAT AAGTTCTCT ATGTACAGCTACAATAGGG aaaaac -5’
5'- gatcc TTCTCCGAACGTGTCACGT TTCAAGAGA ACGTGACACGTTCGGAGAA tttttgaat -3’
3’- ctagg AAGAGGCTTGCACAGTGCA AAGTTCTCT TGCACTGTGCAAGCCTCTT aaaaac -5’
Template Constructs for Short hairpins RNA used in
Lentivector Construction
Figure 6. As inserted in the expression vector PSIH1-H1copGFP, top and bottom
strands of the short-hairpins generators.
The expression of the ShRNA is controlled by the H1 promoter and transcribed by RNA
polymerase III. The loop segment allows a stem-loop structure build. Antisense segments of the
short-hairpins hybridize to the cell‟s sense mRNA strand to yield a duplex that is processed by
cellular silencing machinery (Dicer and RISC). The terminator sequence ends the transcription.
The copGFP stretch of nucleotides (not shown) precedes the H1 promoter and is itself flanked by a
CMV promoter (not shown). The ShENK was chosen as being specific to target and bind
complementarily, 19 nucleotides of the rat ppENK, hereby inducing a stable knockdown of ENK
protein. The vector expressing the ShSCR was deemed to act as a control surgical experiment,
given its lack of homology and complementarity in the transcriptome of the rat. (ShENK;
Enkephalin short-hairpin, ShSCR; Scrambled short-hairpin)
Adapted from: “pSIH-H1 shRNA Cloning and Lentivector Expression system”;
System Biosciences, http://www.systembio.com/downloads/Manual_pSIH-H1_4-080514_Web.pdf
Sh
SC
R
S
hE
NK
ShENK and ShSCR template
insert
37
2.1.2.1 DNA cloning; a brief summary
Since the advent of “recombinant DNA technology” or DNA cloning, groups have
generated and amplified identical DNA. By piecing together various DNA sequences, as
molecules coming from different sources, the term “recombinant DNA” was coined
(Lodish, 2008). Simply put, recombinant DNA is comprised of a DNA fragment and a
vector which are ligated and then introduced within a host cell. The DNA is then replicated
alongside with the cellular machinery. The originality of such an idea was revolutionary in
1974 when Stanley Cohen and Herbert Boyer ligated in vitro eukaryotic DNA encoding for
ribosomal RNA to a bacterial plasmid, pSC101. This recombined DNA (DNA fragment
and plasmid) was then introduced into Escherichia Coli (E.Coli) cells through
transformation. After being amplified and replicated by the E.Coli, the plasmids were then
extracted from the bacterial cells. The plasmids were then expressed to show the potency of
the amplification process resulting in the functional expression of the coding sequence for
ribosomal RNA (Morrow et al., 1974). Much of the work with E.Coli cells has since been
reproducibly elucidated in a simple and short method of gene amplification (Beardsley,
1984). Plasmid replication into E.Coli cells has thus become routine work in many labs. In
our case, we have tailored our plasmid propagation to this method and have selected this
method of amplification for the replication of our 4 different third-generation plasmids.
This process was divided experimentally into 3 steps equivalent to 3 days of work; bacterial
transformation, bacterial inoculation and plasmid extraction.
2.1.2.2 Bacterial transformation; day 1
Bacterial transformation for the pSIH-H1-copGFP expression vector, as well as
packaging plasmids (pMD2, pMDL-RRE, pRSV), is the introduction of these into the
highly competent E.Coli, DH5α cells (Invitrogen). The expression plasmids and the
packaging plasmids all hold an ampicillin anti-biotic resistance gene (AmpR) used for
selection in E.Coli cells. As mentioned in the industrial user guide by System Biosciences
for ligating and transforming shRNA constructs, competent E.coli strains harbouring a recA
gene mutation (recA-) are recommended for the propagation of plasmids. This RecA
mutation present in DH5α cells confers greater stability to plasmids in the process of
replication due to lower frequencies of spontaneous mutation deletions and lack of
38
homologous recombination (Tolmachov et al., 2006). Further, the strain also holds an EndA
mutation which protects against endonuclease activity during purification (Carnes, 2012).
DH5α cells were properly grown in cell plating media consisting of Luria Bertani
Medium. We therefore generated LB medium (for 1L of water; 10g Tryptone, 5g Yeast,
10g NaCl and pH adjusted to 7.0; Sigma), added 15g Agar (Sigma), and added ampicillin
(Sigma) to a final concentration of 50 μg/ml. The liquid solution of LB with Agar was then
autoclaved and poured into 100mm petri dishes until solidification. Plates were then ready
to support bacterial growth.
Each plasmid was diluted to a concentration of 100ng/μl prior to the start of the
transformation as resolved with an optical densitometer at the 260nm wavelength. 1 μl of
this dilution was then added to an eppendorf containing 100μl of DH5α cells which were
kept on ice for 30 minutes. In this manner, each plasmid was unique and homogeneous to
the competent cells within its eppendorf. Positive and negative control eppendorfs were
also included, respectively; 5 pg/µl pUC 19 (Invitrogen) and 1μl high grade water were
added to 100μl of DH5α cells for each respective control. Plasmid poration into the DH5α
cell membranes was performed by “heat shock” at 42oC for 30 seconds in a calcium
independent way. This step allowed competent cells to take up foreign naked plasmid DNA
through the membrane of cells (Van Die et al., 1983; Singh et al., 2010). Once the cells
were transformed, the eppendorf containing the bacterial suspension was put on ice for 2
minutes. Near a Bunsen burner‟s flame, 900μl of Super Optimal broth with Catabolite
repression (S.O.C.) were added (2% tryptone, 0.5% yeast extract, 0.4% glucose, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4; Invitrogen) to each eppendorf
containing 100μl of bacterial culture. S.O.C is a superior form of media that is richer in
peptide content than other media and that also contains glucose; a prime carbon source for
bacteria. S.O.C, thus, aids in bacterial survival after the plasmid poration hereby increasing
the transformation effectiveness (Ejsmont et al., 2011). With vigorous shaking (240 rpm),
each eppendorf containing plasmids to amplify (with S.O.C) and the 2 controls were sent to
37oC in a shaking-incubator for 60 minutes. Following agitation, 200μl of each plasmid
were added to solid media plates containing ampicillin. These solid agar plates were then
39
placed in an incubator overnight at 37oC. Resulting colonies were selected the next day to
proceed with bacterial growth.
2.1.2.3 Bacterial inoculation; day 2
The principle behind bacterial culturing is to grow bacterial colonies efficiently in
order to purify greater amounts of plasmid DNA used for further experimentations, in our
case for viral particle production. Many bacterial models for growth have been devised with
consensus on 4 crucial phases; the lag phase (slow growth), the log phase (exponential
growth), a stationary phase (stagnant growth due to nutrient depletion) and the final phase
where bacterial cells start to die off, called the death phase (Zwietering et al., 1990). By
being aware of such a growth curve, culture parameters can be adjusted to optimize for
critical production of bacteria and provide experimental checkpoints.
We have used an industrial bacterial culture plasmid preparation to enhance yield and
quality of plasmid DNA. The first step was to select bacteria to inoculate a starter culture
on selective ampicillin media. The point of the starter culture is to allow the bacteria to
transition from the lag phase to the log phase. There is also an advantage to the starter
culture in that, it acts as an experimental checkpoint in time; past the transformation but
prior to the harvesting of bacterial culture if stored at 4oC.
Following industrial recommendations, a pre-culture or „starter culture‟ for each
plasmid was performed by taking a single isolated colony from the respective plasmid‟s
agar plate with an autoclaved toothpick. The colony and toothpick were placed into 5ml of
LB medium which also contained 1μl/ml of ampicillin. The incubation proceeded at 37oC
in a shaking-incubator for 8 hrs in order to generate high cell density and a bacterial log
phase. The bacteria were diluted 1/500 into LB medium to prevent overgrowth. Thus, the
dilution ratio prescribed inoculation in 250ml of LB with 500μl of starter culture for each
plasmid. The incubation proceeded at 37oC, overnight (16 hrs) in a shaking-incubator.
Inoculations were performed in duplicates for each plasmid present. Positive and negative
controls reflected growth for the pUC 19 plated petri and no change for the water control
40
plate, respectively. From a molecular level, this step confirmed the logarithmic bacterial
cell replication and the stationary phase of plasmid replication. At the end of the 16 hrs to
circumvent bacterial decline, the plasmids were then subjected to extraction.
2.1.2.4 Plasmid extraction; day 3
Plasmid isolation was performed by using an industrial Qiagen Inc commercial
(Maxi) plasmid purification kit, “EndoFree Plasmid Purification Handbook EndoFree
Plasmid Maxi , Mega , Giga Kits”, reducing the probabilities of contamination. Purchasing
a purification system also enhances the quality control available in limiting the
contamination lab dishes when performing endotoxin-sensitive experiments. During the
purification process, as the outer membranes of gram negative bacteria (in our case DH5α
cells) are lysated and shed, the liberation of the outmost lipid layer released endotoxins into
the lysate. Endotoxins are membrane bound molecules with, a lipid hydrophobic group on
one end and a negatively- charged phosphate on the other. Given their nature, these LPS
molecules tend to segregate into complex molecular structures or form micelles (Wicks et
al., 1995). As a result, this is a major concern, as it can interfere with plasmid purification
but also it can interfere with later use of the plasmids, such as in cell culture, as mentioned
in the manufacturer‟s guidelines “The Art of Plasmid Purification”, (Qiagen Inc). Main
concerns relating to endotoxins and LPS molecules, is their interference with transgene
expression by declining the efficiency of transfection (Weber et al., 1996). Endotoxin
contamination has directly been shown to considerably reduce gene expression of plasmid
DNA transfected by calcium phosphate-based delivery into mammalian cells (El-mogy and
Haj-ahmad, 2012). Seeing that these conditions are identical to the ones we used, endotoxin
contamination would have affected our viral production by altering the ratio of plasmids
expressing for the assembly of the full viral particle, thus translating into a possible lower
viral titer. For these reasons, the complete process of purification proceeded in endotoxin-
free conditions.
Though the protocol is standard and made to be easily reproducible, as detailed in the
company handbook, a brief summary will be provided here. After an overnight bacterial
41
growth, picking up from day 2, the bacterial cells were harvested by centrifugation at 6000
x g for 15 min, (~4oC) in a GSA superspeed refrigerated centrifuge (Sorvall, Du Pont). The
alkaline lysis then proceeded with company specific, P1, resuspension in 10ml of buffer
(50 mM Tris·Cl, pH 8.0 at 4°C, 10 mM EDTA and 100 μg/ml RNase A). Lysis proceeded
with company specific, P2, in 10 ml of buffer (200 mM NaOH, 1% SDS (w/v), at room
temperature) for 5 min. The reaction was then neutralized with pre-chilled buffer (10ml),
P3 (3.0 M potassium acetate, 15–25°C, pH 5.5), by vigorous shaking. The lysate was then
cleared with specific filter cartridges (Qiagen). After inserting the plunger into the
cartridge, the cell lysate was filtered through the outlet nozzle into a 50 ml tube. Endotoxins
were removed from the lysate just obtained by adding 2.5ml of endotoxin removal buffer
(ER buffer), contents of which were inverted for 10 times and chilled on ice for 30 minutes.
Qiagen-tip 500, a resin containing column which binds plasmid DNA, was washed through
prior the application of the lysate with a QBT equilibration buffer (750 mM NaCl; 15–
25°C, 50 mM MOPS at pH 7.0, 15% isopropanol and 0.15% Triton X-100 (v/v)). After the
wash, the lysate was applied and allowed to flow down the column by gravity. The lysate
was then washed twice by washing buffer QC, similar in contents to the QBT buffer only
with a greater amount of NaCl (1.0 M). Finally the lysate was eluted from the resin, with 15
ml of QN elution buffer (1.6 M NaCl, 50 mM MOPS at pH 7.0) into a 50ml falcon
(pyrogen-free; BD) conical tube as recommended. The DNA was finally precipitated by
adding 10.5 ml of isopropanol (room temperature; Sigma) to the same falcon. The total
solution and falcon were then centrifuged in a refrigerated (at 4°C) swinging bucket
centrifuge (Beckman Coulter) at 5000 x g for 60 min. The supernatant was decanted and
the remaining DNA pellet was washed with 5ml of 70% ethanol and then re-centrifuged
again with the same parameters as before. The supernatant was once then decanted and left
to air-dry for 10 minutes. Finally, the pellet was re-diluted in TE buffer (Tris-EDTA,
endotoxin-free buffer) in quantities that could adequately contain the purified DNA. The
concentration was determined with an optical densitometer at the 260nm wavelength.
Plasmid sequencing was outsourced, but made use of specific primer sequences coded on
the backbone of each plasmid. This concluded the purification process of plasmids.
42
2.1.3 Lentivirus production
With a few modifications to the protocol for lentiviral production put forward by
System Biosciences and based on literature itself (Dull et al., 1998; Salmon and Trono,
2006; Tiscornia et al., 2006), the protocol utilized 293TN cells in a third-generation viral
system for viral growth (see table 3 for summary). To produce virulent vector particles, the
293TN cell line has generally 2 main advantages (over other cells) which include: the
expression of the SV40 large T antigen (SV40 lTa) and a Neomycin resistance (NeoR)
gene, both useful in the high-titer production of lentiviral particles (Mendenhall et al.,
2012). In our case, however, we have extensively benefitted from the expression of lTA
only. The NeoR, was not useful in our study, seeing that cells were not selected, by drugs
such as G148 (Invivogen) (Cone and Mulligan, 1984).
Into our producer 293TN cell line, packaging plasmids and expression vector pSIH-
H1-copGFP, are transiently transfected to produce viral particles (Dull et al., 1998). The
packaging plasmids were obtained from Addgene and cells grew in high-glucose DMEM
(Invitrogen). Other standard steps included the addition of 10% FBS-inactivated
(Invitrogen) for the growth of the cells until transfection. From initial seeding to the
transfection day, the cells were passaged regularly by always providing and replenishing
new media to the cells. For optimal viral production, cells were always passaged before
they reached a confluency of 80% in CellBIND 100mm culture dishes with padded lips
(Corning). However, the day before transfection, cells were split 1:3 so as to provide
approximately 3 million cells per petri dish.
Calcium phosphate transfections like we have performed in our protocol, have
hallmarked a cheap way and simple way for the introduction of DNA into mammalian cells
for the past 30 years (Graham and Eb, 1973). This simple process is comprised of an
insoluble precipitate of Ca(PO4) (exact structure changes in solution) and the adsorption of
DNA, which are then taken together into the cell after multiple invaginations of cell
membranes within approximately an hour, for concise review, see (Jordan and Wurm,
2004). Transfection of the expression vector containing the shRNA templates (shENK and
shSCR) as well as the packaging plasmids were transfected as scheduled, with standard
43
calcium phosphate and allowed to incubate overnight. The ratios were as follows; 20μg
pSIH-H1-copGFP for the expression vector and 10μg pMDLg/pRRE, 5μg pRSV-Rev, and
6μg pMD2.G for the 3 packaging plasmids.
For what follows the day after transfection, we introduced changes in the
methodology. We disregarded the washing of cells with Opti-MEM and instead replenished
the cells with fresh medium. Opti-MEM is comprised of HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) and sodium bicarbonate buffers, NaCl, MgSO4, CaCl2, KCl,
NaH2PO4, hypoxanthine, thymidine, sodium pyruvate, l-glutamine, and trace elements,
which allow cells to grow on minimal elements (Invitrogen). Also, instead of using 2%
FBS, we used 10% FBS for the remainder for the entire protocol. As scheduled, viral
particles were collected at 24 and 48 hrs post-transfection and were purified by utilizing the
double filtration method; 5 min centrifugation at 5000 rpm followed by syringe adapted
0.22 μm pore filtration (Corning). However, concentration of viral particles was modified
to 20,000 rpm (rav = 49 200 g, rmin = 29 900 g) in a Beckman SW-32 for 90 minutes at 4oC
(will be explained later). Further, the viruses were repipetted and resuspended in 500 μl of
PBS on ice for 30 minutes. Particles were then further concentrated by using Amicon Ultra
100K filters (Millipore) for 15 minutes at 4000 g. Viruses were then stored in a standard
4oC refrigerator and directly used for transduction on HeLa cells to determine the viral titer.
Various dilutions of 1/1, 1/10 and 1/100 were used to transduce 100,000 HeLa cells for 48
hrs without disruption in various wells across a 24 flat base well tissue culture plate
(Sarstedt). Finally, cells were pelleted by 4000 rpm centrifugation for 5 min and
resuspended in 400 μl of PBS for titration purposes. The percentage of cells expressing the
reporter copGFP were counted in a FACS analyzer and thus allowed for the viral
preparation of an adequate titer. For in vivo injections, the viral titer was deemed adequate
for experimentation if and only if production were batches above 1.0 x 108 TU/ml. Major
methodological steps are summarized in table 3.
44
Table 3- Summary of major steps conducive to efficient lentiviral particle production
Step Brief Technical Description
1 293 TN and HeLa cell defrost (cell seeding)
2 Growth and cell spliting of 293 TN cells & HeLa cells
3 Day BEFORE transfection( Ca(PO4)
):
Plating of 3 million cells in 100mm petri dish
4 Day OF transfection ( Ca(PO4)
): With ~70-85% cell confluency, transfection of
third generation plasmid mix
5 16 Hour incubation and medium change prior medium volume collection
6 24 Hour medium volume collection and 4oC preservation
7 48 Hour medium volume collection
8 Concentration of viral particles via ultracentrifugation ( rav = 49 200g, rmin = 29 900g
with an SW-32 rotor)
9 Purification of viral particles through centrifugation in Ultra Amicon Columns
(4000G)
10 Viral application on HeLa cells for viral titration (diluted & undiluted comparisons)
11 HeLa cells pelleted at 4000rpm and resuspended in 500 μl of PBS
12 Viral titration for FACS analysis
45
2.2 PC12 cell differentiation
2.2.1 Cell culture
The principle behind the cell culture component of our study was to elaborate an in
vitro system of RNA silencing capable of foreshadowing the effects of an in vivo
knockdown of Enkephalin (ENK). As ENK is silenced in vitro, one can, through various
RT-PCR gene amplification cycles, determine the relative diminution of ENK mRNA.
Given that any cellular line in culture is devoid of the complexities found in the rat brain,
(blood-brain-barrier, cerebrospinal fluid circuitry, immunoreactive cells, neuronal
population bias, specificity for neurons expressing ENK), the transduction of a specific cell
line allows selecting for a quasi ideal situation where the complications that might arise in
vivo by lentiviral delivery are conditionally controlled. Therefore, in producing lentiviral
particles we have also tried to differentiate PC12 cells so that they may express a similar
ENK as is found in the rat brain, allowing the lentivirus to repress homologous mRNA.
The undifferentiated rat adrenal pheochromocytoma cells, PC12 cells, used in this
study were originally purchased from ATCC. As originally described by (Greene and
Tischler, 1976) and later by (Byrd et al., 1987)(Byrd and Lichtin, 1987) PC12 cells were
grown in 5% CO2 and 37oC in the incubator. The cells were further supplied RPMI-1640
medium (Invitrogen) and supplemented with an additional 10% Horse Serum (Sigma) and
5% Fetal Bovine Serum (FBS) HyClone (ThermoScientific), both heat-inactivated at 56oC
for 30 minutes.
In our study, the growth factors came from the 5% Fetal Bovine Serum (FBS) and
10% Horse Serum (HS), both which were used in the original literature (Byrd and Alho,
1987). They both supply hormonal and peptidic growth factors that aid the stimulated
proliferation, survival and support the process of differentiation of many cell types in
culture dishes. FBS, as opposed to HS, has a much more universal role in supplementing
different cell types and therefore has resulted in greater popularity given its content;
transport carrying hormones, minerals and trace elements, lipoproteins, soluble attachment
factors, detoxifying factors and low gamma-globulin content (Gstraunthaler, 2003). In
46
order to further eliminate immune reactions, both serums were heat-inactivated (at 56oC for
30 minutes).
Clonal instability as observed and reported by other teams, acted as a limiting factor
in that cells had to be used with the least amount of cell passages (Fujita et al., 1989;
Slotkin and Seidler, 2009). In light of this, selected cells were subjected to treatments 3
days after being defrosted and seeded. Once defrosted, cells were provided with fresh
medium in 100 mm Petri dishes. After having grown for 2 days, cells were split into 6 well
(10mm, Sarstedt) tissue culture plates with lids (Sarstedt). Within each well, a density of 1
x 107 cells per 3ml of medium was plated. As the design required undifferentiated cells to
control treatments, 2 wells out of a 6-well plate were devoted as „control wells‟. Implicitly,
the remaining 4 wells were dedicated to various differentiation concentrations of sodium
butyrate (NaB) (Sigma) ranging from 3mM to 12mM. Experiments with NaB
differentiation proceeded in this 6-well setup with a minimum of 2 plates, at all times. The
vigor of this second plate (control plate) will be described later, the purpose of which was
foremost dependent on the clonal stability of the PC12 cells.
