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이학석사학위논문

Adeno-associated virus 를 매개로 한

bPAC 과 PI3Kγ 유전자의 발현 조절과

이를 통한 신경 가소성 연구

Studies on Synaptic Plasticiy by

Genetic Manipulation via

Adeno-Associated Virus

Expressing bPAC and PI3Kγ

2013 년 6 월 13 일

서울대학교 대학원

생명과학부

백 기 철

Studies on Synaptic Plasticity by

Genetic Manipulation via Adeno-Associated Virus


Adeno-Associated Virus 를 이용한

bPAC 과 PI3Kγ 유전자의 발현 조절과


지도교수 강 봉 균

이 논문을 이학석사 학위논문으로 제출함

2013 년 6 월

서울대학교 대학원

생명과학부

백 기 철

백 기 철 의 이학석사 학위논문을 인준함

2013 년 6 월

위 원 장 공 영 윤 (인)

부위원장 강 봉 균 (인)

위 원 최 석 우 (인)





Advisor : Professor Bong-Kiun Kaang, Ph.D.

A dissertation submitted to the

Graduate Faculty of Seoul National University in

partial fulfillment of the requirement for the

Degree of Master of Science

Gi-Chul Baek

Department of Biological Sciences

Graduate School of Natural Sciences

Seoul National University

Contents

Page

List of Figures ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1

Abstract ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 4

Introduction ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 6

Methods ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 11

Results ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 19

Figures ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 27

Discussion ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 63

References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 68

국문초록 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 75

1

List of Figures

Page

Figure 1. Both euPACα and euPACβ fail to increase intracellular

cAMP concentration by laser activation of the enzyme. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 28

Figure 2. cAMP manipulation by photoactivation of euPAC was

ineffectual. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 30

Figure 3. Representative images of tdTomato tagged Beggiatoa PAC

(bPAC) expression in ACC. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 32

Figure 4. Individual intracellular cAMP concentration (pmol/ml) after

photoactivation of bPAC for 5 minutes in the intensity of 35mW mm-2.

∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 34


photoactivation of bPAC for 5 minutes in the intensity of 35mW mm-2.

∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 36

Figure 6. Summation data in figure 4 and 5 according to bPAC

construct. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 38

Figure 7. Overall results of cAMP concentration changes between

2

different PAC species. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 40

Figure 8. Representative images of flag tagged p110γ or tdTomato

expression in the mouse hippocampal CA1 area. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 42

Figure 9. Basal anxiety levels of p110γ or tdTomato expressed mice in

the elevated plus maze (EPM) test. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 44

Figure 10. Basal locomotion in the open field test (OFT). ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 46

Figure 11. Contextual fear memory of p110γ or tdTomato expressed

mice. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 48

Figure 12. Spatial Learning in the Morris Water Maze test. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 50

Figure 13. Probe test on day 6 in the Morris water maze test. ∙∙∙∙∙∙∙∙∙∙∙ 52

Figure 14. Probe test on day 9 in the Morris water maze test. ∙∙∙∙∙∙∙∙∙∙∙ 54

Figure 15. Basal synaptic properties of p110 γ or tdTomato expressed

mice. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 56

Figure 16. High-frequency stimulation induced long-term potentiation

in Schaffer collateral to CA1 synapses. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 58

Figure 17. LFS-LTD was disrupted by excessive p110γ in the

Hippocampus CA1 Area.. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 60

3

Figure 18. Depotentiation induced by LFS subsequent to three trains of

theta-burst stimulation. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 62

4

Abstract





Gi-Chul Baek

Department of Biological Sciences

The Graduate School

Seoul National University

Adeno-associated viruses (AAVs) are commonly and widely

used method for introduction of the foreign genes into the host of

various species because of its stable expression and safety. Here, we

took advantages of AAV mediated genetic expression, combined with

optogenetics technique, to manipulate specific proteins or intracellular

signaling with spatiotemporal precision in order to examine synaptic

plasticity in rodent brain.

5

In the first experiment, we constructed AAV2/1 serotyped

vectors carrying either euPACs or bPAC, a cyclase which activated by

light. After delivering PAC DNA via AAV into ACC region of mice

brain, we validated the functionality of PACs using cAMP enzyme-

immuno assay. Although euPACs failed to increase intracellular cAMP

levels after photoactivation of euPAC expressed ACC slices, bPAC

successfully made significance increase in the cAMP levels.

In the second experiment, we constructed p110γ, a catalytic

subunit of PI3Kγ, expression vectors driven by Ca2+/calmodulin

dependent protein kinase II α (CaMKIIα) promoter and over-expressed

p110γ in the hippocampal CA1 region. We then examined behavioral

and electrophysiological alterations in p110γ over-expressed mice. It

revealed that over-expression of p110γ caused disruption of NMDA

receptor-dependent long-term depression (LTD) and thus, resulted in

reduced performance in hippocampal dependent spatial navigation

learning.

Key Words: Adeno-Associated Virus (AAV), Photoactivated

adenylyl cyclase (PAC), Phosphoinositide 3-kinases (PI3Ks), cyclic

adenosine monophosphate (cAMP), Optogenetics, Gain-of-function

Student Number: 2011-23252

6

Introduction


Nowadays, viruses are the most popular means of delivering

vectors carrying genes encoding desired proteins. There are two types

of widely used viruses, lenti and adeno-associated viruses (AAV).

These two viral vectors are capable of delivery genes to the host cells

with mild immune responses and express over a long period of time.

Although lenti viruses can be produced by more simple procedures and

packaged with larger size of genes, AAV is preferred because they

mostly do not integrate into host genomes and produced in higher viral

titers.

