R E S E A R CH AR T I C L E

Characterization of the axon initial segment of mice substantianigra dopaminergic neurons

Cristian Gonz�alez-Cabrera1 | Rodrigo Meza1,2† | Lorena Ulloa1† |

Paulina Merino-Sep�ulveda1 | Valentina Luco1 | Ana Sanhueza1 |

Alejandro O~nate-Ponce1 | J. Paul Bolam3 | Pablo Henny1

1Laboratorio de Neuroanatomía,

Departamento de Anatomía, and Centro

Interdisciplinario de Neurociencia, NeuroUC,

Escuela de Medicina, Pontificia Universidad

Cat�olica de Chile, Santiago, Chile

2Departamento de Fisiología, Facultad de

Ciencias Biol�ogicas, Pontificia Universidad

Cat�olica de Chile, Santiago, Chile

3MRC Brain Network Dynamics Unit,

Department of Pharmacology, University of

Oxford, Oxford, United Kingdom

Correspondence

Pablo Henny and Cristian Gonz�alez-

Cabrera, Laboratorio de Neuroanatomía,

Departamento de Anatomía, and Centro

Interdisciplinario de Neurociencia,

NeuroUC, Escuela de Medicina, Pontificia

Universidad Cat�olica de Chile, Av.

Libertador Bernardo O’Higgins 340,

Santiago 8330023, Chile.

Email: phenny@uc.cl; crgonzalezc@uc.cl

Funding information

FONDECYT, Grant/Award Number:

1141170 and 3160763; Programa de

Investigaci�on Asociativa – Anillo, Grant/

Award Number: 11-09

AbstractThe axon initial segment (AIS) is the site of initiation of action potentials and influences action

potential waveform, firing pattern, and rate. In view of the fundamental aspects of motor function

and behavior that depend on the firing of substantia nigra pars compacta (SNc) dopaminergic neu-

rons, we identified and characterized their AIS in the mouse. Immunostaining for tyrosine

hydroxylase (TH), sodium channels (Nav) and ankyrin-G (Ank-G) was used to visualize the AIS of

dopaminergic neurons. Reconstructions of sampled AIS of dopaminergic neurons revealed variable

lengths (12–60 lm) and diameters (0.2–0.8 lm), and an average of 50% reduction in diameter

between their widest and thinnest parts. Ultrastructural analysis revealed submembranous local-

ization of Ank-G at nodes of Ranvier and AIS. Serial ultrathin section analysis and 3D

reconstructions revealed that Ank-G colocalized with TH only at the AIS. Few cases of synaptic

innervation of the AIS of dopaminergic neurons were observed. mRNA in situ hybridization of

brain-specific Nav subunits revealed the expression of Nav1.2 by most SNc neurons and a small

proportion expressing Nav1.6. The presence of sodium channels, along with the submembranous

location of Ank-G is consistent with the role of AIS in action potential generation. Differences in

the size of the AIS likely underlie differences in firing pattern, while the tapering diameter of AIS

may define a trigger zone for action potentials. Finally, the conspicuous expression of Nav1.2 by

the majority of dopaminergic neurons may explain their high threshold for firing and their low dis-

charge rate.

K E YWORD S

axon initial segment, dopamine, substantia nigra, RRID: AB_2289736, RRID: AB_2619897, RRID:

AB_2040204, RRID: SCR_014329, RRID: SCR_002526, RRID: AB_2201518, RRID: SCR_002716,

RRID: nlx_84100, RRID: nlx_84530, RRID: OMICS_02343, RRID: AB_514497

1 | INTRODUCTION

Substantia nigra pars compacta (SNc) and ventral tegmental area dopami-

nergic neurons participate in various brain processes including movement,

motivation and preference formation (Redgrave, Gurney, & Reynolds,

2008; Schultz, 2007; Wise, 2009). Conversely, their dysfunction lies at the

core of many disorders including Parkinson’s disease, schizophrenia, and

addiction (Albin, Young, & Penney, 1989; Galvan & Wichmann, 2008;

Nieoullon, 2002; Wise, 2009). Dopamine release modulates the excitabil-

ity of postsynaptic neurons and triggers cellular changes that underlie

long-term changes in behavior (Tritsch & Sabatini, 2012). Its release is

tightly related to the frequency, pattern, and pauses of action potential

firing of the dopaminergic neurons from which it originates (Heien &

Wightman, 2006; Schultz, 2007). Thus, in order to understand how and

when dopamine is released, it is necessary to understand the structural

basis that underlies firing of dopaminergic neurons (Henny et al., 2012).