Powdered sodium butyrate was diluted in 2.27 ml of PBS to a final concentration of
1M hereby generating the stock solution from which all following concentrations of sodium
butyrate were derived. For cells used as controls, fresh media (lacking sodium butyrate)
was replenished every 48hrs. For cells differentiated with sodium butyrate, media was also
replenished every 48 hrs but concentrations were adjusted from stock and added to regular
medium. A dose-dependent differentiation pattern was generated for each well. The
following final concentrations: 3mM, 6mM and 12mM were attributed to different separate
wells. Original literature suggests 6mM (Byrd et al., 1987) as being the most functional
concentration for proper differentiation; hence, that concentration was repeated in 2
different duplicate wells. Medium changes occurred every 48hrs for both control and
cellular differentiation patterns and cells were lysed after 6 days of treatment with or
without sodium butyrate depending on the respective predestination of each well. Thus, for
treatments with sodium butyrate, cells in specific wells received 3 identical sessions of
sodium butyrate differentiation at the previously mentioned concentrations (Fig. 7).
47
Pheochromocytoma Cell Differentiation Schedule
Figure 7. Figurative representation of sodium butyrate (NaB) pheochromocytoma cells
(PC12) differentiation schedule.
PC12 cells were seeded on day 1 and subjected to neuronal differentiation in either 3 μM, 6 μM or
12 μM NaB concentrations. NaB was directed diluted into the RPMI-1640 cell medium which also
contained 10% Horse serum. Every 48 hrs, after seeding, cells were centrifuged and re-
differentiated with the respective NaB concentrations. After the span of 3 differentiation sessions,
cells were lysed and subjected to reverse transcriptase PCR (RT-PCR) for cDNA generation from
extracted mRNA transcripts. From the total extracted RNA from PC12 cells, and control RNA
(total rat brain RNA) 3 primers were used to amplify by PCR; endogenous opioids enkephalin
(ENK) and dynorphin (DYN) and housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Adapted from: (Kung et al., 2010)
48
2.2.2 Semi-quantitative RT-PCR
In order to obtain a high copy of DNA from a fractionated cell, PCR amplification
can be used to obtain an abundant amount of that DNA. In a “2-step” manner, RT-PCR
(reverse transcriptase- PCR) starts with an mRNA segment of a gene to end up with a
cDNA copy of that gene, and then later proceed to standard amplification of the cDNA by
PCR. By using an oligo-dt primer on the poly-A-tail 3‟ side (and random hexamers) of that
targeted mRNA, reverse transcription will generate a first strand of cDNA. After isolation,
the cDNA can be flanked and amplified (Lodish, 2008). This allows amplifying any gene
of interest found within a cell subject to be analysed, provided the reverse-transcribed first-
strand cDNA can anneal to primers elongated by Taq polymerase. It is easy to design
primers that will amplify the region targeted on the mRNA strand (Lodish, 2008). In a
study such like ours, where the goal is to inhibit the expression of ENK mRNA, adequate
expression levels of the ENK transcript are required to later assess the effectiveness of
repression in vitro. When the cells are fully differentiated, application of lentiviruses with
an expressing shRNA, allows the targeted repression in cell culture of the region targeted
by the specificity of the shRNA. Therefore RT-PCR allows one to completely generate an
abundant amount of cDNA in differentiated cells which would be later transduced and in
„control‟ cells where no virus would be administered. Designed this way, the RT-PCR
endorses a semi-quantitative knockdown efficacy comparison for an in vitro session of
repression. The RT-PCR was approached by dividing the experiment into 3 fundamental
steps, relying on 3 kits manufactured by Roche for this purpose.
2.2.3 Total RNA extraction
RNA isolation and purification relied solely on the “High Pure RNA Isolation kit”
(Roche). This step was necessary in generating the necessary mRNA transcripts that were
later reverse transcribed when the cell was lysed to reveal its contents. Briefly, seeing that
all cells differentiated or undifferentiated were in suspension, all cells were lysed after the 3
sessions of differentions were complete. Each well was collected and subjected to
centrifugation. Old cell media was discarded and cells were resuspended in 200 μl of PBS
and 400 μl of lysis buffer (4.5 M guanidin hydrochloride, 50 mM Tris-HCl and 30% Triton
X-100 (w/v), pH 6.6) was added. After the spin column cycle, the resulting lysates were not
49
stored at -80oC but rather directly used for the reverse transcription reaction. The
concentrations were determined at the 260nm wavelength for absorption by an Eppendorph
Biophotometer.
2.2.4 Reverse transcription
Seeing that the point of reverse transcription was to generate a viable cDNA from a
cellular mRNA, the process of RT-PCR really began with this step. By reverse transcribing
mRNA from NaB differentiated PC12 cells and non-differentiated PC12 cells, the goal was
not only to account for transcript level changes based on NaB treatments but also to
compare this reverse transcription to cells where the level of ENK mRNA was reduced
consistently. For this purpose, a minimum of 2 duplicated 6-well plates with cells were
produced, according to the identical methodology described above (section 2.2.1). The first
plate was thus designed to receive the application of lentiviruses, whereas the second plate
was designed to remain as a control plate to compare the level of knockdown resulting from
the first plate. The knockdown efficacies induced in the first plate would be compared to
the second plate and would thus be an indicator of the gene expression decrease in vitro as
illustrated by reverse transcription. We used this system to obtain results as would be
expected in the rat brain, after lentiviral delivery.
By using “Transcriptor First Strand cDNA Synthesis Kit” (Roche) we used 1 μg of
lysate (total PC12 RNA) for 20 μl of total reaction volume as recommended. Both NaB
differentiated cells and undifferentiated cells were subjected to RNA isolation. From our
undifferentiated cells, 1 μg RNA was extracted in order to be used as an internal control.
Cells subjected to NaB differentiation, were lysed by their respective concentrations and 1
μg of RNA was also extracted according to each treatment. Finally, as an experimental
control to our PC12 total RNA extract, 1 μg of total rat brain RNA (Clonetech) was treated
in parallel. For both, the extracted RNA and control RNA, 10 μM of oligo-dt primers and
3.2μg of random primers were added to the reaction mix (completed with PCR grade water
to 13 μl when applicable). The remaining 7µl came from a master mix (21 μl) that was
pooled for the 3 types of RNA from which individual 7 μl aliquots were discarded
afterwards, for each type of RNA. The master mix featured proportional additions for
50
differentiated, undifferentiated and control RNA; reverse transcriptase (30 Units), protector
RNase inhibitor (60 Units), transcription RT buffer (1X), dNTP mix (1mM each). After the
incubation at 55oC for 30 min and after the inactivation of the Transcriptor Reverse
Transcriptase at 85oC for 5 min, the cDNA synthesis reaction mixes (both, extracted cDNA
and the total rat cDNA) were put on ice until the primers were diluted and ready for the
PCR reaction.
2.2.5 Primer dilution
Primers (Integrated DNA Technologies) for the housekeeping gene GAPDH, as an
internal control, were used in sense (GAPDH, F: 5' GCT CTC TGC TCC TCC CTG TTC
TAGA -3') and anti-sense primers (GAPDH, R: 5' CCA GGC GGC ATG TCA GAT
CCAC -3') expected to yield 814bp on gel. The same primers for ENK and DYN were used
in the sense and anti-sense for samples, the extracted cDNA and the total rat brain RNA.
The ENK sense primer; (ENK, F: 5' TGG GCG GGG CTC AGG AAA GA -3') ENK anti-
sense primer; (ENK, R: 5' TGC TCA CGG GGG ATG GAG CA -3) expected to yield
998bp on gel. DYN sense primer; (DYN, F: 5' ACC GAG TCA CCA CCT TGA AC- 3'),
DYN anti-sense primer (DYN, R: 5' CCT GTC CTT GTG TTC CCT GT -3') expected to
yield 635bp on gel. According to this experimental design, each type of cDNA had 3 sets of
sense and anti-sense primers; ENK, DYN and GAPDH. More specifically, each primer mix
contained 2μl of cDNA (RT product), forward & reverse primers diluted to 0.8µM, and
14μl of PCR grade water to complete the primer dilution.
2.2.6 PCR amplification
The final step out of 3 utilizes the „Standard PCR Procedure‟ found in the “FastStart
Taq DNA Polymerase” guide to cDNA amplification. To the primer dilution which
contains the reverse transcribed cDNA is added the following „master mix‟. The original
supplied recipe as described includes, doubly distilled H2O, 10x PCR buffer, 25mM MgCl2
solution, 10mM of each deoxy-nucleotide and 2 Units of FastStart Taq DNA Polymerase.
In addition to the original recipe, we have also added 5μl of Acetamide, useful in reducing
the non-specific binding of primers in the set-up phase of the PCR amplification. As a
whole, the „master mix‟ was added to the 6 different primer dilutions previously defined
51
and sent for thermal cycling in a Biometra T-Gradient PCR System. The temperature
profile for the cycler was used as preset in the guidelines for the „Applied Biosystems
GeneAmp PCR System 9600‟ with the exception that the elongation step at 72 oC was
scheduled for 1 minute. Primer annealing for all 3 genes (GAPDH, ENK, and DYN)
happened at 56 oC. All samples were loaded into the PCR machine and allowed to cycle
through denaturation of the double stranded DNA, annealing of the primers and elongation.
The course of the experiment typically lasts one night, for 35 cycles maximum. The
samples (10 μl of the PCR product with 2μl DNA loading dye (Sigma)) were then loaded
onto a 2% agarose gel incorporated with Ethidium Bromide.
2.2.7 Gel organization
A standard 19-1114bp DNA molecular weight marker (Roche, Marker VIII) was used
alongside extraction products; RNA extractions from control PC12 (undifferentiated) cells,
differentiated PC12 cells and total rat brain RNA (Clonetech) were resolved on gel. Total
rat brain RNA (Clonetech) acted as an additional positive control, since it held the contents
of the entire rat brain transcriptome, including RNA for endogenous opioids, ENK and
DYN. The gel was organized in the following manner, for total rat brain RNA (Clonetech;
lanes 1-3), for control PC12 cells (undifferentiated cells; lanes 4-6) and for differentiated
PC12 cells (at either 3 μM, 6 μM or 12 μM of NaB; lanes 7-15). Entire RNA from each
treatment was systematically primed in a triplet set of 3 genes and ordered in the same
order for all 3 treatments. The first lane in the 3 gene-triplet was for the housekeeping gene,
GAPDH (lanes labelled G), second lane in the 3-gene triplet, for endogenous opioid
Dynorphin (lanes labelled D) and third lane and last lane in the 3 gene-triplet, endogenous
opioid Enkephalin (lanes labelled E). Ordered this way, each different treatment exhibited a
set of 3 comprehensive mRNA extractions which included a positive control gene (the
housekeeping gene, GAPDH) expected to appear in all treatments and reflecting the basal
mRNA levels to be contrasted with the 2 opioids.
52
2.3 In vivo targeting of ENK mRNA with an shRNA lentivector
2.3.1 Animals
According to animal ethics committee requirements given by the Canadian Council
of Animal Care (CCAC) and “Comité de la Protection des Animaux du Centre Hospitalier
Universitaire de Québec (CPAC-CHUQ)” careful attention was paid to the safety and
health of our animals from reception to euthanasia. In the context of this study, 42 Sprague-
Dawley rats from Charles River (Saint-Constant, Québec, Canada) were individually
housed so as to monitor their water and sucrose consumption. Animals were ordered during
the first week of experiments (see figure 8 for place in experiment timeline), in order to
allow for facility habituation and acclimation as well as handling by personnel. In the
context to simulate a behavioural protocols these were given in 2 separate bottles, so as to
allow the animals to freely choose between either beverages. The animals were also given
freedom to access rat food at any time. The animals followed 12 hour light and dark cycles
(7:30 pm to 7:30 am) at room temperature (~22oC). The water and sucrose bottles
alternated in position above the cage so as to avoid location bias for each beverage. Rats
were weighed daily and new weights recorded so as to insure regular weight gain over
time. The entire timeline of experiments for in vivo and in vitro experiments is available in
figure 8.
53
SCHEMATIC REPRESENTATION FOR THE
ENTIRE PRODUCTION PROTOCOL OF
LENTIVIRAL PARTICLES
Figure 8. Schematic representation of the timeline with requirements for efficient
viral production.
Upon reception, rats were acclimated to novel environments starting roughly 2 weeks before
surgical interventions. Although transportation of the rats during this period was performed to
simulate potential behavioural studies, awaiting brain surgeries, in week 4, starting with day 7.
Seeing that an efficient third generation lentiviral production protocol comprises one expression
vector plasmid and 3 packaging (accessory but required) plasmids, plasmid amplification
proceeded in the first week. Bacterial transformation, followed by bacterial replication and finally
plasmid extraction had to be executed prior to transfection (T) (day 0) of these plasmids into
producer cells. Cell seeding (week 1) of producer cell lines 293TN and HeLa cells were necessary
for the production of viral particles and viral titer determination, respectively. Concentration (C)
of viral particles for in vivo injections was performed on day 3. Dilutions (D) of these freshly
produced viruses were plated over HeLa cells, 48 hrs prior the FACS analysis (F) on day 6. Upon
successful titer determination, rats were subjected to 4 days of surgery. Post-Operative (P.O)
(day 11 and 12) care was administered for at least 2 days. In order to allow the virus to adequately
knockdown mRNA, at least 21 days were provided prior euthanasia.
54
2.3.2 Pre-surgical stereotaxic treatments
As part of the pre-treatment surgical procedure after general anaesthesia,
Bupivacaine (Marcaine), a local anesthetic (2.5 mg/100ml) was injected subcutaneously in
the head. This injection insured long lasting, rapid and effective relief to withstand the
exposure of the skull. Pressure points in contact from the rat onto the stereotaxical surgical
apparatus (David Kopf Instruments) were coated with topical Lidocaine 4% (Maxilene)
cream. On edge with the stereotaxic frame, these points included the incisive bar (rat
adapter) and the ear bars, respectively, where the rodent teeth were gripped and where the
ear channels were anchored. Usually, general anesthesia represses the natural moistening of
the rat eyeballs during the surgery. The dehydration and soreness was therefore relived by
applying a lubricating Lacri-Lube (Allergan) gel over ocular surfaces hereby protecting the
eyeballs during surgery. Both in pre-surgical and post-surgical settings, a sterile and
isotonic mixture of NaCl (0.9%) and an anti-inflammatory analgesic (Anafen 10mg/100ml)
were also injected subcutaneously in the flanks of each rat. Throughout the surgeries, a
heated mat, maintained the animals‟ body temperature so as to prevent hypothermia and
favour regular heart rate.
2.3.3 Stereotaxic anaesthesia
Animals were deeply anaesthesized with ketamine-xylazine (80 and 10 mg/kg,
respectively, I.P.) or alternatively with isofluorane and placed in the stereotaxic instrument.
Throughout, the surgery reflexes were checked through pinching and doses of ketamine
were administered in 30% increments of the original dose. Alternative sedation using
isoflurane gas acted as a safer option for animal sedation when compared to ketamine-
xylazine intraperitoneal injections. With the vaporizer at OFF, liquid isoflurane (Abbott,
100ml) was loaded. The initial induction for rats was performed at 3% isoflurane (v/v) in
an oxygen-only mixture in the induction chamber for 5 min. Afterwards; the animals were
placed in a nosecone (gas mask for rats) with the vaporizer set to 2%-3% isoflurane. With
the nosecone still in contact with each rat, pre-surgical preparations and precautions (see
1.3.4) followed. Each rat was placed on the stereotaxic instrument and respiration was
monitored as well as sedation reflex markers. The vaporizer concentrations were adjusted
accordingly for each rat ranging 1.5%-3.5% on average.
55
2.3.4 Stereotaxic surgeries
Following pre-surgical preparations and precautions (sections 2.3.4, 2.3.5), rats
were placed in a stereotaxic surgical apparatus at -3.3 mm incisor bar and their skull was
exposed. Rats elected for surgeries in the basolateral nucleus of the amygdala (BLA) were
injected at coordinates 4.8mm laterally from the midline, 2.4mm caudally from the bregma
and 8.7mm into the brain (bilateral dorsal injections) as measured from the dura mater. For
rats injected in the BLA, a Hamilton syringe (Model 7001, 7000 series 1μL microliter
syringe with a 25 gauge needle) loaded with either (1μl) of shENK or (1μl) shSCR viral
preparation (homogenous bilateral injections per rat), was gripped directly on the
stereotaxic instrument onto the probe holder. The needle was very slowly propelled into the
brain and stalled at the right depth for 5 minutes prior injection. The viral preparation was
injected over the span of 10 minutes.
For rats elected for nucleus accumbens (NAc) surgeries, burr holes were drilled
bilaterally through the dura mater of the skull at coordinates, 0.90 mm laterally from the
midline and 1.0 mm rostral from the bregma. These surgeries utilized a Hamilton syringe
(Model 7002, 7000 series 2μL microliter syringe with a 25 gauge needle) loaded with (2μl)
shENK viral preparation which was gripped in a microsyringe injector (UltraMicroPump II
by World Precision Instruments) for mechanical automated injections. In contrast to the
manually handled syringe for the injections in the BLA, an automated microsyringe injector
was used for the accumbal surgeries. The syringe plunger was gripped on the pump in a
plunger button holder with the syringe collar and graduated barrel clasped in the bottom
clamp of the injector. The utility of using such an instrument was due to the Micro4
Controller which allowed a standardised and mechanical delivery of the viral preparation.
As such, the syringe was plunged through the holes to 7.6 mm into the brain as measured
from the dura mater. For NAc injections, the syringe was also very slowly propelled into
the brain and left idle for 5 minutes before injecting the viral preparation over 7 minutes.
In accumbal and amygdaloidal injections, the syringe was only removed after a final
wait of 5 minutes after injection. This insured the viral prep would minimally drag back
with the reverse suction produced by the needle. The process restarted on the contralateral
side, injecting the identical viral preparation with sh-template as the ipsilateral side, in the
56
same manner. Once both sides were injected, the animal was removed from the stereotaxic
instrument and the skull incision was sterilized and hydrated with saline (0.9%) before
sealing the incision with microsurgical sutures.
An additional 2 rats were designated to control for the expression and integrity of
GAD65 mRNA in striatal injections, (ENK mRNA rich area). These injections consisted of
bilateral, shENK expressing vectors exclusively, following the surgical techniques
previously mentioned. Burr holes were drilled following the same guidelines as the NAc
and BLA surgeries, at different coordinates for each rat. The first rat had coordinates at 0.8
mm laterally from the midline and 1.8 mm rostral from the bregma. The second rat had
coordinates at 1.0 m laterally from the midline and 1.5 mm rostral from the bregma. For
both rats, the syringe was plunged through the holes to 6.5 mm into the brain as measured
from the dura mater. A variable volume of ENK shRNA expressing vector was also
injected in the left hemisphere (1μl), as opposed to the right hemisphere (2μl). The
injections were performed manually, just as for the BLA surgeries. In both hemispheres and
for each subject, the syringe was propelled into the brain (over 3-5 minutes) and left idle for
5 minutes, before injecting the viral preparation over 5 minutes. After a final wait of 5
minutes the syringe was removed. The variation in volume helped to assess volume
variance and viral diffusion in both hemispheres of the brain as well as reinforcing our
stereotaxic methods. Euthanasia and perfusion proceeded in the same manner for all the rats
used in this study.
2.3.5 Animal euthanasia
Twenty-one days after the injections of viral preparations within the brain of the
animals (see figure 8), the animals were subjected to the euthanasia protocol. In order to
preserve the integrity of the tissue and the location of the proteins within the nuclei of the
brain, the tissue was fixated with paraformaldehyde (PFA). After a near lethal dosage of
ketamine-xylasine sedation (0.1ml / 100gr), the animals were ready to be opened and
perfused. Briefly, a perfusion pump flow rate of 20 ml/min allowed animals to be perfused.
The animals were incised at the rib cage and along the thoracic cavity in order to expose the
heart. The atrium was punctured before the insertion of the needle, hereby favouring the
pre-mature outflow of fluids and blood. This step also insured that there was no pressure
57
build-up from the fluid exiting the needle-punctured heart. With a needle first connected to
a bath of saline water (NaCl 0.9%), the left ventricle of the heart was inserted directly
through and guided to the main artery above the aortic arch. This favoured flow and
fixation to the entire brain. Saline was thus perfused for less than 2 minutes, preparing the
rat to receive 4% PFA. In quantities of 300ml-400ml per rat, the valve is switched to PFA
4% and the perfusion process began then. The solution consisted of 4% PFA and a Borax
buffer of 0.1M (9.5 pH). After the perfusion was completed, the brains were removed from
within the rat‟s skull and placed in separate individual vials containing 4% PFA.