We used recombinant AAV1 (rAAV1) vectors pseudotyped

with serotype 2 packaging system which known to generate less

immune responses (Rabinowitz et al., 2004) containing the neuron

specific promoter, the mouse Ca2+/calmodulin protein kinase II α

(CaMKIIα) promoter, for effective expression in rodent neurons

(Mayford et al., 1996; Paterna et al., 2004). Using this AAV mediate

gene delivery system, we could achieve competent expression of

desired proteins in the desired regions of the mouse brain.

7

Photoactivated Adenylyl Cyclase

Recent achievements on controlling of intracellular signaling

have been greatly expanded by optogenetic techniques that render

spatiotemporal precision. Along with channelrhodopsin (ChR), the first

demonstrated single-component optogenetic tools (Boyden et al., 2005),

various kinds of light controllable enzymes, channels and pumps have

been engineered such as halorhodopsin (HR), archaerhosopsin (Arch)

optoXRs and photoactivated adenylyl cyclases (PACs), the cAMP

manipulator that we have interested (Airan et al., 2009; Stierl et al.,

2011; Yizhar et al., 2011).

There has been developed several tools for manipulating cAMP

with spatiotemporal precision. Temporal specificity was achieved by

chemically - SMICUS (spatiotemporal manipulation of intracellular

cAMP using sAC) - or optogenetically - PACs, BlaC, Rh-CT (5HT-1A),

optoXRs - regulated adenylyl cyclase (Airan et al., 2009; Oh et al.,

2010; Ryu et al., 2010; Stierl et al., 2011; Sample et al., 2012). Among

them, optoXRs and Rh-CT are opsin-receptor chimaeras, act through

G-protein signaling. The other two modulators, PACs and BlaC, are

BLUF (blue-light sensing using flavin) photoreceptor linked cyclases

that can directly control cAMP level. There are two kinds of PACs

originated from two different species.

8

Euglena gracilis, a unicellular flagellate protist, utilizes PAC as

a controller of phototaxis, oriented movement in response to the light

stimulus (Ntefidou et al., 2003). Euglena PACs (euPACs) consist of two

sununits, euPACα and euPACβ, forming a heterotetramer (Gomelsky

and Klug, 2002), though single subunit is catalytically active.

Beggiatoa, a genus of bacteria, colonizes under the sea and

metabolizes via sulfide oxidation. By analyzing its genome sequences,

Hegemann’s group found that Beggiatoa have Type-III adenylyl

cyclases liked to blue light-sensing BLUF domain (Stierl et al., 2011).

Recent papers applying PAC showed behavioral and

electrophysiological manipulation using transgenic Drosophila

expressing PAC (Schroder-Lang et al., 2007; Bucher and Buchner,

2009; Stierl et al., 2011), however, there have not been any PAC

application in rodent brain in vivo so that we focused on the validation

of PAC in rodent system.

Phosphoinositide 3-Kinases

Phosphoinositide 3-kinases (PI3Ks) were first discovered by

Lewis Cantley (Whitman et al., 1985) and are involved in various

biological processes such as growth, proliferation and differentiation of

9

the cells. PI3Ks produce various 3-phosphorylated phosphoinositides

(PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(3,4,5)P3) which

function in the trafficking pathway and signal transduction. The PI3K

family is grouped into three different classes, class I, II and III, that

based on their structures and substrates.

The class I PI3Ks are further divided into IA and IB by the

similarity of their sequences and PI3Kγ is the member of the class IB.

The class IB PI3Ks are composed of a heterodimer between the p110γ

catalytic subunit and either p101 or p84 regulatory subunits encoded by

a single gene each (Fry, 1994). Most notably, PI3Kγ, unlike other

classes of the PI3Ks, is exclusively expressed in immune system,

cardiovascular system and brain (Ruckle et al., 2006). The roles of

PI3Kγ is mostly studied in the immunology field because it was first

identified in the research with polyma viruses (Whitman et al., 1985).

Mounting evidence has been revealed that PI3Kγ conducts

requisite roles in synaptic plasticity in various brain regions. Although

the requirement of the PI3Kγ in the induction or expression of LTP is

still debated, PI3Kγ has been implicated it is deeply involved in

multiple types of LTP, such as NMDA receptor or metabotropic

glutamate receptor 1 (mGluR1)-dependent LTP in the hippocampus(van

der Heide et al., 2005; Selbach et al., 2010). Also, PI3Kγ is required for

10

certain forms of long-term depression (LTD). For example, induction of

mGluR1-dependent LTD is required for the PI3K-Akt-mTOR signaling

pathway (Hou and Klann, 2004).

Purpose of the study

Firstly, for the PAC experiment, temporally precise controlling

of intracellular cAMP concentration in vivo can give us great

advantages on studying numerous cAMP-dependent pathway related

brain functions. Moreover, it is possible to combine with another

photoactivated proteins such as channelrhodopsin due to PAC has very

low absorbance above 500nm wavelength. Therefore, we introduced

PACs to rodent system using AAV mediated gene delivery.

Secondly, for the PI3Ks experiment, in line with our previous

PI3Kγ knock-out (pik3cg-/-) study (Kim et al., 2011), we thought gain-

of-function study may also shed light on the role of PI3Kγ in the

hippocampus and, furthermore, give a clue for enhancing memory in

fast changing circumstances.

11

Methods

AAV Production and Stereotactic Viral Injection

All adeno-associated viruses were produced with AAV2/1

serotyped vectors and packaged using HEK293T cells. Viral titers were

determined by quantitative real-time PCR (Prism 7300, Applied

Biosystems, USA) with intercalater SYBR green (TAKARA, Japan).