It is well established that the site of action potential generation in

most central neurons is the axon initial segment (AIS) (Bean, 2007;†These authors contributed equally to this work.

J Comp Neurol. 2017;525:3529–3542. wileyonlinelibrary.com/journal/cne VC 2017Wiley Periodicals, Inc. | 3529

Received: 15 January 2017 | Revised: 8 July 2017 | Accepted: 10 July 2017

DOI: 10.1002/cne.24288

The Journal ofComparative Neurology

Bender & Trussell, 2012; Coombs, Curtis, & Eccles, 1957; Hausser,

Stuart, Racca, & Sakmann, 1995; Palmer & Stuart, 2006). This proximal

region of the axon is characterized by an array of cytoskeletal organ-

elles (Palay, Sotelo, Peters, & Orkand, 1968) and scaffolding proteins

that maintain cell polarity (Sobotzik et al., 2009; Yoshimura & Rasband,

2014) and aggregate voltage-gated ion channels that initiate and termi-

nate action potentials (Bender & Trussell, 2012; Kole & Stuart, 2012).

Furthermore, the AIS can control the firing pattern and action potential

shape (Bean, 2007; Bender & Trussell, 2012; Clark, Goldberg, & Rudy,

2009; Kole & Stuart, 2012), and its structure and location show activity-

dependent plasticity (Evans, Dumitrescu, Kruijssen, Taylor, & Grubb,

2015; Kuba, Ishii, & Ohmori, 2006; Kuba, Oichi, & Ohmori, 2010).

In most dopaminergic neurons, the axon branches off a primary

dendrite (Grace & Bunney, 1983; Tepper, Sawyer, & Groves, 1987) and

just as in other neurons, the AIS is considered to be the site of action

potential initiation (Blythe, Wokosin, Atherton, & Bevan, 2009; Gentet

& Williams, 2007; Grace & Bunney, 1983; Hausser et al., 1995). The

dendritic origin of the axon has both physiological and computational

consequences regarding action potential back-propagation and spatio-

temporal integration of synaptic inputs leading to action potential ini-

tiation (Blythe et al., 2009; Gentet & Williams, 2007; Hausser et al.,

1995). Notwithstanding its evident importance, it is surprising that no

studies have yet provided a structural or molecular characterization of

the AIS of dopaminergic neurons. In order to define the characteristics

of their AIS that may underlie common electrophysiological features, or

indeed, the physiological diversity observed in this population, we

defined the morphological, synaptic, and molecular features of the AIS

of dopaminergic neurons (Brischoux, Chakraborty, Brierley, & Ungless,

2009; Henny et al., 2012; Roeper, 2013).

2 | METHODOLOGY

2.1 | Animal ethics

Experiments that utilized animals were approved by the Ethics Com-

mittees of the School of Medicine of the Pontificia Universidad

Cat�olica de Chile, and of the Comisi�on Nacional de Investigaci�on Cien-

tífica y Tecnol�ogica (CONICYT), both of which conform to the guide-

lines of US National Institutes of Health (NIH). C57BL/6 adult male

mice were obtained from the animal care facility of the Faculty of Bio-

logical Sciences, Pontificia Universidad Cat�olica de Chile. Experiments

involving animals used for ultrastructural analysis (Figure 3) were per-

formed in accordance with the UK Animals (Scientific Procedures) Act,

1986 and Directive 2010/63/EU of the European Parliament and ethi-

cal permission was granted by the University of Oxford Ethical Review

Process. In this case, animals were obtained from the MRC Brain Net-

work Dynamics animal care facility.