2.3.6 Brain harvesting
After a 24-hour post-fixation in PFA, the rat brains were transferred in new vials
containing a 20% solution of sucrose-PFA. This step was entirely dependent on the time it
took for the rat brain to evacuate its water content to the highly concentrated surrounding
medium. The osmolarity was dependent on the quality of the initial perfusion and resulted
on average between 48-72 hrs per brain. The brains were individually frozen on dry ice for
15 min and stored at -80oC for later use. They were then sectioned on a microtome in the
coronal plane, in 30μm thick slices. These slices were placed in an anti-freeze solution
(ethylene glycol 30% and glycerol 20%) and stored in a standard freezer (-20oC) until the
probe hybridization. Whole brain slices were segregated based on different levels within 4
different well of a 24 flat base well tissue culture plate (Sarstedt), containing anti-freeze
solution. During brain harvesting, a rat was designated for GAD65 mRNA hybridization by
a physical manual dent with a sterile blade in the right outer cortex of each brain retained
the positioning and injection-type (shENK with 2μl) in later quantifications and
comparisons.
2.3.7 Immunohistological resolution of lentiviral diffusion and neuronal cell transduction:
a brief summary
Intrinsic properties of the expression vector chosen allow its proper resolution and
localization into coronal sections after immunohistochemical treatments. As mentioned
before, upon injection of the lentiviral preparation, the virus will transduct neuronal cells in
the brain given by the properties of its envelope protein (VSVg), for review, see (Cronin et
58
al., 2005). The RNA will first be reverse-transcribed before being processed by the pre-
integration complex. This insures the conversion from RNA to double-stranded DNA
before the import within the nucleus by the expression of viral genes. Once the DNA is
imported within the nucleus, the DNA can be integrated within the genome of the cell and
transcribed as a regular stretch of DNA (Logan et al., 2002). From the transcription, results
a hairpin which is exported outside of the nucleus.
A handy tool for analysis is that the expression vector plasmid for shENK shSCR
both enclosed the reporter gene copGFP. Indeed, the initial construction of these expression
plasmids contain not only docking sites for polymerases to bind and transcribe, but also a
fused plasmid segment between the cytomegalovirus (CMV) promoter with the reporter
copGFP. The reporter has a 2-fold role, in the titration of the viruses for FACS analysis and
a fundamental role in the immunohistochemical identification of the cells transduced by the
virus.
Briefly, the immunochemical detection of a reporter protein is based on the
selectivity of a primary antibody‟s (1stAB) specificity to recognize an epitope. First, a
blocking solution, bovine albumin serum (BSA; Sigma, St. Louis, MO) was used to shun
non-specific antibody binding to reduce the background signal by blocking hydrophobic,
ionic and electrostatic interactions between proteins (Buchwalow et al., 2011). Usually
immunoglobulin, IgG‟s (but may be IgM‟s) (Fritschy, 2008) will bind the copGFP
translated protein coming from the region of the genome with the integrated viral plasmid.
A secondary antibody (2nd
AB) locates and binds the 1stAB. The 2
ndAB needs to be
carefully selected to be specific enough to bind the 1stAB (binding “anti-1
stAB”) alone and
also be of the same host as the blocking reagent. In our case, the 2nd
AB also contains a
molecule of biotin. Biotin serves as a visual amplification signal when bound to Avidin and
catalyzed by Horse Radish Peroxidase (HRP). This will form an „Avidin-Biotin Complex
(ABC)‟ (VectaStain) resulting from a close bond to a molecule of biotin. As Avidin binds
multiple molecules of biotin (up to 4), the signal amplifies and is resolved by a reaction
with Diaminobenzene (DAB). As such, over a cerebral section and visible to the naked eye,
the staining pattern reveals the pattern of the viral injection given by reporter copGFP.
59
2.3.8 Radioprobe synthesis for ENK and GAD65 mRNA hybridization
In order to assess the level of RNA interference induced by the virus for the mRNA
of ENK, anti-sense radioprobes were designed to hybridize and locate ENK mRNA as
revealed on maximum resolution auto-radiographic film (Biomax MR, Kodak). The
plasmid to synthesize the probe was a kind gift from Dr. Sabol (Yoshikawa and Sabol,
1984). Prior its use for in situ Hybridization, the ENK oligonucleotide was cloned, purified
and synthesized based on our lab‟s procedures (Poulin et al., 2006). The radioprobe for
ENK was designed using 250ng of ENK linearized plasmid in a radioprobe (35
S-UTP)
synthesis mix for 60 minutes at 37oC. The mix contained the following elements: 5X
transcription buffer (6 mM MgCl2, 30-40 mM Tris (pH 7.9), and 10 mM NaCl), 10 mM
DTT, 0.2 mM ATP/GTP/CTP nucleotide mix, 100 µCi of 35
S-UTP, 40 U of RNase
inhibitor and 20 U of the ENK-specific T7 RNA polymerase. Once the probe synthesized,
the probe was mixed in a liquid hybridization solution to be applied and successfully cover
all the brain sections by applying 90μl of the solution stock over each slide with brain
sections. For 1ml, the hybridization solution contained: a house “Solution 1” [518 μl of
formamide, 62 μl of 5 M NaCl, 10 μl of 1 M Tris (pH 8.0), 2 μl of 0.5 M EDTA (pH 8.0),
20 μl of 50 Denhardt‟s solution, 207 μl of 50% dextran sulfate], 50 μl of 10 mg/ml transfer
RNA, 10 μl of 1 M DTT. Finally, 118 μl of DEPC water minus the volume of probe
synthesis mix for 1 slide of brain sections was added. This thus concludes the probe
synthesis for ENK mRNA. The hybridization was performed overnight at 56oC.
As a means of control, anti-sense Glutamic Acid Decarboxylase 65 (GAD65)
radioprobe was designed to hybridize and locate GAD65 mRNA, specific to GABAergic
neurons. GAD65 is a 65 KDa enzyme and is one of 2 isoforms involved in the synthesis the
of γ-Aminobutyric acid neurotransmitter (GABA). GABA works as a major inhibitory
neurotransmitter globally in the brain (Grimes et al., 2003). Various implications of
GABAergic neurons have been assessed for GAD65 mRNA by in situ hybridization in
widespread regions of the rat brain (Bowers et al., 1998; Chen et al., 1998). Though these
studies have focused on attributing function to GAD65, this enzyme isoform has been
found across in many regions of the adult rat brain, including the visual and neuroendocrine
systems (Feldblum et al., 1993). Given the abundance of the GAD65 in all divisions of the
60
amygdala at different intensities, ENK mRNA and GAD65 mRNA was found to be co-
expressed by ENKergic neurons in 3 regions of the amygdala (Poulin et al., 2008). The lack
of disruption of GAD65 mRNA signal when probed for hybridization therefore acts as a
control for our study where ENK mRNA can be attenuated in neurons that express both
mRNA transcripts.
In a very similar fashion to the ENK mRNA probe, the probe for GAD65 was
designed with a plasmid provided by Dr. Tillakaratne (Erlander et al., 1991). The recipe
for the GAD65 radioprobe synthesis steps and mix is identical to the ENK radioprobe with
an appropriate RNA polymerase (T3). The recipe for the 1ml hybridization solution 1 was
identical for both GAD65 and ENK. GAD65 hybridization was also performed overnight at
56oC, but on an adjacent brain section, from the same rat in a separate experiment.
2.3.9 Immunohistochemistry for lentiviral localization: combined protocol
In order to be able to co-localize the viral injection sites within the rat brains as well
as verify the effects of the shRNA viral vector targeting the interference of ENK-mRNA,
both an immunohistochemistry (IHC) and in situ hybridization (ISH) needed to be dually
combined on the same brain sections. As previously mentioned, separately, IHC will reveal
the copGFP from the expression vector whereas the ISH will target the ENK mRNA
endogenously produced in regions where neurons produce ENK. In order to assess the
transduction of our lentiviral vectors as well as identifying ENKergic neurons, we have
combined first IHC and then ISH within the same experimental setting, interlaced, to fit in
the same day.
2.3.10 Solutions required for immunohistochemistry
Under RNase-free conditions, a stock solution of 0.04% triton-PBS 1x was made in
a 1 liter bottle and sent for autoclaving. From this stock solution, 1 % (1 gram/100ml of
solution) of BSA (Sigma, St. Louis, MO) was added to the volume required for each
treatment phase, thus blocking primary and secondary antibody incubations. This volume
was calculated as being 5ml per rat brain, per treatment phase. The solution was then gently
stirred and filtered through a syringe-adapted 0.22 μm filter (Corning). Heparin Sodium
61
Salt (Sigma, St. Louis, MO) was pre-weighed (2.5 mg/ml) for both 1st
AB and 2nd
AB
treatment phases.
2.3.11 Immunohistochemistry for copGFP
Under RNase-free conditions, to preserve mRNA for the ISH integration,
immunohistochemistry was performed on free-floating brain sections in 6 well-plates
(10mm, Sarstedt). Brain sections were rinsed 4 times in PBS 1X for 5 minutes each time at
room temperature. This insured the brain sections were immersed in the phosphate buffered
saline solution to thoroughly cleanse each section from the anti-freeze solution it was
preserved in. We then used the previously prepared [0.04% triton-PBS 1x-1% BSA]
solution to block the rat brains for 20 minutes. The brain sections from each rat were then
transferred to a new 6-well plate so as to incubate brain sections in the 1stAB. The 1
stAB
(1:5,000 rabbit anti-copGFP; Evrogen; catalog No. AB50, lot No. 50201010205) was
incubated together with 2.5 mg/ml Heparin Sodium Salt into the [0.04% triton-PBS 1x-1%
BSA] solution for 2 hrs. The brain sections were then rinsed in PBS 1X, 3 times for 5
minutes each time. In order to detect the 1stAB, the brain sections were once again
transferred in a new 6-well plate in a solution containing the biotinylated 2nd
AB. The 2nd
AB, a donkey anti-rabbit IgG (1:1,000 Jackson Immuno Research Laboratories; lot No.
82417) was added to a solution of previously pre-weighed Heparin Sodium Salt and [0.04%
triton-PBS 1x-1% BSA]. The whole solution was then incubated together with the brain
sections for 1 hour. The brain sections were then washed 3 times in PBS 1X for 5 minutes
each time, preparing the brain sections to be conjugated as a biotinylated complex. Finally,
the avidin-biotin reagent amplifying signal solution (ABC Vectastain; Vector Laboratories)
was mixed with 0.04% triton-PBS 1x solution for 60 minutes. The final step in the IHC part
of the experiment uses 3-3-diaminobenzidine (DAB) to visualise the signal. To complete
the reaction, 3μl of 30% H2O2 was added to a filtered solution of DAB. The brain sections
were then incubated in DAB solution for periods varying between 7 and 9 minutes,
depending on the strength of the visualized signal. To neutralize the reaction from DAB,
the brain sections were then rinsed 3 times in PBS 1X for 5 minutes each time, and then
mounted on positively charged microslides (Surgipath). Once mounted, brain sections were
left to air dry at room temperature. The entire IHC was carried at room temperature and
62
each treatment phase was gently agitated on a standard stirring plate (Simmons et al.,
1989).
2.3.12 In situ hybridization integration
In order to fully complete the combined IHC and ISH within the same experimental
sitting, the mounted brain sections were subjected to 45 minutes of air drying prior the
ENK or GAD65 mRNA pre-hybridization process. The integration of the ISH following
IHC was a result of various adaptations from different authors (Simmons et al., 1989; Hebb
et al., 2004; Poulin et al., 2006). As a typical ISH, it was divided into 3 parts; pre-
hybridization, radioprobe annealing and hybridization and a post-hybridization process.
Much like the IHC in this sequence of experiments, the pre-hybridization was performed
under RNase-free conditions.
Brain sections treated for GAD65 radioprobe were only subjected ISH and IHC
separately. However, all other specifications for the hybridization process proceeded as
explained for the ENK mRNA hybridizations with the appropriate changes mentioned.
2.3.13 Pre-hybridization and radioprobe hybridization
The hybridization process localizes and detects mRNA through an (35
S) radioactive
probe. Thus, in the pre-hybridization phase reagents were utilized so as to permeate the
tissue and allow the radioprobe to reach the targeted mRNA. In a successive and thorough
manner, each mounted microslide was fully dipped vertically in 250ml containers that
contained each required pretreatment solution. First, the slides were dipped in 4% PFA, so
as further fix the tissue. Then slides were dipped in a container of proteinase K (10 g/ml in
100 mM Tris HCl, pH 8.0, and 50 mM EDTA, at 37°C), an endopeptidase that will cleave
all peptidic bonds. Slides were rehydrated in DEPC H2O, and dipped in a buffer useful for
acetylation (100 mM triethanolamine (TEA; pH 8)). Acetylation in 0.25% acetic anhydride
in 100 mM TEA will ensure electrostatic interactions were minimized in order to allow the
probe to bind specifically to the mRNA as opposed to positively-charged sites. A final
dehydration in increasing ethanol concentrations (EtOH; 50%, 70%, 95%, 100%) prepared
63
the tissues for radioprobe hybridization. The slides were left to dry for 30 minutes
following the pre-hybridization procedure.
The radioprobe previously synthesized (see, 1.4.2) and immersed within the
hybridization solution was heated to 65oC for 10 minutes and applied in 90μl quantities
over each microslide. Each microslide was then covered with a micro cover glass. The
slides were then transferred to preheated slide warmers for overnight hybridization at 56oC
(~55-56 oC), for both types of probes, ENK and GAD65.
2.3.14 Post-hybridization
In the post-hybridization procedure, by using a stringency washing methodology,
the goal was to gradually decrease the stringency conditions to a low stringency wash and
increase the signal/noise ratio by decreasing the salinity and increasing the temperature. As
such, the process of dipping the slides into 250ml containers of decreasing stringency
solutions and RNase container allowed the removal of not only the microslide coverslips
but also the unbound probes that may have non-specifically bound to non-ENK mRNA.
Successive post-hybridization washes included 4 dippings in 4X standard saline citrate
(SSC), RNase A (20 g/ml, 37°C) for 60 minutes, 2X SSC, 1X SSC, 0.5X SSC, 0.1 SSC at
60°C for 30 minutes, and dehydration in graded EtOH concentrations similar to the pre-
hybridization gradation. Once dried, the brain sections were deffated & delipidated in
xylene so that the resolution of the emulsion be clear and uniform, prior to be dipped into
NTB2 nuclear emulsion (Kodak; diluted 1:1 with distilled H2O). Each set of slides was
then stored in a dark concealed case and after 7 days of exposure for ENK (10 days for
GAD65), the slides were developed. According to manufacturer‟s guidelines (Kodak Inc,
Rochester NY), the slides were immersed in D19 developer (Kodak), rinsed in H2O, and
fixed in a fixative solution (Kodak). This autoradiographic process was a very crucial step
that resolved the radioactive staining pattern of the probes. In short, the nuclear emulsion
coated each slide with a thin film. As the radioisotopes decayed in the dark, the
photographic emulsion permanently retained the position of the radioactivity; in the final
phase after the emulsion phase, individual silver grains were developed as ionized radiation
(Swamy, 2008). Finally, slides were dehydrated with ethanol, cleared in xylene, and
coverslipped with DPX mounting medium (BDH Laboratories Supply).
64
2.4 Quantification
2.4.1 Data analysis
Reverse-transcribed cDNA from PC12 cells was amplified by PCR and the
generated product was resolved on a gel. We have thus subjected all our NaB
differentiation products onto various 2% agarose gels. Pictures of the gels were taken under
UV light with different exposures times below 1.207 seconds or above 2.952 seconds of
exposure. This accounted for a qualitative measurement as the ethidium bromide staining
was not quantified by any software. However, pictures were taken by WetLab (software
version 3.0). The intensity of each band was estimated by comparison between adjacent
bands. Though these intensities varied with the treatment method of NaB, the results were
correlated with the observations on gel when differentiation occurred.
Brain sections with injection sites were selected and analysed under brightfield or
darkfield depending on the best resolution of the injected virus. These images were
obtained from a Leica DM 4500B microscope and a connected camera, (MicroPublisher 5.0
RTV). The necessary software for the proper acquisition of the images used was Openlab
5.5.0 (Improvision) which allowed the capturing of images with adjustable magnification
and exposure. Depending on the integrity of the IHC staining pattern, the exposure time
varied slightly from one protocol to the next for software capture of the images. A rat brain
atlas (Swanson, 2004) was used to determine the precise level of injection in each brain
slice useful for quantification. These figures were assembled in Word (Microsoft 2007),
cropped with Paint or Photoshop (CS5) and graphs and figures were created with Adobe
Illustrator (CS6 but also with CS5).
ENK and GAD65 mRNA expression appeared as silver grains on quantified on
autoradiographic films (Biomax MR, Kodak) with radioactivity standards (ARC-14688)
carbon-14. These films were quantified and analyzed with optical density (OD)
measurements in regions that matched the staining pattern from IHC. The regions were thus
selected based on the injection pattern of the viral expression vectors. Within the regions,
the injection sites were delimited freehandedly (IMAGE J software) on ENK mRNA
expression films that corresponded to the area injected. As such, structure delimitation
65
matched IHC stained sections of the same brain section. GAD65 expression was also
dependent on the staining pattern of the injected virus; however, an adjacent but
representative brain section was selected, since ISH was performed separately from IHC for
GAD65 mRNA. Brain sections stained by IHC but without discernible neuronal nuclei,
morphology or unquantifiable symmetric staining were excluded from analysis. Only
injected areas where ENK is usually expressed were kept to be quantified; this avoided
selecting brain sections with stained fibers of passage. ISH brain sections were selected
where 35
S was uniformly hybridized over the entire brain section. Sections with folded or
indiscernible brain structures due to radioprobe hybridizations were excluded from the
quantification.
2.4.2 Co-localization of copGFP and ENK mRNA for quantification and analysis
The quantification method was based on the comparison between ipsilateral and
contralateral brain hemispheres where an expression vector was injected. Rats injected with
a vector expressing either the shSCR or with the same vector expressing an shENK were
used to compare one hemisphere to the other. Only rats where one contralateral hemisphere
was untouched (will be referred to as „intact‟) by the virus were selected for comparison.
This meant that one hemisphere needed to have a staining pattern as revealed with IHC that
corresponded to a lack of staining pattern on the contralateral side of the same coronal level
within the same rat (as evaluated by the atlas).
The side where the staining pattern was observed and selected was called the
„ipsilateral side‟ whereas the compared hemisphere was called the „contralateral side‟.
Brains injected with the same vector expressing either shSCR or shENK in the ipsilateral
side, were stained with a copGFP antibody revealing the viral diffusion (injection site) and
matched in similar level to the corresponding „contralateral‟ brain section. Since the point
was to compare the injected side to a “virus-free” area, for the same region injected in the
ipsilateral side; the copGFP staining was selected in the contralateral side when it did not
reveal a viral infusion pattern or in other words, lacked viral staining symmetry. Based on
the copGFP staining, each ipsilateral and contralateral hemisphere was then matched in
similar level for the comparison of ENK mRNA expression on autoradiographic film (see
figure 9 panels A and F for methodology).
66
Downregulation quantification method in injected
hemispheres and control hemispheres
Figure 9. Elucidation of the quantification method used in this study for a representative
brain section with an ShENK vector delivered within Nucleus Accumbens (NAc).
Panel (A) shows the injection sites within the NAc for the represented + 1.70 mm from bregma
(β), and panel (F) shows the contralateral non-injected hemisphere corresponding to the ipsilateral
+ 1.70 mm from bregma of injection. These schematics were adapted from coronal sections from
Swanson (2004). Numbers in brackets, indicate rostral-caudal coordinates relative to bregma in the
rat brain atlas. Panels (b), (d), (g), (i), show copGFP antibody staining by IHC (visualized here by
3,3‟-diaminobenzidine in darkfield microscopy). Panels (c), (e), (h), (j) show ENK mRNA
autoradiographic staining for the identical brain section used as for the IHC; these respectively
being (b), (d), (g), (i). The injected area quantifications, (b&c), (g&h) are delimited by a red
+
+
67
boundary indicating the diffusion of the virus within the brain section. The boundary was
delimited from (b) and applied for quantification, on the ENK mRNA staining for the same brain
section in (c). The boundary delimited by the injection area (b) also served to assess a noninjected
area, the contralateral hemisphere (h). Bottom images (d,e,i,j), are the original images without
delimitations.