All surgical procedures were conducted in sterile conditions

and approved by Institutional Animal Care and Use Committees of

Seoul National University. Wild-type C57BL/6J male mice (8 weeks of

age) were anesthetized by intraperitoneal injection of Ketamine and

arranged in a stereotatic frame (Stoleting Co. USA). Anterior cingulate

cortex (AP : +1.0mm, ML : ±0.35mm, DV : -1.7mm) for the PAC

experiment or hippocampal CA1 region (AP : -1.8mm, ML : ±1.5mm,

DV : -1.7mm) for the PI3Kγ experiment was targeted and equivalent

amount of AAVs (2.0×109 particles for euglena PAC, 1.5×109 particles

for Beggiatoa PAC and 3.5×109 particles for flag-p110γ and tdTomato)

were delivered bilaterally using a 10μl syringe pump (30 gauge,

Hamilton Co., USA) at a rate of 6.0μl/hr. After additional 10 minutes of

diffusion time, a needle was withdrawn and scalp was sutured by black

silk. AAV vectors were then expressed for 2~3 weeks.

12

Histology, Immunohistochemistry and Fluorescence Imaging

The mice kept under isoflurane anesthesia and transcardially

perfused with pre-chilled 4% paraformaldehyde (PFA) in PBS. Brains

were fixed in 4% PFA at 4°C overnight and then dehydrated in 30%

sucrose-PBS solution at 4°C for 2 days. Coronal brain slices were

prepared (30μm for euPAC-3xflag and flag-p110γ, 50μm for mCherry

tagged bPAC and tdTomato) by cryosection (Leica Ltd., Germany).

50μm sections for mCherry tagged bPAC or tdTomato were mounted

on a slide glass with Vectashield containing DAPI (Vector Lab, USA)

and 30μm sections for euPAC-3xflag or flag-p110γ were stored in 50%

glycerol in PBS at -20°C.

euPAC-3xflag and flag-p110γ were detected with anti-flag M2

monoclonal antibody (Sigma, USA, Cat# F3165, 1:1000) and anti-flag

polyclonal antibody (Sigma, USA, Cat# F7425, 1:1000) respectively.

After overnight incubation at 4°C, alexa-488 fluorescence conjugated

IgG (Invitrogen, USA, Cat# A11001, A11008, 1:500) was treated for 2

hours at room temperature. Prior to treat antibodies, sections were

permeabilized and blocked by incubation on the shaker (100~120rpm)

with the buffer containing 0.2% triton X-100, 2% normal goat serum

and 1% bovine serum albumin in PBS. Primary antibody was treated

overnight at 4°C and secondary antibody was treated for 2 hours at

13

room temperature. After washed with PBS, sections were mounted in

the same manner as described above. Images were acquired using a

IX51 inverted fluorescence microscope (Olympus, Japan).

Laser Application and cyclic-AMP Quantification

400μm brain slices were prepared by VT1000 slicer (Leica Ltd.,

Germany) in ice cold artificial cerebrospinal fluid (ACSF, 124mM

NaCl, 3mM KCl, 26mM NaHCO3, 1.25mM NaH2PO4, 10mM MgSO4,

10mM Glucose, 1mM CaCl2, aerated with 95% O2 and 5% CO2) from

the PAC AAV infused mice. After 30 minutes of incubation in a

complete dark chamber, blue laser was continuously irradiated for 5

minutes to ACC where PAC AAV was injected. Dual optic fibers were

mounted to stereotatic frame so that the light distributes evenly both

hemisphere and close to the tissue as possible. ACC then cut from the

brain slice and snap-frozen in liquid nitrogen right after the 5-min laser

application. (Figure 1. A)

Intracellular cAMP concentration was measured using cAMP

enzyme immunoassay kit (Cayman Chemical Co., USA) and followed

the user instruction. Briefly, frozen tissues were transferred to a tissue

grinder (WHEATON, USA) and homogenized with 150μl of 5%

14

trichloroacetic acid (TCA) in water. Cell debris were removed by

centrifugation at 1500 x g for 10 minutes. TCA was extracted from

supernatants using water-saturated ether. Samples were then heated to

70°C for 5 minutes to remove the residual ether and incubated for 18

hours at 4°C mixed with cAMP tracer and antibody in the plate pre-

coated with mouse monoclonal antiserum. To develop of the plate,

Ellman’s reagent was added and read the plate at a wavelength 412nm

using ELISA reader (Powerwave X, BioTek instruments Inc., USA).

Behavioral Experiment

Male C57BL/6J mice aged 7 weeks were purchased from

Charles River. Mice were housed two or four per cage and maintained

on a 12/12 hour light-dark cycle. Food and water were provided ad

libitum.

Open Field Test : Mice were exposed to the open field box, a square

opaque white box (40 × 40 × 40 cm), for 10 minutes under dim light.

Locomoter activity was monitored and analyzed with EthoVision 3.1

(Noldus, Netherlands).

Elevated Plus Maze Task : The elevated plus maze was made of white

15

Plexiglas and consisted of opposing two open arms and two closed

arms. All arms were 150 × 150 cm (length × width) and closed arms

were enclosed with 20-cm walls. The maze was placed 30 cm above the

floor. Mice were placed in the center of the maze and monitored for 5

minutes under the fluorescent light with EthoVision 3.1 (Noldus,

Netherlands).