2.2 | Immunohistochemistry

In order to define the AIS and therefore the site of action potential ini-

tiation in dopaminergic neurons, we used immunostaining for ankyrin-

G (Ank-G), a scaffolding protein responsible for anchoring voltage-

gated channels sodium (Nav) and other voltage-gated channels, to the

membrane of the AIS and nodes of Ranvier (NR), together with immu-

nolabeling for tyrosine hydroxylase (TH) to identify dopaminergic struc-

tures (Hill et al., 2008; Jones, Korobova, & Svitkina, 2014; King,

Manning, & Trimmer, 2014; Zhou et al., 1998). For the double immuno-

staining, four 25–30 g mice were deeply anesthetized with isoflurane

(Isoflurane USP, Baxter Healthcare, Deerfield, IL) followed by an intra-

peritoneal injection of a mixture of ketamine (75 mg/kg) and xylazine

(5 mg/kg). They were then transcardially perfuse-fixed with �25 ml of

phosphate-buffered saline (PBS, 0.01M phosphate buffer, pH 7.4), fol-

lowed by �50 ml of 4% paraformaldehyde (PFA, w/v) in PBS using a

peristaltic pump (Masterflex 7518–00, Vernon, NY). Brains were post-

fixed in 4% PFA overnight, cryopreserved in 30% sucrose in distilled

water for at least 48 hr, sectioned at 40 lm in the coronal plane on a

freezing-stage microtome (Reichert-Jung Hn 40, Depew, NY), and col-

lected in parallel series.

Sections containing the substantia nigra and adjacent brain regions

including the cortex were blocked with 3% normal horse serum (NHS) in

PBS (v/v, Jackson Immuno Research Laboratories Inc., Westgrove, PA)

and subsequently incubated with goat-anti-Ank-G antibody (1:5,000,

Santa Cruz Biotechnology, Dallas, TX; RRID: AB_2289736) in PBS 1%

NHS for 2–3 days at room temperature. After washes in PBS, the sec-

tions were incubated overnight with guinea pig-anti-TH (1:1,000, Synap-

tic Systems, Goettingen, Germany; RRID: AB_2619897) in PBS, 1% NHS

and 0.3% Triton-X. They were then incubated for 2 hr in Cy3-

conjugated donkey-anti-goat (1:1,000, Jackson Immuno Research Labo-

ratories Inc., Westgrove, PA) and Alexa Fluor 488-conjugated donkey-

anti-guinea pig (1:1,000, Jackson Immuno Research Laboratories Inc.,

Westgrove, PA) secondary antibodies. Double stained sections were

mounted onto glass slides, allowed to dry and covered with mounting

medium (Vectashield, Vector Laboratories, Burlingame, CA). Sections

were washed 33 15 min in PBS between incubations in blocking, pri-

mary antibody and secondary antibody solutions, and before mounting.

For triple staining for TH, Ank-G, and Pan-Nav, four mice were

deeply anesthetized as described above and perfuse-fixed with �25 ml

of PBS, followed by �50 ml 1 or 2% PFA (w/v) in phosphate buffer

(pH 7.4). Brains were post-fixed in 1 or 2% PFA overnight, cryopre-

served, sectioned in the coronal plane at 40 lm and collected in series

as described above. Sections were blocked with 3% NHS in PBS for 2

hr and subsequently incubated with goat-anti-Ank-G antibody

(1:5,000) for 2–3 days at room temperature. They were then incubated

overnight with guinea pig-anti-TH (1:1,000) and rabbit-anti-Pan-Nav

(1:2,000, Alomone Labs, Jerusalem, Israel; RRID: AB_2040204). Finally,

the sections were incubated for 2 hr in Dylight-405—conjugated don-

key-anti-guinea pig antibody (1:1,000, Jackson Immuno Research Labo-

ratories Inc.; Westgrove, PA), AlexaFluor 488-conjugated donkey-anti-

goat-IgG antibody (1:1,000, Jackson Immuno Research Laboratories

Inc., Westgrove, PA), and Cy3-conjugated donkey-anti-rabbit second-

ary antibody (1:1,000, Jackson Immuno Research Laboratories Inc.,

Westgrove, PA). Triple stained sections were mounted on glass slides

in Vectashield mounting medium (Vectashield, Vector Laboratories,

Burlingame, CA). Sections were washed 33 15 min in PBS between

3530 | The Journal ofComparative Neurology

GONZ�ALEZ-CABRERA ET AL.

incubations in blocking, primary antibody and secondary antibody solu-

tions, and before mounting.