The area for the ipsilateral injection for ENK mRNA quantification was thus based
on the same ipsilateral section as revealed by the area of injected virus, resolved by
copGFP. The area of injection in the ipsilateral hemisphere for each quantified brain section
evidently varied depending on the viral diffusion. The ipsilateral area also determined the
area of quantification in “virus free” contralateral hemispheres for ENK mRNA comparison
when symmetry was not found. Further, not only optical density (OD) measurements (sum
of pixels) were collected for contralateral and ipsilateral hemispheres but also area and
integrated density (ID) measurements which are an integrated product of the area quantified
and the sum of pixels for that area. Area-dependent measurements were thus based on areas
of injections. Finally, representative brain sections were selected to be quantified and
tabulated for statistical significance when they obeyed the previous specifications of
quantification.
GAD65 expression was also quantified in this manner on autoradiographic film but
evidently since ISH was performed but not combined with IHC in one experimental sitting;
the staining pattern of IHC for the injection of the expression vector came from adjacent
brain sections at the same coronal levels. Also, GAD65 mRNA expression within targeted
areas of injections was quantified for measurements of OD for injections with a vector
targeting specifically ENK-mRNA.
2.4.3 GAD65 mRNA quantifications in areas injected with an shRNA ENK expressing
vector
Quantification embedding GAD65 mRNA expression analysis stemmed from the
same methodology as was applied to area-bound ENK mRNA quantifications. Though
these rats were rather experimental subjects paving the way for sound stereotaxic surgery,
quantification proceeded as previously mentioned, with the same software and guidelines
for all rats quantified. Just as before, the area was determined by the staining area of IHC
68
against copGFP and translated onto autoradiographic films for the quantification of the
expression of ENK mRNA. Given our GAD65 control system, the area was further
translated from ENK mRNA autoradiographic film hybridization onto autoradiographic
film showing the expression of GAD65 mRNA. Brain sections were selected at similar
levels as were the brain sections hybridized for ENK mRNA.
Brain sections were further selected as those with the manual physical dent on the
right hand side of the outer cortex, designating a hemisphere with a 2 μl injection of shENK
lentiviral solution. It is important to note that though the IHC and ENK ISH were
performed within a particular brain section, GAD65 mRNA was performed on an adjacent
brain section from the same injected rat with the same shENK lentiviral solution. It is only
for this reason that the GAD65 quantification acts as a control for the effects attributed to
the injection of an shENK expressing vector and the possible interference associated with
mere needle-based injections on the attenuation of ENK mRNA.
2.4.4 Statistical analysis
All statistics performed for this study were made with the help of statistical
software Aabel 3 (Gigawiz Ltd). Knockdown quantifications of ENK mRNA targeting
expression vectors were analyzed based on the raw values of OD, ID and area (applicable
to targeted areas of injections) after the biomax MR films were standardized in μCi. For the
injected hemispheres and contralateral hemispheres, OD measurements for ENK expression
within the targeted areas (BLA, NAc) were compared by using either a paired or unpaired
student t-test with at least *p < 0.05 nominal significance. For each hemisphere, whether
injected or contralateral, the groups were divided based on the injected expression vector
expressing either an shSCR or an shENK and compared with an unpaired t-test.
Comparisons between injected hemispheres and contralateral hemispheres used a paired t-
test. Thus for each OD analysed, the grouping was based on the expressing hairpin and on
the injected side. To adjust values and comparisons for multiple tests, the Bonferroni
correction was applied, adjusting the threshold of significance to p<0.025, where 0.025 =
0.05/2. All errors were reported as SEM to the mean.
69
CHAPTER 3
3 RESULTS
3.1 ShRNA expression vectors
3.1.1 High-titer viral production reproducibility
Given the global objectives of our study, the imminent need for a functional
lentiviral construct was a crucial step towards the realization of our goals. To target ENK
mRNA in vivo or in vitro, the requirement was to optimize our lentiviral production
protocol so as to create high-titer stocks of infectious viral particles with consistent
accuracy. Infection of HeLa cells with lentiviruses expressing either an ENK shRNA or a
SCR shRNA have yielded consistent production titers, these being; 1.22 x 108 TU/ml, 1.32
x 108 TU/ml, 1.62 x 10
8 TU/ml for the 3 different production batches of shENK expressing
vector used in this study and 1.33 x 108 TU/ml for the shSCR batch used in amygdaloid
injections. These data indicate that technical adaptations in the methodology of the viral
production protocol have rendered adequate and similar viral infectivity; reproducible from
one batch of experimental injections to the next as reported by the percentage of HeLa cells
with stable expression of lentiviral-associated GFP-tagged protein.
3.2 Cellular culture results analysis
3.2.1 PC12 differentiation analysis overview
The usefulness of a cell line inducible for chromaffin-like differentiation such as the
PC12 cells grown in culture, served to validate an in vitro approach for the targeting of
ENK mRNA. This experiment was designed to corroborate in vitro, the specificity and
efficiency of the virally mediated ENK ShRNA expression and to estimate the degree of
knockdown for in vivo experimentation. Implicitly required, was a cell line with existing
ENK mRNA to study the effects of lentiviral mediated ENK mRNA downregulation. It was
70
decided to differentiate PC12 cells with sodium butyrate (NaB) in order to obtain a
proenkephalin transcript resulting in ENK related peptides (Met5-enkephalin-Arg
6-Gly
7-
Leu8), as demonstrated by Byrd et al, 1987. From a basal and immature cellular state of
PC12 cells, the goal was to induce these cells into mature adrenal chromaffin cells, thereby
shifting the opioid output of the cell; from a mainly dynorphinergic expression (immature
PC12 cells) to the increased expression of ENK, which is a biochemical marker of
chromaffin cells (Margioris et al., 1992). Our results, in accordance to Byrd et al 1987,
showed that undifferentiated PC12 cells contained barely detectable levels of ENK mRNA
and have thus been validated as comparative controls to cells who received NaB. However,
the opioid shift sought after in differentiated cells was not attained in either biochemical or
morphological endpoints. Our findings are reported here.
3.2.2 Cellular decline & anti-proliferative effects
The administration of NaB, caused effective disruption of cellular division, as
observed in the cell culture petri dishes, for all 3 types of diluted concentrations of NaB; 3
μM, 6 μM and 12 μM. For all concentrations of NaB administered, as noticed early
between the first session, (4 days of differentiation) and the second session, (after 6 days),
the cell count remained the same or slightly declined. A more dramatic drop in cell count
was observed for wells designated for differentiation between the second session of
differentiation and the third session, (8 days of differentiation).
Qualitatively, the cell decline was visually noticeable in the culture dish in all
experiments performed with PC12 cells revealing that 200,000 to 550,000 cells remained
on average, by the third session of differentiation for all concentrations of NaB. Control
cells also exhibited a decline, however less steep than differentiated cells. As reported
visually, control wells showed cell count decline estimated at 25% from the initial cell
seeding. Neither control cells nor differentiated cells reached confluency since they were
passaged (with medium replacement) on the day of differentiation, which was every 48 hrs.
These observations manifested a noticeable cell count decline as the sessions of
differentiation progressed, thereby suggesting lack of cellular proliferation or even cell
death. We report the steep cell decline by observations of lighter pellets during routine
71
passaging and cell centrifugation, but also as verified by cell count in a Bright-Line
Hemacytometer (Sigma). Cellular counts were compared to initial cell seeding at a density
of 1 x 107 cells per well in 3ml of medium.
3.2.3 Shape, maturational & morphological concerns for PC12 cells
Both differentiated and non-differentiated cells remained in an omnipresent state of
suspension, as observed in vitro with minority (sub-population) of cells adhering to the
plastic dish. Independent of the concentration of NaB, control and differentiated PC12 cells
remained in suspension throughout the entirety of the experiments until extraction of RNA.
Cells showed little cell clustering and seldom cell-substratum adhesion. Furthermore, cells
preserved an overall round shape in between sessions of differentiations with control cells,
thus exhibiting a more preserved morphology throughout every passage. However,
declining cellular populations, cellular debris and possible effects induced by NaB made it
difficult to notice the shape of cells by the third session of differentiation at all
concentrations of NaB administered.
3.2.4 Agarose gel analysis of opioid mRNA from PC12 cells
Total RNA extracted from PC12 cells was amplified through RT-PCR and loaded
on agarose gel electrophoresis for all treatments, as previously described. We failed to
resolve any RNA on gel from the total rat brain RNA (figure 10) after EtBr UV exposition.
This gel showed no RNA for the Clonetech PCR products, suggesting that the RNA from
Clonetech was either old or partly unstable. Following morphological observations,
biochemical extraction of RNA, priming and RT-PCR cells showed the presence of
housekeeping gene GAPDH at approximately 814 bp (as expected) consistently for all
concentrations (3µM, 6µM and 12µM) of NaB, as well as PC12 non-differentiated cells
(figure 10). As expected, the visibility of the bands remains similar and highly consistent
for GAPDH at all concentrations of NaB assayed and in PC12 undifferentiated cells.
DYN was resolved at approximately 683 bp (as expected) below GAPDH for all
cells differentiated with NaB. However the visibility of DYN was resolved to increase in
intensity with increasing concentrations of NaB administrated (figure 10). Qualitatively,
72
PC12 cells differentiated at 3 μM show a very faint and lowest expression level that can be
noticed for DYN. By visually examining the gel, the expression increases by 2- to 3- fold
higher for 6 μM differentiated cells and approximately 4- to 6- fold higher in 12 μM
differentiated cells. For cells untreated with NaB the transcript expression level for ENK,
was not resolved at 998bp (as expected) but unexpectedly also at any concentration of NaB
administered. Contrary to postulated literature and fundamental work with PC12 cells and
NaB, differentiation, these results show a dose-dependent gradient for DYN mRNA rather
than ENK mRNA.
73
Relative RT-PCR analysis shows marked increase for
Dynorphin mRNA expression
Figure 10. Most representative agarose gel of relative RT-PCR analysis for ENK
and DYN mRNA expression after sodium butyrate cell differentiation.
Agarose gel (2%) analysis resolved at 2.952 (secs) under UV exposure. Gel shows the effects of
cell differentiation after cell fractionation and endogenous peptide mRNA extractions and
amplification. All lanes were hybridized with the same appropriate primers accordingly; G, D,
E is respectively, GAPDH, DYN, ENK. Control RNA, (Clonetech, total rat brain RNA, lanes 1-
3) shows no bands when hybridized with the same primers as for lanes differentiated with NaB.
PC12 control cells (lanes 4-6) were left untreated by NaB and only show endogenous gene
expression GAPDH (lane 4). This gel depicts a marked increase for DYN content after 1 week,
comprised of 3 differentiation sessions with 12μM of NaB (lane 14). This gel also shows that
with increasing concentrations of NaB differentiation, (lanes 7-9; 3μM, lanes 11-12; 6μM, lanes
13-15; 12μM) ENK and gene control GAPDH mRNA transcripts levels remain unaffected.
Roche marker 8 (resolving 19-1114bp) was applied on the gel, prior and after RT-PCR products
were applied. G, D and E are 814bp, 683bp and 998bp in length, respectively.
[NaB ]
DYN
NN
Lanes: 1 2 3 4 5 6 7 8 9 10-12 13-15
Clonetech
CTLmRNA
mrnmm
RNA
74
3.2.5 Attempts at experimental troubleshooting
In light of unsuccessful extractions with ENK mRNA transcripts, the following
technical measures were modified from the original protocol in order to cater for possible
solutions. These measures did not unfortunately enhance the growth of our cells or prepare
cells for repeated differentiation. At different experimental levels, the following corrections
were adopted.
Measures involving the use of new primers for ENK mRNA with testing of
different annealing primer temperatures in the PCR reaction targeted the extraction step.
Our ENK primers previously described yielding lengths of 998 base pairs were annealed
originally at 56 oC and modified to 54
oC, with no further success to resolve ENK mRNA
transcripts from differentiated cells. Although our 998bp primer yielding ENK transcripts
were assayed in the preliminary phases with successful resolution of ENK mRNA at 56 oC,
to circumvent this source of bias in our cell culture we have also tried to resolve ENK
mRNA extracted from PC12 differentiated cells with different primer sets. These primers
(forward: 5‟- TGG CTC GTA GCG CTT GGG TC -3‟ & reverse: 5‟-TCG TCT TCC AGC
TGG GGG CTT-3‟) were implemented in the core of our methodology with all other
elements preserved other than the actual primers. However, no further ENK mRNA
transcripts from differentiated cells were obtained on gel with different annealing primers,
thereby defining greater scrutiny for the source of error.
In the culture dish, in order to attempt to salvage the prominent cell loss, we have
performed a few experiments with wells seeded at a density of 2 x 107 cells per 4ml of
medium as opposed to an initial cell density of 1 x 107 cells with everything else preserved
from the original protocol. After 3 sessions of differentiation, 640,000 remained typically in
petri dishes as averaged for all 3 types of NaB differentiations. Control cells experienced, at
most a, 30% cell count decline. Though the initial goal was to compensate for the abrupt
loss of cell count by the end of the differentiation cycle, no substantial improvement in cell
count was observed.
75
In order to eliminate buffer bias in the wells of our cells, we have also tried to
repeat the differentiation schedule and methodology with powdered NaB diluted directly in
the cell culture medium. The final concentration of NaB in media was adjusted to the
required same final concentrations as was performed with NaB diluted in PBS; 3mM, 6mM
and 12mM, depending on the designation of each separate well. Experiments proceeded as
scheduled according to the entire planned methodology. However, following RNA
extraction, no further ENK mRNA transcripts from differentiated cells were obtained on
gel with the effects of NaB standardized by the medium. This showed the role of PBS as a
negligible component in the results of our experiment.
In order to account for the plastic quality of our 6-well tissue culture plates
(Sarstedt), we have instead seeded and grown cells on our CELLBIND 100mm culture
dishes with padded lips (Corning). These were the same plates used for the successful
growth of our 293TN and HeLa cells used in the viral production protocol of our study.
One plate contained cells never to be differentiated (control cells), whereas the other
contained cells to be differentiated at 6mM. The only changes to the initial cell culture
protocol, other than the actual plastic culture dishes, were that cells were seeded at an initial
density of 3.3 million cells and grown in 10ml of RPMI-1640 medium, prepared
identically as before with 10% Horse Serum and 5% FBS. However, after 3 sessions of
differentiation with 6mM NaB, the cells did not show signs of improved growth but rather
showed the same cellular decline as seen in the 6-well tissue plates. Control cells also
experienced a similar cell count decline as was reported in the 6-well tissue plates. RNA
was extracted using the same protocols as previously described with no difference in the
expression patterns of ENK. Seeing that no ENK was obtained from PC12 cells
differentiated with NaB, future experimentations with PC12 cells were abrogated. Further
focus and efforts were amended in the in vivo methodology only.
3.3 In vivo delivery of ShRNA expression vectors
3.3.1 Injection sites revealed by immunohistochemical analysis
76
To evaluate ENK mRNA downregulation in the NAc and in the amygdala,
lentiviruses were injected bilaterally in the rat brain, and IHC staining for reporter copGFP
(expressed by the vector), revealed injection sites within each brain section. Detailed IHC
analysis of brain sections demonstrated that bilateral injections with unique targeting
coordinates (AP -2.4, ML ± 4.8, DV -8.7) showed viral spreading in the CEA but not in the
BLA. More specifically, spread of transduced cells (by the same vector expressing either an
shSCR or an shENK) spanned the lateral part of the CEA (CEAl) and the ventral medial
division of the CEA (CEAm) with few additional injection sites also bordering the nearby
ventral caudate putamen (CP) (see figure 11, panels B, D, F). These are ENK-rich areas
with a colocalizing viral diffusion staining pattern for the vector delivery. The cell spread
of injection sites for all coronal sections mainly diffused over 4 coronal levels, spanning
roughly 1.78 mm to 2.85mm according to the atlas (Swanson, 2004).
77
Schematic representation of quantified viral delivery injection
sites, as resolved by ENK mRNA hybridization
and cop-GFP immunohistochemistry (IHC)
Figure 11. Representative brain sections quantified for viral injection of ShSCR and
ShENK into the nucleus accumbens (NAc) and central amygdala (CEA).
(A) & (B) show the injection sites within the NAc and CEA respectively, as adapted on coronal
sections from Swanson (2004). The levels over each section correspond to the levels in the rat
brain atlas. Numbers in brackets indicate rostral-caudal coordinates relative to bregma. Black and
orange squares respectively discriminate ShENK and ShSCR vector delivery sites across the
quantified coronal levels. (C) & (D) show copGFP antibody staining by IHC (visualized here by
ShSCR ShSCR
shSCR
shSCR
+ + +
- - - -
78
3,3‟-diaminobenzidine in darkfield microscopy) for representative sections from each region. (E)
& (F) show ENK mRNA autoradiographic staining for the identical brain section used as for the
IHC. Regions quantified include; ventral NAc (core & shell), the central amygdalar lateral part of
the CEA (CEAl), medial division of the CEA (CEAm) and some minimal viral spillover in the
ventral caudate putamen (CP) closest to the CEA. Please note that solely a vector expressing
ShENK was injected in the NAc, whereas injections in the CEA contained either, a vector
expressing ShSCR or a vector targeting ENK mRNA depending on each subject rat.
Injection sites in the NAc, as revealed by IHC, confirmed that the NAc was mainly
targeted. Most of the staining in the NAc showed corresponding injection sites in the dorsal
part of the nucleus (figure 11, panels A, C, E). According to the rat brain atlas (Swanson,
2004), transduction of the cells around the injection site spanned 0.45mm to 1.7mm from
bregma. Although both the core and shell were injected and resolved, most of the staining
was found in the dorsal part of the NAc, closer to the core than to the shell (figure 12),
resulting in viral diffusion within ENK rich areas, as stained for the vector delivery (only
shENK expressing vector in this case). Some offshoot injections in the basal forebrain were
occasionally found, lateral to the medial septum but were quantified (data not shown).
Overall, these injections showed evident and expansive viral stains in injected hemispheres,
and hereby endowed our commission to proceed with in-depth quantification.
79
Targeted injections in the NAc show mainly
dorsal injection sites
Figure 12. Injections in the NAc core and shell. The following figure shows the sites
obtained from injections in the nucleus accumbens.
Panels (A), (B), (C), show copGFP antibody staining by IHC (visualized here by 3,3‟-
diaminobenzidine in darkfield microscopy) after 3 weeks of stable genomic neuronal integration.
Panel (A) shows structures targeted by the virus, and as depicted, most of the staining pattern is
resolved in the NAc shell. In contrast to a different rat (B),the staining pattern is resolved in the
dorsal part of the NAc at the core. (C) The staining pattern resolved here also shows viral spread
over the dorsal part of the NAc and into the CP, resolving a more rostral injection. Accumbal
divisions were delimited based on the Paxinos and Watson (1986) demarcations.
80
In both the amygdala and NAc, the viral spread localized over ENKergic neurons,
thus permitting the elaboration of an advanced method of quantification. However, of the
viral spread closest to the targeted amygdalar site of injection, staining resolved by IHC
revealed the presence of virus „around‟ the BLA, alongside the external capsule (EC)
(figure 13). As the EC is anatomically restricted and poor in ENKergic neurons
(Palomares-Castillo et al., 2012), analysis was concentrated on actual infected cells.
Quantifiable spreading was thus mainly limited to injection sites in the CEA, being
adjacent to the targeted site (figure 11). Also from these coordinates, injections resulted in a
minor staining pattern in the laterodorsal CP by an shENK expressing vector. Overall,
amygdalar injections reveal an obstruction in the targeting of our region of interest as
manifested by the viral spreading bordering the external capsule. These results further
imply that the cortical afferents reaching the amygdala need to be circumvented to reach
our predetermined region of interest.
81
Targeted injections for the BLA,
show mainly medial and lateral exclusion sites,
delimited by the external capsule
82
Figure 13. Injections targeting the basolateral amygdala.
The above figure shows the location of injection sites targeted for the BLA obtained as
resolved by immunohistochemistry for a vector expressing copGFP and an shRNA. Each
panel represents 4 different rats with anatomical areas defined by the Swanson (2004) atlas.
Panel (A) shows injection sites within bilateral hemispheres of the same rat, sliced within the
same coronal plane. In both hemispheres, injection sites seem to originate in the ventral CP
and are resolved dorsomedially to the BLA alongside and partially within the medial EC.
Panel (B) shows a unilateral injection site dorsolaterally alongside and partially within the
AMC, delimiting the anatomical rostral shape of the BLA. Panel (C) shows a viral spread in
the CP and an injection site also located dorsolaterally to the caudal amygdala. In yet
another rostral section, panel (D) shows the viral spread located almost exclusively within
the curvature of the EC, stemming laterally from the CP to the dorsolateral amygdala. Red
arrowheads show the passage of the needle, whereas white arrow heads show potential
needle end points.