Morris Water Maze Task and Reversal Learning : The water maze was

a circular opaque grey tank, 140 cm in diameter, 100 cm in height,

filled with water (22~23°C) to a depth of 30 cm. The maze was

surrounded by four distinct visual cues and all procedures were

performed under the dim light. Circular, 10 cm in diameter, Perspex

platform was positioned in the middle of the virtual quadrant (35 cm

distance to the edge of the maze, 35 cm distance to the center of the

maze) and submerged about 1.5cm below the surface of the water. Each

training session consisted of four 60-second trials with four different

starting points which was randomly selected and equally distributed in

four quadrants. For training, mice were underwent one session per day.

When mice were reached the platform, mice were allowed to stay on

the platform for 20 seconds and were then transferred to a holding-cage

for 60 seconds. If the mouse failed to locate the platform within 60

seconds, it was manually guided to the platform. For probe test on day

16

6, mice were allowed to 60 seconds free-swimming without the

platform. For reversal learning, the platform was moved to the quadrant

opposite the location on previous days and mice were then retrained for

2 days. A single 60-seconds probe trial was performed again in day 9.

All experiments were recorded and tracked with Ethovision 3.1

(Noldus, Nethelands).

Contextual Fear Conditioning : Contextual fear memory was measured

in a standard chamber (Freeze Frame, Coulbourn) after 4 days of

handling. Mice were placed in the fear chamber for 5 minutes. After 2

minutes, mice were then given three consecutive foot shocks (2s,

0.24mA, 1 minute interval). After 24 hours, mice were placed again in

the chamber for memory retrieval.

Electrophysiology

Hippocampal Slice Preparation : 400μm transverse hippocampal slices

were prepared from 12 weeks (27 days after AAV infusion) C57BL/6J

mice. Animals were anaesthetized with isoflurane and decapitated for

the brain removal. Brains were then placed in ice cold artificial

cerebrospinal fluid (ACSF, 124mM NaCl, 3mM KCl, 26mM NaHCO3,

1.25mM NaH2PO4, 10mM MgSO4, 10mM Glucose, 1mM CaCl2,

17

aerated with 95% O2 and 5% CO2). The hippocampus was removed

from the brain and sliced by VT1000 slicer (Leica Ltd., Germany),

while maintained in a cold ice. Hippocampal slices were transferred to

ACSF containing 2mM MgSO4 and 2mM CaCl2, where they were

allowed to recover at 32°C for 30 minutes and then maintained at

26~28°C before recordings were made.

Slice Electrophysiology : Extracellular recordings were performed in a

interface chamber (Campden instrument Ltd, UK) maintained at 32°C.

ACSF (124mM NaCl, 3mM KCl, 26mM NaHCO3, 1.25mM NaH2PO4,

2mM MgSO4, 10mM glucose, 2mM CaCl2, aerated as above.) perfused

continuously at a rate of 2ml/min. Standard extracellular recordings

were performed in the CA1 region of hippocampal slices to measure

the slope of evoked filed excitatory postsynaptic potentials (fEPSPs).

Measured fEPSP responses were magnified 100 times by Axopatch

200B amplifier (Molecular Devices, USA) and digitized with a

Digidata 1322A A/D board at a sampling rate of 10kHz. Recordings

were monitored and analyzed using WinLTP program (WinLTP Ltd.,

UK).

Synaptic Plasticity : A bipolar stimulating electrode was placed in the

stratum radiatum of the CA1 region and the baseline responses were set

at 5~20μA (0.1 ms pulse width) of stimulation intensity, depending on

18

basal fEPSP responses. Following a stable baseline period of at least 20

minutes, LTP of LTD were induced by high-frequency stimulations

(100Hz, 1s) or low-frequency stimulations (1Hz, 15min), respectively.

In terms of depotentiation experiments, LTP was induced by 3 trains of

theta-burst stimulation at 10 seconds of inter-train interval. After 30

minutes of the LTP induction, low-frequency stimulation was (2Hz,

10min) were given to make it depotentiated.

Statistical Analysis : All experimental procedures were tested in an

interleaving way between experimental group and control group under

the blind condition. Data are presented as mean ± SEM and statistical

significance was determined using Student’s t-tests; the level of

significance was set at p < 0.05.

19

Results

Euglena PAC (euPAC) failed to elevate intracellular cAMP levels

after photoactivation ex vivo.

Cyclic adenosine monophosphate (cAMP) is a second

messenger that transduces important biological processes. Most notably

in neuroscience, cAMP activates protein kinase A (PKA) (Meinkoth et

al., 1993) which mediates various cognitive functions (Kandel, 2012).

To validate the feasibility of in vivo application of PACs, we

constructed genes encoding each euPAC subunits fused to a 3xflag tag.

These genes were packaged to AAV for achieving effective delivery to

the desired brain regions and stable expression for a longer period of

time. Since there were plenteous reports that anterior cingulate cortex

(ACC) is involved in remote memory which requires adenylyl cyclase

for its maintenance (Frankland et al., 2004; Shan et al., 2008; Goshen et

al., 2011), we opted for ACC to test functioning of PACs. Laser

irradiation was performed in the form of brain slices to save coast and

time for optic fiber implantation surgery. 30 minutes incubation in dark

place, 5 minutes photoactivation was applied and cut ACC off (Figure 1.

A). cAMP concentration was determined using a commercial assay kit,

which based on the competitive enzyme-immuno reaction between

20

cAMP-acetylcholinesterase and intracellular cAMP for binding to the

limited number of cAMP-specific antibodies pre-coated to the test plate.