All antibodies used in this study were routinely tested for optimal

staining using serial dilutions from 1:50 to 1:50,000. No immunostain-

ing was observed if primary or secondary antibodies were omitted

from the procedure. No immunostaining was observed either if primary

antibodies were incubated with non-correspondent secondary

antibodies.

2.2.1 | Antibody characterization

The anti-Ank-G anti-PanNav and anti-TH and antibodies have been uti-

lized in previous publications to respectively identify AIS (Ank-G and

PanNav) and dopaminergic structures (TH), as cited in Table 1. Our

results showed the expected neuronal labeling patterns, as based on

the literature using these or other anti-Ank-G (Puthussery, Venkatara-

mani, Gayet-Primo, Smith, & Taylor, 2013; Van Wart, Trimmer, & Mat-

thews, 2007; Wang & Sun, 2012), anti-PanNav (Ban, Smith, &

Markham, 2015; Moore et al., 2009), and anti-TH (Fu et al., 2012;

Henny et al., 2012; Hioki et al., 2010; Kantor et al., 2015), antibodies in

every case.

2.3 | Imaging

Imaging was performed using an epiflourescent microscope (Nikon Ecli-

ple Ci, Tokio, Japan) equipped with a camera (Microfire, Optronics,

Goleta, CA), a motorized x-y-z stage, transmitted light and filters suita-

ble to the two or three fluorescent markers, or a laser-scanning confo-

cal microscope (Nikon Eclipse C2, Tokio, Japan) mounted on a Nikon

Eclipse Ti-E inverted platform and equipped with lasers and appropri-

ate filters for Dylight-405, Alexa-488, and Cy3 fluorophores. Individual

or stacks of confocal images were acquired with a 603 oil immersion

lens (1.4 NA) at 2,048 3 2,048 pixels with a 0.1 mm/pixel resolution, at

0.2I5 mm steps in the z axis, imaged with a Nikon C2 camera, and

viewed offline with the NIS-Elements C program (Nikon software,

Tokio, Japan; RRID: SCR_014329).

2.4 | 3D reconstructions and structural analysis

Images of Ank-G1/TH1 profiles, that is, the AIS of dopaminergic

neurons, from three animals were acquired following the use of a

pseudo-random sampling procedure on the epifluorescent micro-

scope with the aid of the Stereo Investigator software (MBF bio-

science, Williston, VT; RRID: SCR_002526). Contours of the

substantia nigra from a double-labeled series were delimited using a

low magnification 103 objective, defined on the basis on TH immu-

nostaining (Fu et al., 2012). The 50 3 50 lm2 frames disposed sys-

tematically on a 150 3 150 lm2 grid size were randomly placed over

the substantia nigra of each of the serial sections. Then Ank-G1/

TH1 profiles were examined on each of the frames using a high

magnification 1003 oil objective (1.4 NA). Each time an Ank-G1/

TH1 profile was found within or touching the limits of the frame,

and found to be entirely confined to that section (i.e., not cut at the

top or bottom of the section), a z-stack of images was acquired in

such a manner to include the entire profile. Stacks were taken using

0.25 lm steps at 1,596 3 1,198 pixels with 0.075 lm/pixel

resolution.