83
3.3.2 Representative rats and quantifiable coronal sections
To be able to account for the downregulation of ENK mRNA, ENK shRNA
expressing vector was injected bilaterally in 8 rats in the NAc and bilaterally in 16 rats
targeted for the BLA. In order to control for the specificity of the ENK shRNA, 12 rats
received bilateral injections targeted for the BLA, with the SCR shRNA lentivector.
Careful selection of representative and quantifiable injections have yielded: 4 rats injected
in the CEA with either an ENK shRNA expressing vector (n=1) or an SCR shRNA
expressing vector (n=3), 3 rats injected in the NAc with an ENK shRNA expressing vector
(n=3) and 1 rat in the Caudate Putamen (CP) with an ENK shRNA expressing vector (n=1).
A resulting staining pattern was found in the dorsal CP, an ENK-rich region as revealed by
in situ hybridization, and has been useful in the quantification and validation of the
efficiency of our lentiviral system, and was therefore kept.
Given that amygdalar injections targeted the CEA (figure 11), and that NAc (figure
12) injections targeted more than the desired dorsal shell, the selected rats injected with an
shSCR or shENK expressing vector were quantified and grouped for knockdown
determination. Criteria for the selection of representative stereotaxic injection sites was
based on the visibility of sites within the same coronal sections by; (1) successful IHC
unilateral staining and (2) preserved uniform mRNA ENK expression through in situ
hybridization. In order to be able to compare an injection site to a non-injected site, rats
analysed and quantified were selected where only one of the 2 hemispheres injected
bilaterally showed a clear and unique staining pattern. The uniqueness of this injection
pattern served as a comparison to the other hemisphere within the same coronal plane.
Injection patterns were compared when the levels were similar and when non-symmetric
staining was present in the compared hemisphere. Inherently, this reduced the number of
rats and section comparisons, in which the staining pattern displayed symmetry across
bilateral coronal sections. In order to extract the full suggestive value of these injections,
injection site quantifications came from the less rostrally injected rat.
84
3.4 Quantification of ENK mRNA from ENKergic Neurons
-Through concurring copGFP revelation of injected shRNA expression vectors and
ISH
As a result of the selection process, results have revealed distinctive injection sites
by IHC staining and successful ENK mRNA hybridization. For injection site
quantifications, measurements were reported in μCi/gram of tissue, as retained from ISH
experiments on Biomax, Maximum Resolution film (Kodak).
3.4.1 Injection sites and area quantifications
As table 4 shows, the averaged OD for the delivery of a vector expressing an shSCR
in the CEA, shows similar values for the injected and respective contralateral (intact)
hemispheres; of 1.910 μCi/gr and 2.1576 μCi/gr respectively. A greater difference is
observed for injections with the same vector expressing shENK, within the CEA, in
averaged OD values of 0.9806 μCi/gr and 2.002 μCi/gr respectively for injected and
respective contralateral comparisons. The same pronounced difference in ENK expression
is also observed in the NAc and CP when compared to the contralateral intact hemisphere.
The injected side with an shENK expressing vector shows similar effects; 0.8281 μCi/gr
and 0.2549 μCi/gr respectively for the CP and NAc and 2.7943 μCi/gr and 0.7472 μCi/gr
for the contralateral intact hemispheres of the CP and NAc respectively. Noteworthy is that
the OD in the contralateral intact hemispheres is always greater than the injected
hemisphere, suggesting that the areas selected for comparisons in the contralateral intact
hemispheres, indeed expressed ENK and can be used as benchmark values. Solely based on
OD, these results suggest a downregulation of 51.02 % to 70.36 % for ENK mRNA
ascribed to the effects of the ENK shRNA expressing vector when compared to the
contralateral intact hemisphere. Also, the hemispheres with the shSCR expressing vector
injections show that 91.4% of the total ENK expression is preserved in the respective
injected ipsilateral hemisphere, overall. When analysed for „integrated density‟ (ID), the
proportionality of results are significant and follow those obtained from OD measurements
(data not shown).
85
Table 4- Selected measurements from ENK expression for regions where an shENK was
found (by copGFP) staining are illustrated here (mean ± standard error).
The injection site was compared to the contralateral hemisphereare expressed as mean ±
S.E.M. The area (in pixels per pixels) was the same for both compared hemispheres (ipsi
and contralateral hemispheres).
Averaged knockdown values as quantified per region from ENK mRNA
in situ hybridization for areas of injections
Region
Injected
Injection
Type
Optical Density (μCi/gr)
±S.E.M
Area (px2)
Injection Site Intact contralateral
Site
CE
A
(n =
4) S
hS
CR
1.9710
±0.267
2.1576
±0.231 2326.17
ShE
NK
0.9806
±0.212
2.002
±0.451 1783.00
CP
(n =
1)
ShE
NK
0.8281 2.7943 2467.00
NA
c
(n =
3) 0.2549
±0.0545
0.7472
±0.272 3793.25
86
3.4.2 Markedly important reductions in ENK expression mediated by an shENK expressing
vector but not the shSCR expressing vector
Results from in situ hybridization of ENK mRNA in regions where an shENK
expressing vector was delivered induced an overall attenuation of ENK mRNA (62.42%
downregulated, as averaged). These results are summarized for total area quantifications in
figure 14 and will be discussed in this present section. In accordance with our primary goal
to assess the efficiency of our lentiviral system, we have pooled brain regions injected with
an ENK shRNA expressing vector and compared those to brain regions injected with an
SCR shRNA expressing vector in terms of ENK expression. For each injection type, we
have further also compared brain regions injected with an shRNA expressing vector to
„virus-free‟ contralateral hemispheres. All statistical comparisons were made by using
either an unpaired t-test or a paired t-test as appropriately defined by the Bonferroni
stringent correction (p < 0.025 [0.05/2] for 2 comparisons).
3.4.2.1 Optical density measurements in the targeted area injected with the viral
solution
In order to account for differences between shENK injected rats and shSCR injected
rats, an unpaired student t-test was used to compare, the optical density values for ENK
mRNA expression of virally injected areas. As figure 14 shows, a significant reduction of
ENK mRNA is observed in shENK injected rats when compared to shSCR injected rats,
(t(6) = 4.45; Pbon = 0.004), even after applying the stringent Bonferroni correction. On the
other hand, comparisons between the „intact‟ contralateral hemispheres of shENK injected
rats and „intact‟ contralateral hemispheres of shSCR injected rats, indicated no effects, as
they did not survive the Bonferroni correction ( t(6) = 0.93; p = 0.386 n.s.). These results
show that ENK mRNA is significantly attenuated specifically by shENK injected rats and
shows no specificity for the shSCR expressing vector to downregulate ENK mRNA.
When comparing ipsilateral and contralateral hemispheres within individual rats, a
paired t-test assessed the difference in ENK mRNA expression between shRNA delivered
hemispheres and respective contralateral hemispheres. In this case, comparisons revealed
that injected hemispheres with an shENK expressing vector showed significant
87
attenuations of ENK mRNA expression when compared to their intact contralateral
hemispheres, ( t(4) = 2.95; Pbon = 0.021) (figure 14), even after the application of the
Bonferroni correction. As expected, hemispheres injected with a vector expressing an
shSCR also did not show significantly decreased expression of ENK mRNA when
compared to their intact contralateral hemispheres ( t(2) = 2.72; Pbon > 0.05). Significance
is therefore confirmed for the shENK expressing vector, only in injected hemispheres, as
given by the Bonferroni correction.
Though the Bonferroni correction engenders a stringent comparison for multiple
tests, paired and unpaired t-tests showed synonymous conclusions. Taken together, these
results showed similar conclusions as in table 4, given the suggestive role of the shENK
expressing vector towards the attenuation of ENK mRNA in shENK delivered
hemispheres. When shSCR injected hemispheres are compared to intact hemispheres we
see that the same viral vector which harbors either expression cassette does not hinder the
expression of ENK mRNA.
88
ENK mRNA expression within targeted
areas of injections
Figure 14. Optical density measurements of ENK mRNA expression in the area
injected with the viral solution.
As resolved by radioactive in situ hybridization, the optical density values for virally injected areas
were averaged for injections with; a vector expressing a scrambled shRNA (shSCR) or injections with
the same vector targeting ENK mRNA (shENK). These measurements were collected in different
regions namely, the central amygdala (CEA), the nucleus accumbens (NAc) and caudate putamen
(CP). The optical densities for injected sites, in μCi/gram of tissue, were compared by injection type
and injected hemispheres. A significant difference is observed between shENK injected hemispheres
and shSCR injected hemispheres (across rats), showing the significant reduction of ENK mRNA; as
observed in the injected hemispheres of shENK ( t(6) = 4.45; **p < 0.01). An equally significant
reduction of ENK mRNA is observed in the injected hemispheres of shENK injected rats, when
compared to their respective „intact‟, (within the same rat) contralateral hemispheres ( t(4) = 2.95; *p
< 0.05). Comparisons between the „intact‟ contralateral hemispheres (across rats) of shENK and
shSCR injected rats did not show significance ( t(6) = 0.93; p = 0.386 n.s.). ENK expression also
remained unchanged when shSCR injected hemispheres were compared with their respective (within
the same rat), intact contralateral hemispheres ( t(2) = 2.72; P > 0.05, paired t-test). Results are means
with ± SEM and the Bonferroni correction was applied for paired and unpaired t-tests. Statistical
significance was defined as; **P < 0.01, *P < 0.05.
89
3.4.3 Specification for shRNA expression vector in vivo quantification control with GAD65
In view of our experimental design, the design of in vivo ENK expression
attenuation called for an appropriate control that could also be quantified alongside in vivo.
Given the properties of GAD65 mRNA co-expression in ENKergic neurons of the striatum,
we have also quantified its expression on autoradiographic film (figure 15). This has been
done for only one rat brain injected with an ENK shRNA expressing vector in the striatum
(AP +1.5, ML ± 1.0, DV -6.5), hereby contending for a concise in vivo control. The other
rat injected in the striatum designed for GAD65 mRNA quantification, did not abide by our
standards of quantification showing overly symmetric viral stains or unquantifiable signal.
One must remember however, that these shENK injections were designed for quantification
purposes with the GAD65 probe and act as a control. The coordinates of injection were
distinct to also take account for precision of coordinates.
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ENK mRNA and GAD65 mRNA expression for
ShENK injected rats
Figure 15. Representative brain sections demonstrating viral delivery of the
lentiviral vector expressing an ShENK in the striatum.
Panel (A) shows localized injection sites via the staining pattern of the reporter copGFP
after 3 weeks of stable genomic neuronal integration, in lightfield microscopy. Red
contour illustrates the extent of the injection. The same brain section (B), with the same
area of viral delivery (red contour, as for the staining pattern in A), is observed for a brain
section with a hybridized ENK mRNA radioprobe. Hybridization indicates that for the
region where the virus was delivered, the lack of ENK mRNA expression correlates with
the area of copGFP staining in panel A. An adjacent brain section (C), shows GAD65
mRNA probe hybridization within the same rat, in the same hemisphere of injection. The
area corresponding to the copGFP staining pattern (A) was also transposed onto this
coronal section (black contour). Unlike for the lack of ENK mRNA hybridization (B)
within the area of injection, the hybridization pattern for GAD65 was uniform. For
illustrative purposes, only, the contour in panel A includes the entire staining pattern of
the virus as resolved by IHC with disregard to neurons transduced. However as panels B
&C show, quantifications discriminated for striatal neurons most likely to produce
endogenous ENK mRNA.
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3.4.3.1 GAD65 mRNA quantifications reveals no differences for integrated density
and optical density measurements
An unpaired student t-test comparison of the GAD65 expression by OD
quantifications, within the striatum injected with an shENK expressing vector, did not show
significant difference (t (12) = 0.907; p = 0.382). This intact bilateral maintenance shows a
similar level of GAD65 mRNA expression between both: injected hemispheres and
contralateral hemispheres (figure 16). GAD65 mRNA hybridization was only performed in
a rat injected with shENK expressing vector but quantifications proceeded between
ispilateral and contralateral hemispheres in the same manner without a Bonferroni
correction. These results not only show a similar expression of GAD65 mRNA within both
hemispheres but more importantly also suggest no disruption of GABA synthesis by mere
injections or by the effects of the shENK lentivector for targeting anomalous mRNA. These
results ultimately confirm the validation of GAD65 mRNA quantification as a valid control
to our quantification system.
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GAD65 mRNA expression within targeted
areas of injections
Figure 16. The following figure shows optical density measurements for GAD65
mRNA expression in targeted areas, injected with a vector expressing an shENK.
As resolved by radioactive in situ hybridization, the values correspond to the occupied
area by the viral injections. These were averaged for striatal injections with a lentiviral vector
targeting ENK mRNA (ShENK expressing vector). The optical density for the injected
targeted sites, in μCi/gram of tissue, were compared to the contralateral, intact hemispheres
with a congruent area of comparison between both hemispheres, ipsilateral and contralateral
sides, respectively. Comparisons between injected and respective contralateral, intact
hemispheres did not show significant difference in GAD65 mRNA expression (t (12) =
0.907; p = 0.382), thereby demonstrating that GABA expression was not affected by the
knockdown of ENK mRNA.
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CHAPTER 4
4 DISCUSSION
The following study demonstrates the specific, effective and long-term
downregulation of ENK mRNA in 2 major ENK expressing regions, infected by a lentiviral
vector expressing an shENK. Given the ultimate goals of this study, we have first
developed and optimized a viable production of lentiviral particles that have efficiently
been delivered and transduced onto cells. Select technical optimizations have led to a
reproducible production protocol generating a routine average of 1.39 x 108 TU/ml for
shENK expressing vectors. In the second part of our study, we have approached the
downregulation of in vitro ENK mRNA by differentiating PC12 cells, with NaB, into ENK
secreting cells. In differentiating PC12 cells in vitro, however, we have not observed cell-
to-substratum adhesion nor the expression of ENK mRNA, as would be expected from a
cell line inducible for chromaffin differentiation (Byrd and Alho, 1987). Instead, we have
biochemically extracted increasing quantities of DYN mRNA from differentiated cell
proportional to the concentrations of the differentiating agent, NaB. Nevertheless, given the
orientation of our main objective, we have assayed the transduction, genomic integration
and shRNA expression of the lentiviral vector in the rat brain. Validating our 3rd
objective
after stereotaxic delivery, our study demonstrates a distinguished attenuation averaged to
62.4% for the ENK transcript in ENKergic nuclei infected with an shENK lentiviral
expressing vector. Evidence from our injections with the same vector yet expressing a non-
specific shRNA (shSCR) confirmed the specificity of the shENK. Injection sites were
quantified in a systematic and orderly method of analysis utilizing a combined form of IHC
for the reporter protein and ISH for ENK mRNA. Our in vivo results acquiesced our main
goal objectives by successfully attenuating ENK mRNA expression. As a whole, this study
ultimately characterizes a local and partial depletion of ENK mRNA promoting the use of
this vector system in behavioural stress studies and lack-of-function studies for ENK‟s
primary action in projected nuclei.
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4.1 Methodological development of lentiviral production
The 2 main goals achieved in the successful and infectious production of lentiviral
particles were; (1) the efficient production per volume as delivered of high-titer lentiviral
particles and, (2) the attenuation of ENK mRNA in ENK-specific differentiated neurons,
with a lentivector expressing an shENK. The following sections are thus a compilation of
select technical optimizations and individual amendments that have led to a reproducible
methodology for the transfection, concentration and titration steps of our lentiviral
production protocol.
4.1.1 Optimization of selected steps for a high-titer viral production and in-between batch
reproducibility
Seeing that our main purpose was the in vivo validation of an ENK mRNA
knockdown in the rat brain, we have sought to optimize this methodology by first
establishing a reproducible protocol of production. We have therefore devised the scientific
base of our lentiviral production from pioneer and recent studies which sought to produce
high-titer infectious viral particles in a reproducible manner (Dull et al., 1998; Salmon and
Trono, 2006; Tiscornia et al., 2006). Initially, we have pasted the recipe for viral production
from assorted parts of these studies and compiled them together to create a production
protocol. After each viral batch production, obtaining a titer of at least 1 x 108 TU/ml
comprised an absolute and experimental checkpoint comprising of a minimal cut-off titer to
proceed with stereotaxic injections. As it was the case prior to optimization, if a production
batch did not pass the TU/units threshold, stereotaxic surgeries were stalled and viral
production recapitulated from transfection. More often than not, the resulting rescheduling
engendered organizational losses. We thus proceeded and succeeded to the optimization of
selected steps in order to transform irregular low viral titers into high-titer -viral
productions (above 1 x 108 TU/ml) by efficient packaging and concentration of viral
particles. Evidently, given many minor and major optimized steps, only the most important
ones will be discussed here. Many of these optimizations were technical in nature, gained
from acquired experience while working with the 293TN cell line.
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4.1.2 Preliminary technical notes and considerations
Preliminary optimizations were performed after familiarization to the 293TN cell
line in cell culture to enhance transfectability and viral production. These were simple
measures that complemented empirical evidence but that did not always require
accompanied support from the literature. However, general precautionary measures in the
upkeep of the cell line such as maintaining a constant optimal growth by spliting cells 1:3
or 1:4 every 3-4 days increased transfection efficiencies (Segall and Sutton, 2003). Much
like other labs, we have also kept cells 50-80% confluent (Johnson et al., 2003; Planelles,
2003), 24hrs prior to transfection of plasmids, thereby concluding that systematically
plating 3 million cells in our petri dishes was adequate. This increased transfection
efficiency by validating that neither too few nor too many cells would occlude the vital
space and jeopardize our plasmid ratios. This also kept cells in a continuous log-phase,
which increased survival and permissivity to transfection. Objectively, many of these
survival and growth features were particular specificities of the sub-strain of the 293TN cell
line we have used. In other words, given the often subtle and minor specificities of cell
lines in general, we accustomed our methodology to determine the optimal time for the
confluency of cells and growth. Experience has also taught that though other labs have
advocated the use of coating plastic culture dishes with Poly-L-lysine in order to enhance
cell adherence and yield higher vector output (Johnson et al., 2003; Tiscornia et al., 2006),
we have discontinued the use of Poly-L-lysine coated plates after having tried it. In our
hands, cells showed adequate adherence without the ligand and no noticeable cell loss in
between passaging.
4.1.3 A functional transfection foreshadows the generation of high-titer particles
The production of high-titer viral particles is intimately related to the transfection
efficiency of the plasmids into our lentiviral vector producing cell line (293TN), thus
comprising a titer-limiting step which warrants analysis. In our early days of viral
production, after transfection, we have washed cells twice with Opti-MEM containing
HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Invitrogen), before
replacing the culture dish with the regular medium. Opti-MEM as opposed to DMEM,
contains no glucose, more buffers and allows for reduced serum concentration that can still
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make cells grow. The point of the HEPES buffer as we used it then, was to maintain the pH
(recommended between 7.05-7.15) of the medium for the cells since, the application of the
transfection precipitate-product (Ca(PO4)-DNA) lowers the pH of the medium, which in
turn, jeopardizes the integrity of the precipitate necessary for transfection for review, see
(Jordan and Wurm, 2004). As many factors affect the solubility of the precipitate prior and
during transfection (Chowdhury et al., 2003; Jordan and Wurm, 2004), variations of as little
as 0.05 pH units in the DNA-Ca(PO4) solution, considerately affect precipitate size, and
lowering transfection efficiency for review, see (Wright, 2009). Given these pH
fluctuations, washing the cells with Opti-MEM seemed like a better medium, at that time
since it theoretically appeared to restore the pH of the culture dish (re-establish the higher
extracellular [Ca2+
]), for review, see (Jordan and Wurm, 2004).
However, in our experiments, we have not observed that post-treatment of cells
after transfection with washes of Opti-MEM improved the viral titer obtained at the end of
a typical production batch of cells. This may be attributed to the sub-strain of cells we have
used and its robust permissivity to our transfection cocktail which allowed the uptake of
most DNA-precipitate complexes with very little residues. Calcium phosphate transfections
are reputed to be efficient and inexpensive but subtle factors can render them less efficient.
Given that no noticeable advantages were reported, we have therefore discontinued rinsing
our cells altogether and retracted the use of Opti-MEM medium in all steps of cell culture.
Further changes, amendments and optimizations involved concentration and titration
methods of the viral particles.