In the first test, we continuously irradiated 473nm blue laser at

the intensity of 6mW mm-2 for 5 minutes. The result, however, was

unsuccessful. The cAMP concentration of either euPACβ or euPACβ

with tdTomato expressed group showed no detectable changes after

irradiation although euPACα seemed to have an activity of increasing

cAMP concentration (Figure 1. B) , it was far less than the previous

study that showed 20-fold enhancement after 5 minutes irradiation in a

single oocyte expressing euPACs (Schroder-Lang et al., 2007).

In the second test, we raised the intensity of the laser up to

30mW mm-2 to deliver the higher photoenergy, yet all groups including

tdTomato co-expression groups showed no cAMP level changes at all

(Figure 1. C). We lastly tried with maximized laser intensity of 35mW

mm-2, however, either subunits of euPACs failed to elevate the

intracellular cAMP concentrations at the level of physiologically

meaningful (Figure 2. A and B). We concluded that euPACs are

unusable to optogenetically manipulate cAMP levels in mice brain.

There are three possibilities that might cause of failure. (i) euPACs did

not work in rodent system (ii) 3xflag tagging disturbed the enzymatic

reaction or photoactivation of euPACs. (iii) cAMP was degraded much

21

faster caused by the damages when make a brain slice.

Beggiatoa PAC (bPAC) successfully increased intracellular cAMP

levels after photoactivation ex vivo.

To eliminate the possibilities that caused failure of euPACs, we

changed origin of PAC to Beggiatoa (bPAC), a bacteria living in sulfur

rich environments. (bPAC was a generous gift from Dr. Peter

Hegemann, Addgene plasmid 28135) Superiority of bPAC to euPACs

was first reported in 2011 and they used dissociated hippocampal

neuron culture as well as transgenic Drosophila to show the advantages

of bPAC (Stierl et al., 2011). According to this study, bPAC has several

merits compared to euPACs. bPAC is smaller, shows higher cyclase

activity and lower half-saturating light intensity thus needs less light

than euPACs. We also added a linker (A.A sequence : GGSGGS)

between PAC enzyme and red fluorescence protein, mCherry, to the

either side of bPAC for instant visualization using fluorescence

microscopy. Lastly, we gave more incubation time before laser

stimulation for self-recovery.

We conducted the experiment as the same manner of the last

experiment with euPACs, and observed manifest increase of cAMP

22

concentration after photoactivation although they somewhat exhibited

variations of the increased amount among individuals (Figure 4 and 5).

After confirming the expression pattern of either bPAC in ACC region,

we speculated that the variations came from majorly the difference of

bPAC expression. Both mCherry-bPAC and bPAC-mCherry showed an

adenylyl cyclase activity by photoactivation increasing the cAMP

concentration up to 3-fold from the initial level (Figure 6). Overall,

there were no differences between two subunits of euPAC or two

constructs of bPAC. On the other hand, unlike euPACs, bPAC displayed

much prominent cyclase activity driven by photostimulation suggesting

that bPAC can be a useful tool for temporally precise manipulation of

intracellular cAMP concentration in vivo (Figure 7).

p110γ Overexpression in the Hippocampal CA1 region and Basal

Behavior.

p110γ was driven by CaMKIIα promoter for neuron-specific

expression. Along with red fluorescence proteins, tdTomato, flag

tagged p110γ was expressed in both soma and dendrites of the neurons

(Figure 10). We specifically targeted hippocampal CA1 region to

examine the role of p110γ in hippocampal-dependent learning and

23

memory (Tsien et al., 1996). As the first step to explore the roles of

p110γ, we conducted a series of behavioral experiments by which basal

anxiety and locomotion can be measured.

In the elevated plus maze (EPM) test, which is well established

method to assess anxiety level in rodent, especially used as a anxiolytic

drug screening, p110γ over-expression group showed normal level of

anxiety compared to control group (Figure 11). Also the level of

anxiety and locomotion in p110γ over-expression group was normal in

open filed test (OFT) showing over 80% of the time spent in peripheral

area of the 40cm X 40cm field (Figure 12. A, B) confirming that

exogenous expression of p110γ or tdTomato does not affect normal

rodent behaviors such as anxiety and locomotion.

Hippocampal Learning of p110γ Overexpressed mice

We next investigated the effect of p110γ in the hippocampal

dependent learning. Our previous study reported that freezing level

without pre-exposure to the context in contextual fear conditioning was

reduced and time spent in previous quadrant where platform was

located in the reversal learning session of the Morris water maze was

increased in pik3cg-/- mice meaning that PI3Kγ has partial roles in

24

hippocampus-dependent learning. Arising from these evidences, we

naturally expected that fear memory formation to the novel context and

spatial learning when the location of platform was changed would be

enhanced by abundance of p110γ.

To examine this possibility, we conducted contextual fear

conditioning and Morris water maze task under the same protocols as

previous report (Kim et al., 2011). In the contextual fear memory,

which is widely used to measure hippocampus dependent fear memory

formation, the p110γ over-expression group showed equivalent freezing

level compared to the control group (Figure 13) This suggests that

plentiful p110γ in the hippocampus does not reinforce fear memory

formation. However, spatial learning in Morris water maze test was

significantly disrupted by p110γ over-expression (Figure 14). In

addition, p110γ over-expression group exhibited the tendency to spent

more time in the previous target quadrant and less number of platform

crossing during the probe 1 test on day 6 (Figure 15. A, B) although

reversal learning performance was indistinguishable between two

groups during the probe 2 test on day 9 (Figure 16. A, B). This results

were totally opposite to the initial anticipation and implicate the

obvious disruption in the neural process, so that we sought to examine

the electrophysiological aspects of the p110γ over-expression in the

25

hippocampus.