The double-labeled profiles were traced offline using vector-based

3D reconstruction (Ascoli, 2006) with the Neurolucida software (MBF

bioscience, Williston, VT; RRD: SCR_001775). On the final AIS recon-

structions, a shrinkage correction factor on the z axis was applied due

to the shrinkage that occurs during histological processing (50%

approximately for this study) (Henny, Brown, Micklem, Magill, & Bolam,

2014; Henny & Jones, 2006). Quantitative data for anatomical parame-

ters including length, surface area, volume, and minimum, maximum

and average diameters were obtained using the Neurolucida Explorer

software (MBF Bioscience, Williston, VT).

TABLE 1 Primary antibodies used in this study

AntigenHostspecies Source Cat. # Immunogen

Selected publicationsusing same antibody

Ankyrin-G Gt Santa Cruz sc-31778RRID: AB_2289736

Human Ankyrin-G, near theC-terminus peptide, (NCBIaccession: Q12955)

Puthussery, Venkataramani, Gayet-Primo,Smith, & Taylor, 2013

Tyrosinehydroxylase

GP SySy 213104RRID: AB_2619897

Purified recombinant protein ofrat tyrosine hydroxylase, aminoacids 1–163, (NCBI accession:AAB59722)

Kantor et al., 2015

Pan-Nav Rb Alomone Labs ASC-003RRID: AB_2040204

Peptide corresponding to aminoacid residues 1501–1518,(NCBI accession: P04774) ofrat Nav 1.1

Ban, Smith, & Markham, 2015

Tyrosinehydroxylase

Ms Millipore MAB318. clone LNC1, RRID: AB 2201528

Tyrosine hydroxylase purifiedfrom PC12 cells. Recognizesan epitope on the outside ofthe regulatory N-terminus

Hioki et al. 2010

Digoxigenin – RocheDiagnostics

Anti-digoxigenin-APFab fragmentsRRID: Ab_514497

Digoxigenin Gonzalez-Cabrera, Garrido-Charad,Roth, & Marin, 2015

GONZ�ALEZ-CABRERA ET AL. The Journal ofComparative Neurology

| 3531

2.5 | Electron microscopy and ultrastructural analysis

Three 25–30 g adult mice were deeply anaesthetized with ketamine

and xylazine as described above and perfused transcardially with 20 ml

of PBS followed by 4% PFA/0.25%glutaraldehyde in PBS. Brains were

dissected, kept in 4% PFA for 1 day, cut coronally at 50 lm using a

Leica vibratome (VT1000S; Leica Microsystems, Wetzlar, Germany)

and collected in series. Sections were then equilibrated overnight in a

cryoprotectant solution. Individual sections were placed in crafted foil

boats, dried with filter paper, placed over liquid nitrogen to freeze for

30 s and thawed for 1–2 min at room temperature. This cycle was

repeated three times to enhance antibody penetration. Sections were

then washed in PBS 33 15 min, blocked in 10% normal donkey serum

(NDS) in PBS for 3 hr, and then incubated overnight in 1:500 goat-anti-

Ank-G in 1% NDS/PBS. The next day they were washed 33 15 min in

PBS and incubated for 2 hr in 1:200 dilution of 1.4 nm gold-conjugated

rabbit-anti-goat secondary antibody (Cat#2005, Nanoprobes, Yaphank,

NY) in 1% NDS/PBS, washed in PBS 33 15 min and then 33 15 min

in acetate buffer (0.1 M sodium acetate 3-hydrate). Individual sections

were silver intensified for 4–5 min (HQ Silver kit, Nanoprobes;

Yaphank, NY) to reveal Ank-G immunoreactive sites, washed 23 10

min in acetate buffer and then 33 15 min in PBS. Sections were then

incubated overnight in mouse-anti-TH (dilution 1:1,000; MAB318,

Millipore, Billerica, MA; RRID: AB_2201518) in NHS 1% and washed in

PBS 33 15 min the next day. Sections were incubated for 2–3 hr in

biotin-conjugated horse-anti-mouse (1:200 dilution; Cat# BA-2000,

Vector Laboratories, Burlingame, CA), washed in PBS 33 15 min to be

then incubated for 3 hr in avidin-biotin-peroxidase complex (ABC)

(Vectastain, ABC Elite kit, Vector Laboratories, Burlingame, CA) pre-

pared in Tris buffer (0.05 M, pH 7.4), and washed in Tris buffer 33 15

min. Sections were finally placed in wells containing 2.5 ml of diamino-

benzidine (DAB) solution (0.025% DAB in Tris buffer) to which 50 ll of

1% H2O2 was added for 4–5 min to reveal TH immunoreactivity.