4.1.4 Purification and concentration methodology
After supernatant collection and filtration, the requirement for the high-titer
concentration in vivo is an imminent requirement given the complexity of the rat brain and
the propensity of different cells, the vast possibility of transducible neurons and
transducible neurons that express ENK mRNA. By experimental consideration, we have
reduced a former 27 000 rpm cycle of centrifugation to a 20 000 rpm cycle of
centrifugation with the SW-32 rotor (or corresponding gravitational force with the SW-28
rotor, when the SW-32 was unavailable). Although viral titers of 1.0 x (105 -10
6) TU/ml
have been observed in the cell culture medium after peak viral production (Burns et al.,
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1993; Mátrai et al., 2010), by robust physical concentration and ultracentrifugation, our
higher nominal titer of at least 1.0 x 108 TU/ml was achieved. This titer comprises a
recommended titer for in vivo work and for harder to transduct cells, that can be readily
attained by a standard ultracentrifugation (Rosenblad and Lundberg, 2003).
As table 5 shows, different g forces for rotors similar to the SW-32 rotor have
concentrated viral particles with preserved high-titer. The VSV envelope, given its
monomeric glycoprotein structure, as was used in our case, has allowed us to concentrate
our particles further by protecting viral particles from hydrodynamic particle turbulence
and longer lengths of centrifugation cycles (Burns et al., 1993; Segura et al., 2006). This
particular feature allowed us to modify our protocol and incorporate a slower cycle of
centrifugation with greater flexibility. Across the literature, acceptable ultracentrifugation
cycles to generate higher vector titers have been reported between (1-3) x 105 and up to
x106 g forces with times ranging between 1-4h per cycle at 4
oC for review, see (Segura et
al., 2006; Sakuma et al., 2012). However, each combination of speed and length of
centrifugation affects the output of titer collected. At the other side of the spectrum, there is
an inverse relationship between time and speed; very long cycles of centrifugation with
very low speed centrifugation methods not only collect larger volume capacity but can also
collect high-titer virus (VandenDriessche et al., 2002; Johnson et al., 2003). In our case,
though the VSVg is equipped to withstand the shearing force of ultraspeed centrifugations
as comprised in one of its feature points, by lowering the speed of centrifugation, we
captured enough force to pellet viral particles in a shorter amount of time. By reducing
both speed and time within an acceptable range of the centrifugation spectrum, less
shearing force is placed on pelleted viral particles. Conveniently this change saves time, but
this optimization also concurs with a lower minimal centrifugation speed that other groups
have also performed in the lower part of the spectrum of centrifugations in the literature
(Nadeau, 2003), generating similar cut-off titers. The reduction in speed of centrifugation
accompanied reproducibility in our protocol which validated a primary goal but also aided
in the formation of high-titer particles.
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Adapted from (Segura et al., 2006)
4.1.5 Selection of different producer and titration cell lines, enhances the production of
lentiviral particles
Evidently, the selection of producer and titering cell lines bears a direct link to the
viral titer output produced. Many selections of cell lines across the literature have produced
and titrated viral vectors to consequently show the efficiency of a production protocol by
the strength of the viral titer. As these next 3 sections will demonstrate, we have achieved a
high-viral titer after optimization of our production protocol, by concisely selecting and
combining 2 different cell lines in our most reproducible results. For the production of
viruses, the 293TN comprised an ideal selection over other cell lines.
Table 5-Different centrifugation times and parameters for different viral preparations.
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Although the parental kidney producer cell line like the HEK 293 is a popular
choice for the production of lentiviral particles (Hommel et al., 2003), we have supported
the use of its more robust derivation, the 293TN cell line as a producer cell line. The
expression of the SV40 lTa, is an endogenous feature of the 293TN producing cell line,
which has promoted selection of the latter for production. In comparison to the HEK293
cell line which does not express the lTa, the endogenous expression of the lTa in the 293T-
derived cell lines, has been shown to produce a 10-fold higher lentiviral vector titer (Gama-
Norton et al., 2011). From a general standpoint in cells expressing the SV40 lTa, it has
been shown that the antigen seems to promote DNA synthesis and transcription in general
and indirect ways (Ahuja et al., 2005). The best utility feature in our case is that the lTa
complements with the SV40 origin of replication domain on the backbone of the ENK
pSIH-H1-copGFP expression vector which forms the viral genome as mentioned in the
System Bioscience, user manual 2010. Therefore, in cells such as the 293TN, the
endogenous expression of the lTa, guarantees that the lTa can bind the vector‟s SV40 origin
of replication to promote transcription of the expression vector upon initial transfection, as
was shown for another HIV-derived virus (Lu et al., 2004). Although other studies argue
that the lTa and its analogous SV40 binding domain on the vector can promote the
episomal replication of the plasmid (Bonci et al., 2003; Wanisch and Yáñez-Muñoz, 2009),
our vector backbone terminates transcription with a poly-adenylation signal (System
Bioscience, user manual 2010) which further promotes the formation of the short-hairpin
and precipitates the RNA silencing mechanism. The SV40 T antigen found in the 293TN
cells and the cell line‟s robust flexibility in the culture dish, thus promotes its experimental
choice as a producer cell line competent for the creation of virions. Although, the 293TN
cell line was suitable prior to optimizations for titering, we have now opted to estimate
vector expression in HeLa cells.
4.1.6 HeLa cell titration
Our choice of titering cell line was one of many other human cell lines available
(HEK 293 and derivatives, HCT116 and HT1080 derivative HeLa, but also TE 671,
HEPG2, K562 and U937) that can be easily transduced by lentiviruses with the VSVg
envelope with high efficiency (Cui and Chang, 2003; Mao et al., 2008; Barde et al., 2010).
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Some degree of consideration must be attributed in the general sense for the choice of
titering cell line based on the method of titration, the desired in vivo output cells targeted
for transduction and the reproducibility of titrations required (excellent study here, (Sastry
et al., 2002)). In one instance, the Trono group have advocated the HCT116 cell line over
the HeLa cell line for DNA titer determination (Barde et al., 2010) given a greater genome
stability. In our case, GFP titer determination by FACS is highly dependent on the inherent
GFP transgene expression by an active promoter after integration in the genome of the cell
line. In this regard, the choice of a HeLa cell line over a 293TN cell line for titering is not
obvious given the robustness and flexibility of the 293TN cell line.
In one comparative study the 293 cell line showed better transduction efficiency and
functional titer (for 4, and 14 days after transduction) than the HeLa cell line for a lentiviral
delivery of a GFP transgene with a CMV promoter (Sastry et al., 2002). The higher
transduction efficiency in 293 cells over HeLa cells concurred in both, GFP and DNA
titering methods. Moreover, in another study that assayed the CMV, PGK and EF1-α
promoters in the 293T, HT1080, HOS and HeLa cell lines for titering methods, the CMV
promoter in the 293T cell line showed the highest transgene activity (Mao et al., 2008).
Interestingly, when the 293T, HOS and HeLa cell lines were compared for GFP expression
by their EF1-α promoters, HeLa cells showed the highest GFP expression. What these
studies show is that though the CMV promoter works for both 293T and HeLa cell lines,
for titering purposes the 293T cell line shows more promoter activity and improved
expression of the GFP transgene. However, these blatant features may overcompensate the
titer obtained, making it harder to realistically relate the titer to an in vivo, neuronal system.
4.1.7 A stricter measure of strength provided by the HeLa cell line
Regardless of the method of production or titration, the underlining basis of our
study lies in the adequate and efficient downregulation of the ENK protein, evidently with a
large enough TU/ml to transduct neuronal cells in vivo. Any cell line that would have been
used for titration in our protocol would have to express and „pass‟ the ultimate cut-off titer
we have concurrently accepted to meet by exceeding the minimum threshold of 1.0 x 108
TU/ml. Given that the 293TN cell line is optimal for viral production and overall enhanced
transcription, with the expression of the lTa, it depicts somewhat an „inflated‟ titer during
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titering, given the enhanced transduction and promoter activity. By choosing a cell line like
the HeLa cell line which just like neuronal cells does not express enhanced features for the
transcription of the of the GFP transgene after vector integration, we have proposed a
stricter requirement of strength prior to in vivo vector delivery. Seeing that our minimum
threshold of 1.0 x 108
TU/ml, does not change with the titering cell line, meeting it in a
HeLa cell line, depicts a closer to reality titer. Furthermore, just as many other teams have
supported viral production in 293T cells yet titration in HeLa cells (Farson et al., 2001;
Abordo-Adesida et al., 2005), it is easier to validate and compare their studies to ours. It is
also important to realize that the mere titration in a different cell line from the producing
cell line, acts as an additional screening element in itself, also. By performing titration in a
different cell line from the producing cell line, we can apprehend neuronal transduction by
observing the results of the transduction in the titering cell line. In the case of the HeLa cell
line, this can thus provide a glimpse of the ultimate transduction in vivo, the ultimate
destination which bears a complete physiological system with augmented variables.
As we have demonstrated, the optimization of a viral production protocol can be
achieved by properly dissecting and optimizing key steps of the methodology. In doing so,
we have achieved the reproducible concentration of high-titer viral particles with a
threshold cut-off useful for in vivo experimentations. Given the expression of a short-
hairpin specific to the ENK mRNA transcript, the next challenge was to quantify the
virulence and efficiency of our generated viral titers. We have thus devised a differentiation
schedule of PC12 cells, designed to receive the delivery of lentiviral particles and
downregulate the expression of ENK mRNA in vitro. The quantification of this system by
RT-PCR could thus be used to adequately measure the effects of a lentiviral application
onto more complex systems, such as the in vivo rat brain.
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4.2 In vitro knockdown system: PC12 rat pheochromocytoma
cell line
4.2.1 Adequate and representative in vitro system
In order to verify the effectiveness and specificity of our lentiviral preparation, a
discriminate system of quantification needed to be selected representative of the silencing
mechanism elicited by the lentiviral expression of the shRNA. Although, many teams have
utilized various in vitro techniques to quantify a knockdown prior to in vivo delivery, our
method of choice needed to adequately represent the attenuation prior being assayed in
vivo, and needed to be arbitrarily reproduced in multiple experimental protocols. ENK
peptides, as secreted and present inside the central nervous system in most brain structures,
(Hökfelt et al., 1977; Miller and Pickel, 1980; Khachaturian et al., 1983; Harlan et al.,
1987) have scarcely been expressed in vitro, with neuronal phenotype and peptidic opioid
consistency. Therefore studies with ENK secreting neurons in vitro have been mainly
limited to primary cell cultures (Falk et al., 2006). However, given the complexities
required for the upkeep of a primary culture to mimic the in vivo variables such as extended
viral incubation times and delivery after a lentiviral application, a more robust alternative
was found. As chromaffin cells were very early on associated with the synthesis of opioids
(Weisinger, 1995), a tumorigenic and transformed cell line derived from the adrenal
medulla has been selected as an alternative system with appropriate flexibility for our
purpose (see section 1.1.4).
4.2.1.1 The PC12 in vitro system
By utilizing the PC12 cell line, capable of opioid secretion under the strict
framework of a differentiation schedule, the scarcity of methods expressing in vitro ENK
has been remedied. However, even if these cells have been branded by many as “premiere
model cells” for review, see (Fujita et al., 1989) for malleable differentiation in the study of
neuronal differentiation, this immediate novelty implies cautionary methodical planning.
Pharmacological manipulation utilizing various differentiating agents, have shown to direct
maturational outcomes in either of 2 major cellular end points, from the basal chromaffin-
like and immature state PC12 cells find themselves in. On one hand, early work with PC12
103
cells first established the cell line to be inducible by nerve growth factor (NGF)
differentiation towards sympathetic neuronal maturation (Greene and Tischler, 1976). On
the other hand, later studies with certain short-chain fatty acids (SCFAs) and sodium
butyrate (NaB) have promoted the differentiation of PC12 cells into mature chromaffin
cells able to secrete catecholamines and ENK (Byrd et al., 1987; Mally et al., 2004).
Whereas both types of cellular endpoints have been classified based on obvious different
morphologies, our results indicate otherwise. A brief understanding of the peculiarities of
the differentiation process will solicit an understanding of our results.
4.2.1.2 Inducible maturational effects by specific differentiation agents tailor
bidirectional outcomes for PC12 cells
The differentiation of PC12 cells, in either direction is essential in order to correctly
use this cell line. Untreated PC12 cells display very limited levels of proenkephalin mRNA
when analysed by northern blot (Byrd et al., 1987) yet synthesized and secreted DYN (Karl
et al., 1996).
When applying NGF, neuron-like differentiation accompanies biochemical, synaptic
and morphological features of neuronal cells that transform the immature PC12 cells into
non-dividing and neurite outgrowing cells (Schubert et al., 1977; Gunning et al., 1981; Lee
et al., 2012). Although at first glance, the differentiation of PC12 cells into a neuron-like
lineage may seem suitable to the in vivo orientation of our study, 2 points need be
considered. The first is that the cells need to be constantly differentiated with NGF,
otherwise, they start reversing to an undifferentiated cellular state within just 24hrs (Greene
and Tischler, 1976). The second and most important factor is that NGF-treated cells express
very little Met-ENK when compared to untreated PC12 cells (Mizrachi et al., 1990). Given
these 2 factors, lentiviral vectors expressing an shENK would show unnoticeable
attenuation in PC12 cells differentiated with NGF while incorporating a laborious
requirement to keep them differentiated without interfering with the silencing mechanism
of mRNA.
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In differentiating cells toward the mature chromaffin route, NaB leads to a
permanent cell product by simply incorporating pre-diluted NaB into medium changes,
which were performed every second day. This method of differentiation increases the
expression of ENK mRNA and has been viewed as the „restoration‟ of gene expression to
an adrenal-like state (Byrd and Alho, 1987). More specifically, this model of differentiation
is defined by the reversal of the tumorigenic phenotype (malignant pheochromocytoma
cell phenotype) to a more mature and characteristic chromaffin phenotype (Pastan and
Willingham, 1978; Byrd and Lichtin, 1987; Margioris et al., 1992). Morphological
characteristics include; „flattened‟ cell morphology (from a round bright state), a constant
„monolayer‟ of cells, cell-to-substratum adhesion, cell to cell adhesions and also reported,
the increase in size and number of the secreted catecholamine storage granules (Naranjo et
al., 1986; Byrd and Alho, 1987; Byrd et al., 1987; Mizrachi et al., 1990). In addition to
these results, other teams have also concurred evidence for the maturational process with
biochemical markers, showing that an increasing gradient of ENK expression exists, as
differentiation proceeds along the chromaffin pathway (Mizrachi et al., 1990). By
differentiating PC12 cells with NaB, the Byrd group not only links gene expression and
phenotype with NaB differentiation in PC12 cells but also implies a reversal of gene
expression, thereby defining a functional role for the differentiation agent. They support
this view by showing the specific induction of ENK mRNA that marks the process of
differentiation (biochemical) and morphological cell state changes (cellular phenotype),
both comprised in the maturational process toward chromaffin cells. In the analysis of our
results, this specific „link‟ has very crucial and designated corollaries. Given the erratic and
unexpected experimental conclusions we have observed, a sequential yet progressive
interpretation of our data will be presented. The following sections compound the results of
various studies, which build on the fundamental principles evinced by the Byrd and
Margioris teams.
4.2.1.3 The overall role on transcription by NaB is tied to cellular arrest
Alongside the maturational effects in the PC12 line reported by the Byrd group,
NaB has a broad role on the whole transcriptome of the cell that has shed light on the
interpretation of our results. NaB has been reported to be a potent histone deacetylase
105
(HDAC) inhibitor, hereby altering gene expression, inhibiting cell proliferation, preventing
the onset of neoplasia and even promoting „death signals‟ for the induction of apoptosis
(Davie, 2003; Chen et al., 2004; Xu et al., 2007; Cayo et al., 2009). The control of
transcription is mediated by the reversible changes induced to the chromatin by acetylation
through histone acetyltransferases (HATs) and deacetylation through HDACs. By virtue, of
being an HDAC inhibitor, NaB inhibits the repression of transcription and the condensation
of chromatin, which allows transcription. In the process of mediating transcription, these
can be direct effects of promoter binding by NaB or these can be secondary effects of
chromatin re-modelling (Xu et al., 2007). Another great example of an HDAC inhibitor,
has showed that by inhibiting HDAC deacetylation, resulting hyperacetylation leads to the
accumulation of acetylation on histones H3 and H4 of p21‟s promoter (Richon et al., 2000).
In turn, the upregulation and expression of cyclin-dependent kinase (CDK) inhibitor p21
(see figure 17), causes growth arrest. By considering the overall role of NaB, as an HDAC
inhibitor, we can provide a preliminary and partial molecular explanation for the cellular
arrest we have observed in vitro. The role of NaB as an HDAC inhibitor also foreshadows a
causal basis for the gradient of DYN mRNA extracted, a theme which will be fully
developed in the coming sections.
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The role of NaB on cell growth inhibition
Figure 17. The inhibition of histone deactylases and cellular outcomes.
Figure shows the effects mediated by the inhibition of histone deacetylases (HDACs) by an
HDAC inhibitor (HDACi), such as NaB. For our purposes, we are mainly concerned with the
signalling cascades related to the „growth arrest‟, the apoptotic pathways (intrinsic and
extrinsic) as they have been reported on PC12 cells induced by NaB, (Cayo et al., 2009). Figure
modified from (Xu et al., 2007).
107
4.2.2 Validity of controls and technical considerations
As we begin to dissect the experimental turnout of our differentiation schedule on
our line of PC12 cells, it is important to assess the validity of our controls. As figure 10
shows, there are no primers bound for any of the first 3 lanes (lanes 1,2,3, figure 10) i.e, the
lanes belonging to the total rat brain RNA control (from Clontech). Even if those lanes
don‟t indicate any binding for GAPDH, DYN and ENK on that particular gel, all 3 types of
primers assayed do indeed show binding to the cDNA for each of their specific genes. This
is representative for GAPDH in the PC12 control lanes and in the lanes with all
concentrations of NaB assayed. Primer binding was also successful for DYN in the RNA
extractions which resolved it, mostly for the 6mM (lanes 10-13) and 12mM (lanes 13-15)
differentiations. Lastly, the primer binding was also successful for ENK mRNA, and on
other gels assayed during this study (data not shown).
The fact that no GAPDH, DYN or ENK cDNA is resolved on the gel from the total
rat brain control mRNA does not mean an error in technical terms attributed to the reverse
transcription step, to the primer dilution, or to the PCR amplification step. This is because
all experiments were performed sequentially for all types of RNA without a distinct setting
or pattern change for either. Starting with the „cDNA synthesis‟ protocol, to each type of
extracted RNA and including the total rat brain RNA, the random primers, oligo-dt primers
and the water were added systematically and sequentially (section 2.1.4). A similar
sequential and systematic procedure was also performed for the PCR amplification
comprising 3 individual tube contents for the primer-specific cDNA stretches and a pooled
mix with the contents of the PCR amplification. Following, a consistent serial methodology
for all steps of the PCR amplification protocol, excludes these reasons for the absence of
the total rat brain RNA positive control. More likely, the absence of this control is due to a
possible degradation or expiration of the rat brain RNA, resulting from either being over
handled with repeated freezing and unthawing episodes or simply that the RNA degraded
with time, as is suggested by manufacturer‟s “Bench Guide, Chapter 3”, (Qiagen).
Our cellular control, being the undifferentiated PC12 cells, shows a similar intensity
of GAPDH, which validates primer binding for that gene of the total RNA extracted from
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PC12 cells. The similarity in transcript intensity across all bands for GAPDH confirms the
use of this gene as a good internal standard given that NaB shows no effect in altering
mRNA levels in either undifferentiated and differentiated cells. Also, GAPDH concurs with
the unaltered mRNA level for another housekeeping gene, β-actin, upon NaB
differentiation (Byrd and Alho, 1987). These concurring observations based on 2 different
housekeeping genes show that NaB has a pattern of activation that does not affect the
genetic loci of either gene, hereby validating either housekeeping gene as a reference for
gene expression.
The absence of a band for ENK amplified DNA is not an anomaly for the PC12
undifferentiated cells as was for the total rat brain RNA lack of ENK. Rather, this concurs
with the literature (Byrd et al., 1987). The absence of a band for DYN amplified DNA
however, contradicts the results obtained by other teams for untreated PC12 cells. Other
teams have shown that untreated PC12 cells expressed the prodynorphin transcript and its 8
kDa peptide product, postulating the latter as being the prodynorphin (Karl et al., 1996). In
contrast, our results show the absence of DYN with no treatment (control) and the presence
of DYN at highest intensity with the highest concentration of NaB administered. This thus
establishes a dose-response for NaB and alludes to effects that target an increase in the
transcription of DYN mRNA.