Electrophysiological Properties of p110γ Overexpressed mice

We lastly looked into the electrophysiological properties of

p110γ overexpression in the Schaffer collateral-commissural (SC-CA1)

pathway. Since the spatial learning performance in the Morris water-

maze task executed on the 24th day from the AAV infusion was

significantly decreased in p110γ overexpression mice, we carried out

all of the electrophysiological experiment after 24 days of AAV

infusion to verify synaptic alterations.

Basal synaptic transmission was measured over different

stimulus intensities ranged to 30μA and analyzed in fiber volley (FV)

amplitude versus field excitatory post-synaptic potential (fEPSP) slope.

(Figure 17) Paired-pulse facilitation (PPF) ratio was also measured to

assess pre-synaptic components (Figure 17) and all of the measured

basal synaptic properties were unchanged in p110γ over-expressed mice

compared to tdTomato expressing control mice indicating that the

excessive amount of p110γ did not affect basal synaptic strength and

pre-synaptic neurotransmitter release.

We then investigated synaptic plasticity models in p110γ over-

expression mice. LTP induced by high-frequency stimulation (HFS, 100

26

Hz single pulse, 1s) showed no difference between p110γ over-

expression group and tdTomato control group (Figure 18). LTD in

p110γ over-expressed group, however, was barely induced by low-

frequency stimulation (LFS, 1 Hz 900 pulses) (Figure 19) and

depotentiation induced by LFS (2 Hz 1200 pulses) to the previously

potentiated pathway was completely impaired in p110γ over-expressed

mice (Figure 20). It is suggesting that distinguishable performance of

spatial learning in Morris water maze was due to the impaired LTD of

the p110γ over-expression in the hippocampus CA1 area.

27

Figure 1. Both euPACα and euPACβ fail to increase intracellular

cAMP concentration by laser activation of the enzyme. (A) A schematic

procedure of laser activation and tissue preparation. After confirming

the expression of red fluorescence, all brain slices of the light group

were irradiated by 473nm blue laser for 5 minutes. ACC regions were

cut prior to freezing in liquid nitrogen. (B) Photoactivation of either

PAC subunits or tdTomato co-expressed PAC resulted in a tiny

difference in cAMP concentration. Laser intensity was 6mW mm-2.

(PACα n=2, PACβ n=2, PACα + tdTomato n=2, PACβ + tdTomato n=2)

(C) cAMP concentrations (% to dark) when the laser intensity was

increased in 5-fold. All four groups showed no differences in the level

of cAMP after photoactivation.

28

Figure 1.

(This work was done in collaboration with Jae-Ick Kim and Su-Eun Sim.)

29

Figure 2. cAMP manipulation by photoactivation of euPAC was

ineffectual. (A) Relative cAMP concentration after 35mW mm-2 laser

irradiation, which is maximum intensity in our system. It was also

insufficient to increase cAMP levels (PACα n = 4, PACβ n = 3). (B)

Summation of cAMP levels (% to dark) in both euPACα and euPACβ

subunits revealing that euPAC is unusable to optogenetic manipulation

of intracellular cAMP levels in vivo.

30

Figure 2.

31

Figure 3. Representative images of tdTomato tagged Beggiatoa PAC

(bPAC) expression in ACC.

32

Figure 3.

33


photoactivation of bPAC for 5 minutes in the intensity of 35mW mm-2

(mCherry-bPAC; B and C, bPAC-mCherry; A and D). They showed 2

to 3-fold elevation of cAMP concentration by activation of bPAC.

Upper panels are images of each slices expressing bPAC.

34

Figure 4.

35

Figure 5. . Individual intracellular cAMP concentration (pmol/ml) after

photoactivation of bPAC for 5 minutes in the intensity of 35mW mm-2

(mCherry-bPAC; A and C, bPAC-mCherry; B). They showed maximum

4.6-fold increasing of cAMP concentration by activation of bPAC.

Upper panels are images of each slices expressing bPAC.

36

Figure 5.

37

Figure 6. Summation data in figure 4 and 5 according to bPAC

construct. (mCherry-bPAC n = 4, bPAC-mCherry n = 3). bPAC showed

overall increase in cAMP levels. (A) cAMP changes in mCherry-bPAC

expression group (n = 4). (B) cAMP changes in bPAC-mCherry

expression group (n = 3).

38

Figure 6.

39

Figure 7. Overall results of cAMP concentration changes between

different PAC species. Euglena PAC failed to increase cAMP levels

while Beggiotoa PAC effectively increased cAMP concentration by

laser application. (A) Data presented by the type of PAC construct. (B)

Data presented by the type of origin species. bPAC was much more

efficient cAMP synthesizer than euPAC (* p < 0.05, unpaired t-test;

euPAC-light group versus bPAC-light group, ** p < 0.01, paired t-test;

bPAC-dark group versus bPAC-light group). Summation of cAMP

levels (% to dark) in both mCherry-bPAC and bPAC-mCherry

indicating that bPAC was suitable for optogenetic manipulation of

intracellular cAMP levels in vivo.

40

Figure 7.

41

Figure 8. Representative images of flag tagged p110γ or tdTomato

expression in the mouse hippocampal CA1 area. (Blue : DAPI, Green :

Flag-p110γ, Red : tdTomato, imaged 50 days after AAV injection.)

Scale bar represents 500μm.

42

Figure 8.

43

Figure 9. Basal anxiety levels of p110γ or tdTomato expressed mice in

the elevated plus maze (EPM) test. (flag-p110γ n = 12, tdTomato n = 9,

tested 2 weeks after AAV injection.)

44

Figure 9.