Sections were then post-fixed in osmium tetroxide. Briefly, they

were washed in 0.1 M PB (pH 7.4) for 30 min, then 8 min in osmium

tetroxide (1% in PB; Oxkem), washed 5 min in PB. They were then

incubated 15 min in 50% ethanol, 30 min in 1% uranyl acetate (TAAB,

Aldermaston, UK) in 70% ethanol, 15 min in 95% ethanol, 23 10 min

in absolute ethanol, and 23 10 min in propylene oxide (Sigma-Aldrich,

Darmstadt, Germany). Sections were then lifted into durcupan resin

(Durcupan ACM, Fluka AG., Buchs, Switzerland) and left overnight. The

next day sections were placed on microscope slides, coverslipped, and

cured for 3 days at 658C.

The tissue was initially analyzed by light microscopy. Pieces of tis-

sue from the SNc were cut out from the sections and glued to a block

of resin and �50 nm ultrathin were cut using a Leica ultramicrotome

and collected on single-slot pioloform-coated grids (Henny et al., 2012,

2014). The sections were contrasted with lead citrate and analyzed in

an electron microscope (Philips CM10 or CM100). The sections were

examined to assess the immunolabeling for Ank-G and TH identified

by the silver-enhanced immunogold and the peroxidase reaction prod-

uct, respectively. Images were taken to document observations. All

Ank1 and TH1 profiles that were observed were imaged. Five Ank1/

TH1 profiles were sequentially imaged in 30–100 serial ultrathin sec-

tions for 3D reconstruction which was carried out using Reconstruct

software (Synapse web, 1.1.0.0; RRID: SCR_002716). No immunostain-

ing was observed if primary antibodies were omitted from the proce-

dure. No immunostaining was observed either if primary antibodies

were incubated with non-correspondent secondary antibodies.

2.6 | In situ hybridization

2.6.1 | RNA probes primer design

RNA probes were designed using the mouse (Mus musculus) nucleotide

databases (NCBI Nucleotide, RRID: nlx_84100) and the alignment tools

of the NCBI website (NCBI BLAST, RRID: nlx_84530; Primer-BLAST,

RRID: OMICS_02343; http://www.ncbi.nlm.nih.gov). We designed and

commercially synthesized (IDT DNA, USA) specific primer pairs (Table

2) to amplify the complementary DNA (cDNA) sequence corresponding

to each probe. The selected transcript regions for each probe were the

following: Nav1.1: XM_417347, 425bp from nucleotide 515 to 939;

Nav1.2: XM_420906.3, 384bp from nucleotide 117 to 500; Nav1.6:

NM_001168383, 317bp from nucleotide 14 to 330. Each region cho-

sen is located in the coding region of its corresponding mRNA.

2.6.2 | RNA probe generation

Total RNA was isolated from the mesencephalon of postnatal day 5–

15 mice. The brain was removed from the skull and the mesencephalon

was homogenized in 1 mL of RNAsolv Reagent (Omega Biotek, Nor-

cross, GA) using a dounce homogenizer. Single-stranded cDNA was

synthesized using Improm-II reverse transcriptase (Improm-IITM

Reverse transcriptase, Promega, Madison, WI) and the three specific

double-stranded DNA sequences obtained were cloned into amplifica-

tion vectors (p-GEMT easy vector, Promega, Madison, WI). Purified

DNA was commercially sequenced (Sequencing Service, P. Universidad

Cat�olica de Chile) and compared to already published sequences. The

DNA constructs were linearized by enzymatic digestion and used for

subsequent in vitro digoxigenin-labeled RNA transcription. See

Gonz�alez-Cabrera, Garrido-Charad, Roth, and Marin (2015) for the

detailed methodology.