4.2.3 NaB differentiation did not fully induce the chromaffin phenotype expected
The consistent petri dish observations of cellular arrest and even cellular decline
manifested by our butyrate induced PC12 cells shows an effect partially caused by NaB
(but not fully) (Cayo et al., 2009). The cellular arrest that was observed with differentiated
cells was in line with the pioneer studies performed by Byrd et al (Byrd and Alho, 1987;
Byrd et al., 1987) upon which we have based our methodology. However, we have not
observed the remainder morphological characteristic markers reported by this same group,
which are essential for the maturation of the cells (enumerated previously). Given that our
empirical observations contrast pioneering evidence, we suggest that the differentiation of
our PC12 cell line did not achieve the desired morphological end point which accompanies
the secretion of ENK, in vitro but rather, that it may have possibly only „started‟ to
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differentiate towards a chromaffin phenotype. Though this revelation is mostly
morphological, cell-cell, cell-substratum interactions and adhesive properties provide very
important survival cues as well as consequences to signal transduction and the induction of
downstream gene expression. Most of these interactions were not observed especially for
ENK mRNA at the 6mM differentiation.
4.2.4 Unattained morphological end points show a link to unattained biochemical
endpoints
Our results further show that the lack of cellular adhesion over time has
accompanied unexpected biochemical extractions. In the differentiated cells in our study,
the lack of adhesion has posed a serious threat to the normal and sequential induction of
transcription, following the cellular arrest expected by the normal induction of
differentiation (Davie, 2003). In the case of PC12 cells, if unfolding morphological and
biochemical changes are carefully accounted for and quantified, a link easily results that
steers the temporal and organized sequence of events. A study involving the co-culturing of
PC12 cells with other adrenergic in vitro cell lines, namely, bovine adrenal medullary
endothelial cells (BAME cells), (Mizrachi et al., 1990) showed that when undifferentiated
PC12 cells were grown on a monolayer of BAME cells, proteins responsible for adhesion
not only played a role in adhesion but also spawned downstream functional changes in both
cell types, thereby providing a microenvironment favourable for survival. What this study
showed was that the successful differentiation of PC12 cells was marked by sequential
events involving maturational morphological changes (chromaffin phenotype) followed by
a 2.5 fold increase of Met-ENK contents, after 3 days of co-culture. These changes were
also shown to be entirely mediated by the co-culturing effects of cell-cell contacts and cell-
substrata adhesions. In our case we have not observed an increasing gradient of ENK
mRNA at any concentration of NaB administered most likely because cells were not
morphologically adapted while in the dish, as differentiation proceeded. As cells
differentiate toward another cellular type, it is important to keep in mind that preliminary
and ordered cellular events ensure cell survival which substantially magnifies the success
of a differentiation process.
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4.2.5 The effect of NaB for DYN is dose-responsive due to unreversed PC12 phenotype
Our results indicate a clear dose-response to the upregulation of DYN mRNA,
through increasing concentrations of NaB as opposed to the induction of ENK mRNA. This
further supports our view that our PC12 cells merely „started‟ to differentiate along the
chromaffin-like pathway and also supports the lack of morphological markers observed in
the culture dish. As supported by literature, the presence of DYN mRNA can be found in
rat pheochromocytomas because of highly concentrated production whereas, rat chromaffin
cells do not synthesize peptides derived from prodynorphin (Margioris et al., 1992, 1995;
Karl et al., 1996). The implication here, is that the de-differentiation of chromaffin cells to
a tumorigenic pheochromocytoma state (opposite route), induces the expression of the
DYN gene. These studies provide support to our explanations so far. If indeed the process
of differentiation in PC12 cells towards chromaffin phenotype is as suggested by the Byrd
group, by the “reversal of the malignant phenotype”, the expression of DYN mRNA should
decrease as the differentiation process perseveres in the chromaffin pathway. As we have
not observed an increase in ENK mRNA by 3 sessions of differentiation and we have not
observed a decreased expression of DYN mRNA, it is reasonable to think that cells were
closer to an undifferentiated, immature state. This kind of reasoning may provide an
explanation to our results, not just for the mRNA expression of DYN extracted over ENK
mRNA in our results, but also for the morphological changes in PC12 cells. This would
then be in line with the Margioris and Byrd groups. However, the reversal in gene
regulation based on the expression of DYN mRNA, has not been tested yet. Nevertheless,
even if cells were halted on the differentiation path, the increasing dose-response for
extracted DYN mRNA with increasing NaB concentrations shows an overall mechanism of
action for NaB and an effect on the opioid genetic loci within the cell line. The pronounced
cell loss, however, is beyond the effect of the differentiating agent.
4.2.6 We suggest that a positive culture microenvironment provides proper nutrient
absorption and is essential for our PC12 cell line’s biochemical consistency
For much of our study with PC12 cells we have based our methodology on the
recognized lab work of Byrd et al. They report a constant monolayer of cells and no
difficulties in working with the cell line. A major problem in our study was the upkeep of
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the cell line in general, since neither control nor differentiated cells, ever reached
confluence, the latter ensuing a pronounced cell loss by the third session of differentiation.
Confluency can be viewed as an important indicator of the cell culture acclimatization and
an indicator of cell survival within a culture microenvironment. In addition to confluency,
the co-culturing of PC12 and BAME cells (as previously described) showed how crucial
and essential the local „microenvironmental‟ (Gstraunthaler, 2003) requirements for in vitro
and in vivo mammalian cell survival, growth metabolism and/or proliferation, truly is
(figure 18). There are 3 fundamental parts which mimic the physiological state that needed
for a proper mammalian cell survival, in untransformed cells. The microenvironment of the
culture dish provides the space and nutrients so that cells can develop both morphologically
and biochemically. We therefore suggest that in spite of our results, an appreciation of such
interactions within an in vitro setting would not only help the morphological markers
promote the biochemical markers, respectfully, but also enable the absorption of necessary
nutrients.
As part of the acclimatization to the culture‟s „diffuse environment‟, the efficiency
of nutrient absorption has been questioned, given the pronounced cell loss and lack of
confluency. Classic studies with transformed cell lines have shown that when a BHK 21
cell line is transformed by a tumorigenic virus (polyoma virus), the cell line can grow while
in suspension and be responsive to growth factors found in the Fetal Calf Serum (we used
FBS) to traverse the cell cycle. However, the same BHK 21 cells but this time
untransformed, require adherence to respond to the growth factors in the serum (Clarke et
al., 1970). These studies question the ability of our cell line to adapt to the culture
conditions and absorb the right growth factors in the right state. In other words, these
studies further reinforce that the administration of NaB to our PC12 cell line (following our
methodology), most likely triggered differentiation prior to the adaptation of the cells to the
culture dish. As a result, this may have attenuated „survival signals‟ within the
microenvironment of the cells in culture by the lack of absorption of key growth factors.
Had cells been responsive to the environment, this could have possibly enabled cells to
survive overall, and most likely live through the sessions of differentiations. In the context
of our study, these facts suggest that a positive culture environment incorporating the 3
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most essential components (figure 18) would allow PC12 cells to survive, mature and
develop adhesive properties.
Understanding morphological
and biochemical endpoints
Figure 18. Microenvironment requirements of the mammalian cell.
Above figure shows the 3 most essential parts that are essential to the homeostatic behaviour of
cells in vitro and in vivo. These portray the most basic needs for adherent cells to survive and to
respect mitotic events. These are also conducive requirements for the healthy, proliferation,
division or differentiation of cells. (1) “Diffuse environment”. This requirement consists of the
cell medium and the supplemented growth factors provided in the culture serums. (2) “Cell-
ECM adhesion”. These are cell-substratum adhesions with the cell binding to the extra-cellular
matrix (ECM). (3) “Junctional Adhesions”. These are cell-cell adhesions, when cells bind to one
another. Figure obtained from (Gstraunthaler, 2003).
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4.2.7 We suggest that integrin ligand-coated culture plates may help to stabilize our PC12
cell line
Long before in-depth studies on the important requirements for cell viability in
culture, Tischler et al, noticed the better adhesion to collagen-coated culture dishes (as
opposed to plastic culture dishes) for PC12 cells during NGF differentiation (Greene and
Tischler, 1976). In later years, some have even proposed a disadvantage for cells able to
adhere to plastic, suggesting a possible subpopulation bias different from the remainder of
PC12 cells in culture (Banker and Goslin, 1998). Although not absolutely required for
adherence with NaB differentiation, studies especially with NGF differentiation have
broadened the general appreciation for ligand-coated supports and their relation to PC12
cell viability. A recent study has even confirmed the knowledge available concerning the
importance of cell anchorage to precoated dishes with ECM proteins for the coordinated
control and regulation of cellular events (Lee et al., 2012). In studies with undifferentiated
PC12 cells, ECM proteins are equally important with dynamic receptor properties, showing
best binding to laminin (LN) and collagen IV (col IV) but much less binding to fibronectin
(FN) (Tomaselli et al., 1987, 1988). More recently, poly-L-lysine with LN surpassed the
other types of ECM anchorage proteins, as determined electronically (Zhang et al., 2012).
By mediating activation of cell-cell contacts through integrin binding, a more favourable
physiological microenvironment is created, thus fulfilling 2 of the 3 major requirements for
healthy culture adaptation (see figure 18). Integrin anchorage proteins may thus stabilize
and help adapt our PC12 cell line prior the induction for differentiation by NaB.
4.2.8 The availability of growth factors in our cell culture medium may also have
contributed to unexpected cell behaviour
The modulation of downstream signalling for cell cycle events by the ligation of
integrins is a concept that is closely linked to cell adhesion and growth factor signalling
(Aplin et al., 1998). More specifically it is not the mere adhesion of integrins itself that
triggers changes that promote cell cycling, but rather, the synergic collaboration with the
soluble growth factors available.
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Given the discrepancies associated with global animal sera (Mather and Roberts,
1998; Riond et al., 2009; Brunner et al., 2010), it is possible that certain growth factors
lacked. As the importance was stressed earlier, in regards to the absorption of growth
factors in either a suspended cellular state or adherent cell state, it is important to see that
because of a possible serum bias, the lack of cell adhesion may have also resulted. This also
concurs that for adhesion to be present; it requires the synergistic effect of „active‟ growth
factors to accompany the process of adhesion (i.e. MAPK signalling pathway) (Aplin et al.,
1998). We are not the first group to have had difficulties in the upkeep of a cell line and to
have questioned the quality of HS and FBS in the growth and maintenance of our cells
(Peters et al., 2000). Such a group has suggested that individual lots of HS and FBS be
tested through pre-screening. Such a testing could simply include testing each serum in
different cell lines with different concentrations and report the differences in cell growth,
survival or differentiation.
4.2.9 Our results confirm NaB as a potent inhibitor of histone deacetylase
Strictly from the biochemical extraction of DYN mRNA, an independent
mechanism reveals an unspecific effect of NaB as a potent HDAC inhibitor. Although
unexplained, it is only sensible that by being exposed to an HDAC inhibitor, PC12 cells
respond in cellular arrest and the induction of certain genes. Since we know that DYN is
expressed in the PC12 pheochromocytoma cell line, but not in chromaffin cells, this
provides an explanation for the upregulation of DYN with NaB administrations of
increasing concentrations. It is well agreed, however, that there is an overall increase in
gene transcription with the induction of a high concentration of an HDAC inhibitor for a
longer period of time in culture (Xu et al., 2007). Our results confirm the effects of
inhibition of an HDAC; through the persistence of hyperacetylated promoter sequences. By
looking into the methylation and acetylation patterns for the promoters of DYN, we would
be able to confirm this hypothesis, and clarify our results further. Along this tangent,
further experiments could include an assessment of the control of translation (expression
and degradation of mRNA) when subjected to NaB differentiation in spite of chromatin
remodelling. Though multiple other experiments exist to further the study of DYN
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expression, these would not be of interest, in our experimental design and in the scope of
this study.
4.2.10 Final considerations and final notes
Given that our cell line required elements promoting its adaption and adhesion in
culture prior differentiation, the unexpected isolation of DYN mRNA, as opposed to ENK
mRNA, showed that cells did not progress towards the chromaffin route. More importantly
in the grand scheme of our study, this prevented the application of lentiviruses expressing
an shENK. Though it can seem that this erratic behaviour is an isolated case, the pioneer
establishers of the PC12 cell line have attributed these effects to the irradiations that
induced the tumor in rats ( A S. Tischler, Powers, & Alroy, 2004). Although they provide
technical considerations and urge PC12 users to try more than one cell line, we have not
proceeded further. The need for an in vitro validation all together, was also discontinued,
given time constraints, as well as any further possible optimizations involving the pre-
coating of culture dishes with integrin ligands. Seeing that a viable source of ENK mRNA
was nevertheless needed to pursue our ultimate goal, major experimentations were set to
ultimately be performed in vivo. Hence the organizational implications and technical
optimizations were directed for in vivo experimentations.
4.3 In vivo Knockdown system: Stereotaxic injections of shRNA
vectors
At the heart of our study, we have validated our 3rd
and most important objective;
the successful attenuation of ENK mRNA with a characterized quantification of the
attenuated transcript is representative of a nominal titer of at least 1.0 x 108 TU/ml. In vivo,
we have demonstrated that the delivery of an shRNA expressing lentivirus with high
infectious titer, produced and knocked down ENK mRNA expression. The particularity of
the knockdown observed was further validated when compared to the same vector
expressing a non-specific shRNA (scrambled shRNA), which showed synonymous ENK
mRNA expression in injected and contralateral hemispheres, conferring that the expression
vector did not have a global unspecific effect. The specificity of the shENK was confirmed
by an undisturbed local environment around striatal injection sites, for the uniform
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expression of GAD65 mRNA. The interpretation of the quantified ENK knockdown in this
study will be discussed in this section.
4.3.1 Immunohistochemistry in our protocol: usefulness and limitations
One of the most critical tools from this study for quantification purposes was the
combined IHC and ISH within the same experimental sitting, which became necessary. The
requirement to combine both techniques within the same experimental setting was based on
empirical observations that the ENK mRNA radioprobe signal on the resulting
autoradiographic film often appeared „washed off‟ and faint when ISH and IHC were
performed in 2 different settings at 2 different times as is routinely performed (Simmons et
al., 1989; Hebb et al., 2004; Poulin et al., 2006). The faint mRNA signal was hypothesized
to be linked to a partial degradation of mRNA. By shortening the time between first IHC
and then ISH, the preservation of the mRNA radioprobe signal transferred normally onto
the autoradiographic film. The result of this experimental milestone allowed the
quantification of the co-localization (or lack of) between copGFP and ENK mRNA and
insured that autoradiographic films could be routinely quantified with consistent OD
measurements. The reporter protein copGFP is in itself, a very insightful tool with a direct
link to a knockdown other than quantification of mRNA itself. The binding of a polyclonal
antibody (rabbit anti-copGFP) in IHC, delimits the area of injection and the spread of
transduced cells around injection sites. Although a powerful method of analysis, results
obtained from IHC should be carefully considered prior their interpretation.
4.3.2 Preliminary notes and knockdown insight provided by immunohistochemistry
As previously mentioned, following delivery of cells permissive to lentiviral VSVg
transduction, the expression vector integrates within the target cell‟s genome. In our study,
we successfully witnessed the integration of the vector within the genome of neurons, the
latter showing the prolific transcription of the copGFP marker protein hereby facilitating
the detection of infected cells after delivery. Although the staining was resolved by a
reaction of hydrogen peroxide with DAB after the binding of the anti-copGFP antibody in
fixed brain sections, the inherent mechanism of expression is reminiscent of the one
observed in our HeLa cells; transcription by the RNA polymerase II for the CMV promoter
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found on the expression vector (Ghazal and Nelson, 1991). However, as was designed for
our pSIH-H1 expression vector, the expression of the hairpin uses RNA polymerase III for
the expression of the shRNA, under the H1 strong promoter and a (T5) terminal sequence
for the dislodgment of the polymerase III (Abbas-Terki et al., 2002; Qin et al., 2003;
Wiznerowicz and Trono, 2003). Though these 2 polymerases may work together
molecularly (Raha et al., 2010), an actual measurement of the ENK transcript remaining
after viral integration needs be assessed to quantify the knockdown. Therefore the
facilitated detection of infected cells at this level without ISH is merely an aid in the
localization of integrated vectors within the genomes of infected cells. The copGFP
staining pattern defines an area within which, potential downregulation can be localized as
an intermediate step in the full quantification of the attenuated expression.
4.3.3 Viral spreading as witnessed by copGFP visualization shows limited diffusion
Viral diffusion within brain parenchyma is evidently an important factor to consider
when targeting specific regions of the rat brain. As many studies have reported, given the
large size of either HIV-1 or lentiviral particles bearing the VSVg envelope, diffusion
within the brain‟s extracellular space (ECS) is limited. These viral particles range in the
100 nm (Cetin et al., 2006) whereas the available space between cells in the ECS of the
brain is in the range of 40-60 nm, making expansive diffusion unlikely and a haphazard.
These studies nevertheless bear limits showing, that though spreading is very close to the
injection site in general brain parenchyma, many other variables can influence the spread of
the virus. In our experience, these include but are not limited to; injection sites near
ventricles, a tract of nerve fibers or unusual parenchymal regions.
As we have noticed, the delivery of the lentivirus expression vector into the brain
parenchyma spans multiple coronal sections, in both the amygdala and the NAc, as
revealed by viral stain of IHC for copGFP. As demonstrated in figure 11, although multiple
coronal sections have been observed to contain the integrated expression vector, those
closest to the injection site have the highest concentration of signal visualized by the
reaction with DAB, implying the likelihood of multiple infected single neuronal cells at the
core of the injection. Transduced cells within the injection sites and around it, thus
comprise multiple coronal levels and we have considered them part of the region acceptable
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for area transposition. It is thus within this spectrum of transgene expression that we have
selected our brain sections to further proceed with our quantification scheme. Staining was
generally observed near the injection sites, and spreading to more distal areas was minimal
in our case. Concurring with another study that delivered lentivirus within the NAc
(Fernandes et al., 2012), the bulk of our spreading pattern was retained close to the
injection sites. Distal coronal sections with a faint GFP signal have been preferentially
excluded from our analysis, being before or after those quantified in the NAc and
amygdala.
4.3.4 Very efficient transduction and knockdown of ENK mRNA in different ENKergic
populations
According to our results, the highest knockdown for the shENK expressing
lentivirus was measured in striatal structures namely, the dorsal CP (70.36%), followed by
the NAc (65.89%) and followed by quantification in the amygdala, the lateral and medial
CEA (51.02%), as observed by optical density measurements and comparisons to the intact
hemispheres. The attenuation observed for ENK mRNA in different ENKergic populations
shows the prominent transduction of neuronal cell and the indiscriminate expression of the
shENK in infected neurons. Since we have been able to quantify a knockdown in each of
these structures, based on the lack of colocalization of the copGFP and the ENK mRNA,
our method shows a reproducible attenuation pattern, in vivo. Our results further show that
regardless of the region targeted, the ENK transcript can be downregulated, at least to half
of the endogenous expression.
4.3.5 Percentage knockdown of ENK mRNA and ENKergic populations bias
Although the high-titer lentiviral construction we have generated was nearly
identical and within the same range and comprised within the same 108 TU/ml magnitude, a
similar attenuation pattern and percentage may be expected. However, our results show that
the knockdown witnessed depicts different values. As knockdown was quantified in
injected hemispheres, a trend being inversely proportional to the density of ENKergic
neurons is observed in a region such as the CEA with the lowest knockdown ascribed yet
endogenously expressing the highest amount of ENKergic cell bodies (fig 11). This is in
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contrast to the striatal knockdowns which are very much ENKergic also, but not as much as
the CEA.
A brief comparison between the ENKergic neurons of the striatum and amygdala
shows a significant difference in endogenous expression. The striatum has been shown to
endogenously express 2 predominant neuronal population types roughly at ~50% each; one
being ENK neurons expressing the D2 dopamine receptor while the other population being
the DYN neurons expressing the D1 dopamine receptors (Shivers et al., 1986; Steiner and
Gerfen, 1998; Reiner et al., 1999). In the amygdala however, ENK has been shown to have
an abundant expression (Day et al., 1999; Hebb et al., 2004; Poulin et al., 2008) and more
so in the CEA (McDonald, 1998; Swanson, 2003). Neuroanatomical studies have further
divided the CEA into multiple parts with the 3 main parts known as the medial, lateral and
capsular CEA; CEAm, CEAl and CEAc respectively (Cassell et al., 1986; Swanson and
Petrovich, 1998). The CEAl and CEAc have the highest and most concentrated ENKergic
populations of the CEA (Veinante et al., 1997; Poulin et al., 2008).