(This work was done in collaboration with Ye-Seul Lee and Seo-Hee Ahn.)

Open

Closed

Center

Open

Closed

Center

0

20

40

60

80tdTomatop110γ

Tim

e sp

ent (

%)

45

Figure 10. Basal locomotion in the open field test (OFT) (flag-p110γ n

= 14, tdTomato n = 12, tested 15 days after AAV injection.). (A)

Percent time spent in 10cm2, 20cm2 or peripheral area of the field. (B)

Total distance moved (cm) for 100 seconds in the OFT.

46

Figure 10.


47

Figure 11. Contextual fear memory of p110γ or tdTomato expressed

mice. (flag-p110γ n = 14, tdTomato n = 12)

48

Figure 11.


49

Figure 12. Spatial Learning in the Morris Water Maze test. Learning

performance was declined in p110γ over-expressed Mice. Average

escape time traveled to the hidden platform (mean ± s.e.m.) in the

Morris water maze test. (flag-p110γ n = 14, tdTomato n = 12, ** p <

0.01, repeated measures two-way ANOVA)

50

Figure 12.


1 2 3 4 5 7 80

20

40

60 tdTomatop110γ

**

Days

Esca

pe la

tenc

y (s

)

51

Figure 13. Probe test on day 6 in the Morris water maze test. (A) The

averaged percentage of time spent in the target quadrant (TQ, * p <

0.05, repeated measures two-way ANOVA), the opposite quadrant (OQ)

and two adjacent quadrant (AQ) during probe trials given on day 6 of

the MWM task. (B) Total number of crossing the location of platform

(** p < 0.01, unpaired t test) during the probe trials given on day 6 of

the MWM task.

52

Figure 13.


53

Figure 14. Probe test on day 9 in the Morris water maze test. (A) The

averaged percentage of time spent in the new target quadrant (New TQ),

the previous target quadrant (Old TQ) and two adjacent quadrant (AQ)

during probe trials given on day 9, the day after 2-day of reversal

learning, of the MWM task. (B) Total number of crossing the location

of platform during the probe trials given on day 9 of the MWM task.

54

Figure 14.


55

Figure 15. Basal synaptic properties of p110γ or tdTomato expressed

mice. (A) Input-output relationship in p110γ or tdTomato expressed

mice. (B) Paired-pulse ratio at SC-CA1 synapses in p110γ or tdTomato

expressed mice.

56

Figure 15.

(This work was done in collaboration with Po-Jeong Park.)

57

Figure 16. High-frequency stimulation (HFS, single pulse at 100Hz for

1s) induced long-term potentiation in Schaffer collateral to CA1 (SC-

CA1) synapses. (flag-p110γ n = 11, tdTomato n = 12)

58

Figure 16.


59

Figure 17. LFS-LTD was disrupted by excessive p110γ in the

Hippocampus CA1 Area. Low-frequency stimulation (LFS, 900 pulses

at 1Hz) induced long-term depression in SC-CA1 synapses. (flag-p110γ

n = 10, tdTomato n = 12)

60

Figure 17.


61

Figure 18. Depotentiation induced by LFS (1200 pulses at 2Hz)

subsequent to three trains of theta-burst stimulation. (each trains are

composed of 5 bursts, flag-p110γ n = 10, tdTomato n = 12)

62

Figure 18.


63

Discussion

PAC as a tool for cAMP modulator with temporal precision

We demonstrated that not euPACs but bPAC is applicable for

light manipulation of cellular cAMP level in rodents. Either euPACα or

euPACβ expressed brain slices showed no changes in cAMP

concentration whilst either mCherry-bPAC or bPAC-mCherry

expressed brain slices showed 3-fold increase of cAMP concentration

after photoactivation with 473nm blue laser at 35mW mm-2 of

continuous pulses.

Intracellular cAMP level is oscillates approximately within ±20%

range of the medial level from day to day (Eckel-Mahan et al., 2008).

In addition, several studies reported that cAMP increased transiently

about 1.5~2-fold during drug-induced learning or rapid eye movement

(REM) sleep (Cui et al., 2007; Luo et al., 2013). For this reason, we set

a minimal amount of cAMP increment as 2 times higher than initial

cAMP level for efficacious modulation of cAMP signaling. Following

this criterion, we concluded that euPACs are not eligible for a cAMP

modulation tool. Failure of euPACs might caused by relatively larger

size resulted in lower the expression efficiency, so that it was not

enough to make a detectable changes in cAMP concentration.

64

Furthermore, euPACs were tagged with 3xflag, which cannot be

detected before determination of cAMP concentration, it was

impossible to discriminate the “maximal expressed slice” for lessening

a masking effect caused by uninfected, PAC lacking cells.

To overcome these problems, keeping in its basic enzymatic

functions, we adopted PAC from another species. bPAC was great

substitute indeed. AAVs carrying bPAC constructs expressed in much

higher efficiency and mCherry made visualization of bPAC easier

(Hong et al., 2011). With these advantages, we successfully elevated

cAMP level by 3-fold after photoactivation of bPAC, yet it was far less

increment compared to previous studies reporting that almost ~200-fold

increase of cAMP concentration by PAC activation (Schroder-Lang et

al., 2007; Stierl et al., 2011). This discrepancy might caused by

different biological system (rodent brain versus Drosophila or Xenopus)

and PAC introduction method (AAV versus transgenic or direct

delivery using gene gun).

Across the rodent biological systems, cAMP signaling is

widely involved in the regulation of metabolism of nutritive elements

as well as learning and memory in the brain. Here, we introduced

genetically modified and virally packaged bPAC as a tool for cAMP

modulation in mice. Together with other optogenetically controllable

65

tools, bPAC allows us to explore synaptic plasticity with a higher

region and time-specific manner.