TABLE 2 Sequences of PCR primers used to amplify specific cDNA sequences of each marker

Primer Forward Primer Reverse Annealing (8C) Fragment (bp)

Nav 1.1 50-GACTGGGTAGTGGTAGATCTCTG-30 50-AAAAGAGGTGCCTACGGTCTG-30 56 383

Nav 1.2 50-ACTCCGCCAAGGAAGAGAGA-30 50-TTCCCGCATGCATTCAACAC-30 56 544

Nav 1.6 50-TCCTCATTGCGTGAGCAAGT-30 50-CGTGGGTTGTGGTGATGAGA-30 57 518

3532 | The Journal ofComparative Neurology

GONZ�ALEZ-CABRERA ET AL.

2.6.3 | In situ hybridization

Six 25–30 g adult mice were perfused transcardially as described above

using �50 mL of PBS followed by �50 mL of fixative (4% PFA in PBS).

Brains were dissected out, post-fixed in 4% PFA for 12 hr at 48C and

cryoprotected in a 30% sucrose solution in distilled water. Once the

brains sank, they were mounted on a freezing sliding microtome (Leitz

1400) and cut into 60 lm coronal sections. Free-floating sections were

washed three times in PBS (0.01 M phosphate buffer pH 7.4; 0.02%

NaCl in diethyl pyrocarbonate (DEPC) treated water) and treated with

acetylation solution for 10 min (6% hydrogen peroxide; 0.1% Tween-20;

Promega, Madison, WI). The sections were then incubated in proteinase

K solution (Proteinase K 10 lg/mL, Promega, Madison, WI) for 10 min at

room temperature, washed once in PBS-0.1% Tween-20 (PBST), fixed in

4% PFA-PBST for 20 min, washed three times in PBST, and then pre-

hybridized at 658C in hybridization buffer for 3 hr (Gonz�alez-Cabrera

et al., 2015). After incubating in the specific RNA probes for 16–18 hr at

62–658C, the sections were washed twice in solution A (5X saline-

sodium citrate (SSC) pH 5.3; 50% formamide; 1% sodium dodecyl sul-

fate) and three times in solution B (2.5X SSC pH 5.3; 50% formamide;

1% Tween-20) at 658C for 30 min. After two washes in maleic acid

buffer solution (MABT; 100 mMmaleic acid, Sigma; 150 mM NaCl; 0.1%

Tween-20; pH 7.5), the sections were incubated in blocking solution (2%

Blocking Reagent, Roche, Indianapolis, USA; 2% heat inactivated normal

goat serum, in MABT) for 4 hr at room temperature and then incubated

for 16–20 hr at 48C with sheep-anti-digoxigenin-AP Fab fragments (1/

1,000 dilution in MABT; Roche Diagnostics; RRID: AB_514497; Table 2).

Finally, the sections were washed six times in MABT, incubated in alka-

line reaction buffer (100 mM Tris pH 9.5; 50 mMMgCl2; 100 mM NaCl;

1% Tween-20), and developed at room temperature by adding nitro-

blue tetrazolium chloride/5-Bromo-4-chloro-3-indolyl phosphate (NBT/

BCIP) reagent (NBT 375 lg/mL; BCIP 188 lg/ml; Stock Solution, Roche,

Mannheim, Germany).

2.6.4 | Quantification of mRNA positive neurons

The proportion of Nav1.2-expressing and Nav1.6-expressing neurons in

the SNc was established in the following manner. Four sections were

systematically selected across the anterior-posterior extent of the SNc

for each of the parallel series labeled for Nav1.2 and Nav1.6 in each of

three animals. Images at 403 magnification were taken at medial, cen-

tral and lateral aspects of the SNc. All labeled profiles in each image

were counted and the relative proportion of Nav1.2-expressing and

Nav1.6-expressing neurons was established.