Although we did not intend to target the CEAl in our study, the shENK viral spread
over this region shows an attenuating effect to a region expressing a majority of ENKergic
neurons whereas a higher knockdown is observed in the NAc and CP, where arguably,
approximately half as many ENKergic neurons are expressed per region. This difference in
ENK expressing neurons may explain, at least in part, why the knockdown in the CEA was
quantified lower than the knockdown in striatal areas. In other words, given the dense
propensity of more ENKergic neurons, mRNA might still be produced by a minority of
neurons. The lower knockdown seen in the CEAl may allude to the lower limit of a general
knockdown but a greater sample size would be required to validate this point. With
significance however, the shSCR vector integration against the knockdown of the shENK
shows significance in the paired t-test Bonferroni correction. The significant difference can
be observed when comparing between the intact and injected hemispheres. By comparing
the shENK and shSCR delivered hemispheres and the shENK delivered hemisphere vs a
non-injected hemisphere, we ascribe the attenuation of ENK mRNA to the shENK and not
to the expression vector. By statistically comparing the shENK and shSCR delivered
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hemispheres, we can show how insignificant the delivery of the shSCR vector and its
integration in the genome affect the expression of ENK mRNA.
In contrast to quantifications in the CEA, quantifications in the NAc (while in the
ventral striatum), comprise a much more representative knockdown in our case, even if
limited. Primarily by being targeted and injected within the NAc, the population of
ENKergic neurons is consistent, showing a representative consistency when areas of
injection are freehandedly delimited. Given that very little diffusion was observed, the
quantification of knockdown was more reproducible, and more easily averaged between the
multiple hemispheres injected in the NAc. Our injections in the NAc also accompanied a
bigger sample size compared to the other regions quantified, which confers a knockdown %
that may be closer to what would typically be seen, if multiple rats were injected in the
NAc with an shENK expressing lentivector. Hence, quantifications in the NAc are more
representative shENK injections and with more representative accompanying
autoradiographic quantifications. Based on the results of our study and given the features of
quantification in the NAc, the attenuation of ENK mRNA observed reveals the most
representative level of knockdown. Over a striatal population of neurons, this knockdown
may represent the upper limit of ENK knockdown achieved with a batch of lentivirus, with
1.38 x 108 TU/ml.
It is important to remember, that just as we have performed the ENK shRNA
expression yields the efficient, yet not the absolute, attenuation of ENK mRNA. As
pioneering studies with mice deficient for ENK by gene knockout have shown, the full
disruption of the PENK gene, is not lethal (König et al., 1996). What needs to be addressed
however, is whether or not, a partial knockdown will discriminate anxiety-like behaviours
as was done in PPEKO mice. Just as models of anxiety probed for the disruption of the
absolute ENK gene, those same tests can be used to probe the partial attenuation of ENK
mRNA in anxiety-like behaviours (Bilkei-Gorzo et al., 2004). The merit of such a study
would then illustrate whether a knockdown of the ENK transcript to at least at half of the
original endogenous expression, yield a similar component of anxiety. It is only then, that
the true nominal value of a knockdown can be witnessed in a stress-related region infected
by an ENK shRNA expression vector.
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4.3.6 Viral inhibitions of ENKergic innervations
Perhaps the most important and most appreciated feature of a lentiviral delivery
targeting ENK mRNA, is the viral backbone integration within ENKergic neurons and the
expression of a silencing hairpin. Once integrated, ENK mRNA is deleted with high
specificity, interfering ultimately with the ENK projections stemming from transduced
nuclei. With greater specificity than synthetic opioid agonists and antagonists and no half-
life, ENK can be removed from a particular circuit. This is a considerable advantage over
antagonist molecules since as; pioneer in vitro experiments have shown the binding
specificities for the opioid receptors are different for each of the 3 classic receptors
specificity (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). Therefore by
completely activating the endogenous silencing mechanism of the cell, transcript copies of
ENK can be destroyed. This would be particularly useful over an antagonist molecule like
Naloxone, which displays close binding selectivity for MOR as for KOR (Mansour et al.,
1995b).
Though the specificity of our RNA silencing system exceeds that of
pharmacological substrates, one must remember that in contrast to opioid
agonists/antagonists, the integration of the virus in the genome of the cell affects target
projected cells, as opposed to the immediate local soma. As we have noticed, the targeting
of the NAc, infected the shell and the core. Because of its overall simplicity and the
propensity of ENKergic neurons in the NAc (Reiner et al., 1999; Zhou et al., 2003), a
similar knockdown was achieved in both the shell and core. However, the shell of the NAc,
has been shown to project to a ventromedial part of the VP as opposed to the dorsolateral
pallidal subterritory of the VP that the core projects to (Zahm and Heimer, 1990). As we
have induced an artificial downregulation of ENK mRNA, it is important to mention that
this has also been reported under a paradigm of chronic variable stress, where palatable
food was also assessed in the stress response (Christiansen et al., 2011). What the group
noticed was that chronic variable stress increased ENK mRNA in the NAc shell, whereas
sucrose attenuated the ENK mRNA in the lateral shell, agreeing with the administration of
daily palatable food (Kelley et al., 2003). Though the effect of „snacking‟ and comfort food
shows an overlap between stress and reward it is still not well understood in its relation to
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endogenous peptides. In such a scenario, the virally mediated long-term attenuation of the
ENK transcript could reflect the possible role of ENK and its relation to snacking.
However, assessing the sucrose consumption in a chronic stress paradigm in which rats can
habituate to the repeated stress such as immobilization (Dumont et al., 2000), may also be a
useful comparison in trying to develop a “comfort food hypothesis”.
4.3.7 Considerations for area selection subject to quantification from IHC staining
Hypothetically, if qualitative and well defined elements during surgeries such as
identical stereotaxic coordinates, viral volume injected and identical intracranial incubation
time frame (figure 6), they should cater for very little variation when comparing different
rats injected in the same region with the same expression vector. However, when working
with an in vivo system, various limitations arise. Solely based on viral spreading as was
observed, resulting injection sites don‟t always preserve similar surface areas or staining
patterns from one rat to the other. For example in the injections in the CEA as shown in
table 4, regardless of the shRNA expression cassette (shENK vs shSCR), different surface
areas subjected to quantification have resulted. Different surface areas of viral spreads in
between rats and hemispheres were also observed in the NAc, although these were
accounted within the average area quantified in table 4. As surface area is related to volume
injected in the general parenchyma but does not increase beyond 500-700 μm for viable
injections given limited diffusion (Cetin et al., 2006; Osten et al., 2006), the variations in
surface area and staining pattern that we have noticed are in part due to prominent viral
injection tracts above the injection sites, if all other variables are to be left out. This applies
to comparisons in-between rats which showed the most variation or those rats with bilateral
injection sites in which the surface area of the injection sites differed most from one
hemisphere to the other. Though this greatly reduces the number of coronal sections with
symmetric injections sites, our quantification system was stringent enough to quantify
subtle differences in injection patterns. Professedly, in order to support this claim, we
would have to show that the injection tracks, take away volume from the injection site and
evidently quantify the knockdown induced by the volume within the track. Accordingly,
this was quantified, as the staining pattern in the CP illustrates the impact of injection
tracks.
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4.3.8 Injection tracks carry efficient infectious titer of viral solution refluxed from
amygdalar injection sites
Injection tracks are common in a wide spectrum of stereotaxic experiments, where
needles are used to deliver organic material intracranially, since backflow is often noticed
up along the injection track (Saha et al., 2000; Rosenblad and Lundberg, 2003; Vande
Velde et al., 2011). Interestingly, our injection tracks may explain, not only why the surface
area and staining pattern of injection sites sometimes differed in hemispheres injected at the
same level but also the presence of viral stains in the CP; Thus the quantification of the
reflux volume in the CP showed that a knockdown of up to 70.36% as given by an shENK
delivery designed for the amygdala. Essentially, there are 2 main reasons that can explain
why considerable viral volume was found in the CP, enough to be able to quantify it. In the
first place, the staining stemmed from injections targeting the BLA, showing a dorsolateral
orientation of injections tracks that also passed lateral or through the CP (dorsolaterally). In
the second place, reflux from the removal of the needle may have caused intracranial
pressure to backflow freshly injected viral solution. As figure 13 shows, a typical viral stain
reflux departs from the amygdala, making way dorsolaterally towards the CP along the
external or intermediate capsules. Many other tracks have been observed in targeting the
NAc and BLA, as well as in other stereotaxic experiments but the track used to quantify the
CP, occupied enough surface area to be quantified.
Though the knockdown in the CP may be the largest quantified, one must remember
that the knockdown in the CP is the quantification of an injection track and cannot be taken
as the upper limit of lentiviral knockdown with certainty. Therefore the effective percent
knockdown may depict an unrepresentative knockdown for the CP and should not be
viewed as a region where viral solution was administered. Moreover, the singularity of this
quantified region certainly does not define the knockdown as an absolute. Also, the lack of
a comparison to the effects of an injection of an empty vector or an shSCR expressing
vector like we have used, does not provide a control for a baseline in ENK expression
within that rat, at the level of the injection track. The knockdown in the CP only reveals,
that injection tracks bearing a small volume of lentiviral vector expressing an shENK can
124
also be quantified. A repeated and more localized volume like for our NAc injections
shows a better representation of a knockdown.
Injection tracks are often inevitable but they can be minimized, provided
obstructions such as lateral ventricles don‟t get pierced. Managing injection tracks is
generally based on empirical principles of stereotaxic surgery and proper stereotaxic
etiquette, for craniotomy, anesthesia and delivery (Cetin et al., 2006). In essence, viral
delivery of lentiviruses (and recombinant adeno-associated viruses) works best if at least
10-15 minutes is dedicated for each single injection site, much like we have done. If during
surgery, the brain is treated like a „closed container‟ so as to minimize intracranial pressure,
injection tracks can also be minimized. In our case, although we have waited before, during
and after injection totalling approximately 20 minutes for each injection site, the only
reasonable amendment would be to remove the syringe from the brain parenchyma over 5
or 10 additional minutes, as is done in injections with cannulae (Fernandes et al., 2012).
Alternatively, a „double injection technique‟ normally used to separate the large injection
volume in 2 injection batches in studies trying to induce cerebral hemorrhage, may also
reduce reflux (Sansing et al., 2011). Although our volume is several-fold smaller, by
waiting 5 minutes in between delivery batches, they have efficiently reduced intracranial
pressure.
4.3.9 In vivo controls for quantifications
Although, knockdown is ascribed to the shENK expressing vector, the shSCR is just
as equally a necessary measure of control. Concurrently, injections in the CEA validate that
multiple shSCR vector injections which show a consensus of 8.65% conferring
insignificance to the baseline integration of the lentiviral vector within the genome of
control rats. Given its nature, the primary use of the shSCR vector is that by having an
unrelated shRNA, the vector can show successful integration within the backbone of the
cell‟s genome while having its resulting expressed hairpin, indeed expressed but yet
destroyed by the Dicer mechanism. The use of the shSCR further validates that
downregulation by the shENK vector shows effects attributed to the shENK expressing
lentivirus as opposed to general effects that might simply activate the silencing machinery.
125
In a study, such as ours where the purpose is to develop and quantify a knockdown,
the choice of the shSCR as a control vector comprises the best comparison tool to the
effects of the shENK. Caution must be rendered when comparing one region quantified for
a knockdown in ISH to another. The variability of the ENKergic neurons has been carefully
documented to vary greatly, inter-individually (Krishnan et al., 2007). In its proper form, a
comparison of one hemisphere with a downregulated transcript is best compared to its
contralateral hemisphere in order to account for possible inter-individual differences in
ENK transcript. However, as we have demonstrated the comparisons between shENK and
shSCR, injected rats is just as significant. In addition, given that the practicality for in vivo
behavioural analysis is not represented by only having one hemisphere attenuated for ENK
expression but rather for bilateral attenuation, comparisons to shSCR, are a very valid
measure of baseline control for mRNA knockdown studies. Also, bilateral injections with
an shSCR would be used to screen for abnormal behaviours ensued by the mere insertion of
the vector within the genome of neurons. This could be similar to the benefit from placebo
surgeries which probe for the invasive surgery and monitor for behavioural changes.
However, a control for the integrity of injected brains composes an imminent requirement
when performing stereotaxy.
The GAD65 hybridization showed preserved signal in the striatum of rats injected
with an shENK expressing vector as well as in the corresponding un-injected hemispheres.
This control also shows how specific our shENK expressing vector is, for the
downregulation of ENK mRNA specifically; given that all striatal neurons use GABA
(Steiner and Gerfen, 1998), and that ENK and GAD65 colocalize in many structures of the
amygdala also (Poulin et al., 2008). Since the GAD65 mRNA quantifications remained
very similar across both hemispheres quantified, as reported statistically (figures 15 and
16), the specificity of the viral vector is validated. Quantification with the GAD65 mRNA
further showed that injection tracks did not affect the tissues of injected brains.
4.3.10 Anatomical hurdles and limitations
Generating a viable knockdown for ENK mRNA nevertheless accompanies many in
vivo hurdles in achieving the stereotaxical target sites in both the NAc and the amygdala.
Although we have catered our coordinates for the BLA for injections rendered to the
126
amygdala, we have not resolved any injection sites in the BLA that consisted of a
quantifiable knockdown. Rather, our injections show that the CEA directly adjacent to the
BLA was targeted. More specifically, the CEAl was the recipient of most of the injections
administered as delivered for a lentiviral vector expressing an shSCR or the same vector
expressing an shENK. In rats that could not be quantified, the viral spread was localized to
the external capsule, the amygdalar capsule or both (figure 13). The random pockets of
concentrated viral spread alongside the EC may have been the transduction of a few
intercalated paracapsular islands rostrally (Palomares-Castillo et al., 2012) or assorted
intercalated nuclei of the amygdala in lateral parts (Pinard et al., 2012). Given the
lateralization of the viral spread in the amygdala, these results therefore allude to an
obstruction posed by the peri-BLA, strong enough to divert the orientation of the needle.
Indeed, this diversion was caused by the thick fibers of the external capsule which have
been reported to be comprised of afferents from higher-ordered cortical structures (Müller
et al., 2009). As these fibers contour the periamygdalar structures that are the LA and BLA,
synapses of these fibers have been observed originating from afferents of the entorhinal and
perirhinal cortices (Von Bohlen Und Halbach and Albrecht, 2002). Unfortunately, given
the thickness of these afferents, our Hamilton syringe bent to pressure, as opposed to
cutting through these fibers. Since the LA harbours the thicker portion of the capsule, and
as we targeted the posterior BLA, being parasagittal to the LA, the needle may not have
been sturdy enough to pierce the capsule. The cortical afferents to the amygdala thus
diverted the terminal tip of the needle, retaining viral solution within or along the external
capsules of the amygdala. In essence, all that was required was a syringe with the ability to
resist being bent when propelled towards the capsule. Hypothetically, once pierced, the
reverse effect could also have been observed; a delivered virus contained solely within the
nuclei entrapped by the external and amygdalar capsules.
As we have reported, injections destined to target the NAc shell have been assessed
as part of the core and shell, on unilateral hemispheres. Given that the shell is medial and
ventral to the core (according to the Swanson Atlas), injections targeted the more dorsal
part of the NAc as opposed to the ventral region. Additionally, most rats targeted for the
NAc showed delivery of the virus in more rostral sections of the striatum, comprising parts
of the NAc where the core is predominant to the shell (Swanson, 2004). These results are
127
reminiscent of an incisive bar set too low and loose coordinates. Given the well
documented and inherent challenge of stereotaxic surgeries (Kruger et al., 1995), the
authors of the atlas recommend that based on the coordinates provided, each investigator
should refine coordinates in spite of many rat-specific variables. These include the weight,
species, sex, individual animal variability and fixing by the ear bars, along the interaural
line. They also suggest consulting more than one atlas, so that the coordinates can be
averaged by the operant, thus customizing the stereotaxic apparatus or stereotaxic method
to target the same region of interest.
In addition, given the imminent proximity of the lateral ventricles, a small change in
needle orientation would automatically penetrate a ventricle. Therefore, the lack of needle
rigidity also interfered with the targeting of the NAc shell. As the needle was propelled
through the rat brain, intracranial pressure and brain parenchyma influenced the track of the
needle through the NAc. Although the effect is not as pronounced as for the diversion in the
EC around the BLA, a flexible and long needle gauge will be more susceptible to the minor
fluctuations in pressure.
In light of limitations in the NAc and BLA, there are a few solutions at hand that
can be proposed. Given that a major issue in the targeting of the BLA was that our 7000
series experienced much curvature within the brain, a viable solution would be to reduce
the length of the needle, and have a sturdy needle clasp that will stabilize the needle. A very
interesting alternative which fits the description of a more rigid syringe is stereotaxic
surgery by using micropipettes (Cetin et al., 2006). Recently, Hamilton produced a new line
of „Neuro‟ syringes, with an adjustable needle point of up to 20mm and a supportive sleeve
for the syringe. This greatly contrasts our previous 7000 model series, in that, the needle
was not adjustable and comprised of a 69.9mm needle length. Inevitably, a smaller needle
length, the susceptibility to bend the needle by handling or when inserted in the brain is
greatly reduced. The use of Neuro syringes for any further injections may serve this
purpose. Although the NAc shell does not have a set of thick fibers to penetrate, the rigidity
of the neurosyringe would be useful in avoiding the ventricles. After the rat is properly
positioned in the stereotaxic apparatus, so as to position a flat skull, an idea to further target
the shell would be to provide an angle prior to injection. As some teams who have targeted
128
the CEA with a Hamilton syringe in order to avoid the fibers of the internal capsule, they
have performed their injections at a 100
degree angle to the parasagittal plane (Saha et al.,
2000). By providing a 100
degree angle to the parasagittal plane for injections targeting the
NAc shell, the clearance of the ventricles may increase the reproducibility of bilateral
injections.
As can be expected, the greatest limitation in this study is the sample size. Given a
larger sample size, the ENK mRNA downregulated by a vector expressing an shENK in our
selected regions would help determine a threshold of knockdown required for ENK mRNA.
A larger sample size of both shENK and shSCR -injected brains would help determine the
definite value of ENK mRNA leftover. Of course, these would have to be tied to
behavioural conclusions, since ENK has been shown to have dichotomous fluctuations in
different regions under a similar chronic stress paradigm (Dumont et al., 2000; Mansi et al.,
2000), while mediating a portion of the global physiological response to stress. Based on
studies that would show the preliminary fluctuations of ENK mRNA following a stress-
related paradigm, the viral delivery of shENK would help determine which regions of the
brain respond best to anxiety probed behavioural tests by showing resilience or
vulnerability. What would at first be a region most sensitive to an ENK deficit, naturally,
would then be challenged artificially by a transcript attenuation of the same order.
129
Conclusion
Performed nearly 2 decades ago, the global deletion of ENK in mutant mice, shows
the non-lethality of a peptide at the expense of increased anxiety and lowered pain
threshold (König et al., 1996). When it was virally overexpressed in the CEA, however,
ENK what shown to potentiate an overall anxiolytic effect, this time specific a particular
region (Kang et al., 2000). In this study, we have shown that the virally mediated ENK
shRNA expression by a neurotropic third generation lentivirus, can be expected to attenuate
more than half of the original expression of ENK mRNA, synthesized in the ENKergic
populations of the amygdala and the NAc. The facilitated integration of viral particles into
neurons and reverse transcription upon genomic integration clearly shows a robust
attenuation of ENK mRNA that has been quantified. Our results show that of the 3 most
crucial steps in a lentiviral production protocol, 2 of those have a direct bearing on the
nominal titer required for in vivo work; concentration and titration of lentiviral particles.
We have also shown that the combination of the 293TN cell line as an enhanced cell line,
given its expression of SV40 lta, (Gama-Norton et al., 2011) is best matched with another
cell line for titration. Titration in the HeLa cell line is a good choice, since it standardizes
the TU/ml that would otherwise be disproportionately titrated in the same cell line that
produced viral particles. Our in vivo work also shows that delivery in various regions of the
rat brain yields a similar spread of virus, showing that diffusion is not likely. The advantage
of contained and localized injections is that as the lack of co-localization with the reporter
gene (copGFP) is quantified on autoradiographic paper (given radioprobe binding), a
reproducible quantification system is created. The percent value of the knockdown that
ensued was realistic, as the shSCR did not induce a „nonspecific‟ off-target effect. In
addition, the uniform binding of the GAD65 mRNA radioprobe shows that no „specific‟
off-target effect was induced either, by the shENK expressing vector (Rao et al., 2009a,
2009b). These controls therefore depict a complete system of analysis where, an in vitro
system can be bypassed. Although, a case can be made against an in vitro system, having
an additional control outside of the rat CNS would allow to corroborate in vivo results. Our
PC12 system can be precipitated into maturation to chromaffin cells, by the plating of LN
or collagen IV hereby generating an artificial microenvironment, inducible for
130
differentiation under the prerogative of a differentiating agent. Finally, though ENK was
removed from the ENKergic projections destined for the VP and from the CEAm, only
behavioural tests like the startle response, light-dark test, social interaction and elevated-
zero maze can systematically decipher the functions and interactions that involve ENK in
the circuits of fear and anxiety (Bilkei-Gorzo et al., 2004).
131
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