PI3Kγ in spatial learning and memory

Secondly, we investigated the behavioral and electro-

physiological effects of excessive PI3Kγ in the hippocampal CA1

region in mice. Our previous experiments with Pik3cg-/- mice

corroborated that the PI3Kγ is specifically required for NMDA

receptor-dependent LTD and behavioral flexibility during spatial

learning. To further elucidation of the role of PI3Kγ, we used AAV

mediated genetic delivery of p110γ, a catalytic subunit of PI3Kγ, to the

CA1 neurons. Notably, there are methodological differences between

AAV mediated gain-of-function and loss-of-function by genetic

deletion. The most distinctive factor is region and time point specificity.

In the genetic deletion study, we unable to control when and where to

remove p110γ, instead whole body knock-out from the birth. On the

other hand, for gain-of-function study, it is easily manageable to choose

region and time point via AAV.

Taking advantage of AAV, we found that over-expression of

p110γ in CA1 region rather disrupted than enhanced certain type of

66

hippocampal-dependent memory. LTP in p110γ group was normal.

However, LTD and depotentiation of potentiated synapses were

impaired. These results implicated that over-expression of p110γ

somehow hindered its regular functions or other isoforms of PI3Ks

which have diametrically opposed roles. It seems likely that excessive

amount of p110γ disrupted NMDA receptor-dependent LTD, thus,

resulted in inferior spatial learning performance in Morris water

navigation task which requires spatial pattern separation (Sahay et al.,

2011).

LTD and hippocampal dependent long-term memory

Vast amount of evidences has been indicated that hipocampal

LTD is not just a reverse action of LTP rather it also contributes to

hippocampal memory formation and storage like LTP (Lee et al., 2000;

Kemp and Manahan-Vaughan, 2007). Consistent with these views, LTD

in CA1 area may mediate novel spatial features (Manahan-Vaughan and

Braunewell, 1999; Kemp and Manahan-Vaughan, 2004). In addition,

hippocampal LTD is also necessary for long-term storage of spatial

memory, that is, consolidation of the memory (Ge et al., 2010). With

these roles of LTD in mind, we concluded that spatial learning of

67

Morris water maze was declined because of disrupted LTD by p110γ

over-expression. Also, p110γ is not critical for homosynaptic NMDAR

LTD in the hippocampal CA1 area, but instead, may act as a modulator

of NMDAR LTD (Daw et al., 2002; Kim et al., 2011). In this reason,

plentiful of p110γ may not helpful to enhance any kind of cognition at

all, but disrupted finely tuned reaction of the enzymes.

While spatial learning in Morris water maze was compromised,

contextual fear learning was unaffected by excessive p110γ. Fear

memory is mainly expressed in amygdala responding throughout

simple to complex modality. Whereas hippocampus contributes only in

polymodal events (Phillips and LeDoux, 1992). Also, in Pik3cg-/- mice,

learning threshold seemed to be elevated because reduced fear learning

was recovered when the training intensity was increased. In these

reasons, we concluded that p110γ over-expression might have no

impact on contextual fear memory.

68

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국문 초록

Adeno-Associated Virus를 매개로 한

bPAC과 PI3Kγ유전자의 발현 조절과


백 기 철

Adeno-associated viruses (AAV) 는 다양한 생물종에

외래의 유전자를 도입하는 매개체로서 널리 사용되는 실험적

방법으로, 도입된 유전자의 안정적인 발현과 생물학적 안정성

을 가지고 있기 때문에 다른 종류의 바이러스와 비교하여 그

선호도가 매우 높다. 이러한 AAV의 장점과 광유전학을 결합

하여 특정 단백질 (효소) 또는 세포 내 신호 전달 체계를 시

공간적 정확성을 가지고 조절 함으로써 생쥐의 뇌에서 신경

가소성을 연구 하였다.

76

첫 번째 실험에서는 AAV2/1으로 항원형이 결정된

vector에 빛에 의해 활성화되는 cyclase인 euPAC 또는 bPAC

을 삽입 하였다. 생쥐 뇌의 ACC 부위에 PAC 유전자를 AAV

를 이용해 전달한 후 cAMP 효소-면역 반응 분석을 통해

PAC의 기능을 확인 하였다. 비록 euPAC이 발현된 ACC 절편

을 광활성화 시켰을 때 세포 내의 cAMP 수준이 증가되는 것

을 관찰하지 못했지만, bPAC이 발현된 ACC 절편에서는 세포

내 cAMP 수준이 현저하게 증가하는 것을 확인 하였다.

두 번째 실험에서는, PI3Kγ의 촉매 아단위 (subunit)인

p110γ를 칼슘 / 칼모듈린 의존 단백질 인산화 효소 II α

(Ca2+ / calmodulin-dependent protein kinase II α, CaMKIIα)

프로모터에 의해 발현이 조절되는 vector에 삽입하여 이를 해

마체의 CA1 지역에 과발현 시켰다. 이후 행동학적, 전기생리

학적 실험을 통해 p110γ가 과발현 된 생쥐에서 어떠한 변화

가 있는지 알아보았다. 그 결과 p110γ의 과발현은 NMDA 수

용체 의존적인 장기 억압 (long-term depression, LTD)을 손

상 시켰으며 이로 인하여 해마체 의존적인 공간 탐색 학습 능

력이 크게 감소하였다.

disclaimer - seoul national universitybluf (blue-light sensing using flavin)photoreceptor linked...

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