2.7 | Statistical analysis

We performed the single-sample Kolmogorov-Smirnov test to assess

normality of data sets for structural parameters of AIS. As they were

not normally distributed, we used nonparametric statistical testing

throughout. Spearman’s correlation tests were performed using Graph-

Pad Prism 6 (GraphPad Software Inc.). Significance for statistical test

was set at p< .05.

2.8 | Image processing and figures

Individual electron microscopic images were observed with ImageJ

software (NIH, 1.49). Confocal individual images or stack of images

were viewed offline using the NIS-Elements viewer software. Images

for figures were contrasted and color balanced using Creative Suite

Adobe Photoshop (Adobe Inc.). Plates and figures were generated with

Creative Suite Adobe Illustrator (Adobe Inc., San Jose, CA).

3 | RESULTS

3.1 | Identification of the AIS of SNc dopaminergic

neurons

We used double and triple immunostaining to identify the AIS of dopami-

nergic neurons of the mouse SNc. The immunolabeling for Ank-G in the

cortex, as previously reported (Cruz, Lovallo, Stockton, Rasband, & Lewis,

2009), consistently revealed a large number and high density labeled AIS,

and NR (visual cortex (V1) illustrated in Figure 1). As expected from its

role in anchoring Nav channels to AIS and NR, the distribution of Ank-G

expression coincided with the expression of Nav channels revealed by

the Pan-Nav antibody which is targeted against a conserved region of the

Nav subunits (Figure 1a). In the SNc immunolabeling for Ank-G revealed a

lower number and density of labeled AIS and a much higher density of

NR profiles compared to the cortex (Figure 1b,c). As in the cortex, both

AIS and NR also expressed Pan-Nav immunostaining (Figure 1b,c). Within

the SNc, Ank-G and Pan-Nav immunoreactive structures were also identi-

fied as TH1 thus identifying them as AIS of dopaminergic neurons

(Figure 1b,c). As expected on the basis that the axons of dopaminergic

are not myelinated, Ank-G immunoreactivity in the SNc presumed to be

NRs, did not colocalize with TH immunoreactivity (Figure 1b,c).

3.2 | Structural features of the AIS of SNc

dopaminergic neurons

We quantified the size and shape of the AIS in dopaminergic neurons by

reconstructing randomly- and systematically-acquired profiles labeled for

both Ank-G and TH (Ank-G1/TH1) throughout the SNc using Neurolu-

cida and StereoInvestigator software. A depiction of all AIS reconstructed

(Figure 2a) and an example of a representative AIS, as seen from three

orthogonal views (Figure 2b) are shown. Morphological analyses showed

that AIS had a mean length and surface area of 25.8 lm and 29.3 lm2,

respectively, but with considerable variability (Figure 2a–d, Table 3).

Mean diameter was 0.37 lm and the ratio between maximal and minimal

diameter (max/min diameter) was 2.07 (Figure 2e, Table 3). Length signifi-

cantly correlated with surface area (Figure 2c, right top inset), although

not with volume, indicating that longer AIS were not necessarily thicker

(Figure 2d, right bottom inset). This is corroborated by the fact that length

did not correlate with minimal, maximal, or average diameters (Figure 3d,

right top inset). Finally, length did not correlate with maximum/minimum

diameter ratio indicating that decreasing diameter (tapering) is similar for

all AIS, independent of their length.

GONZ�ALEZ-CABRERA ET AL. The Journal ofComparative Neurology

| 3533

3.3 | Ultrastructural characteristics of the AIS of SNcdopaminergic neurons

Tissue double immunostaining using immunogold and immunoperoxidase

for Ank-G and TH, respectively, was used to characterize the ultrastruc-

ture of AIS of dopaminergic neurons in the SNc. As previously observed in

the fluorescently labeled material, Ank-G-immunopositive labeling in the

SNc was also observed in two types of profiles. In the first type, immuno-

gold particles were located just beneath the membrane of unmyelinated

regions of myelinated axonal processes (Figure 3a,b), thus indicating NR

profiles. The second type of structure was larger, more diverse in shape

and devoid of myelin as far as the processes were followed through serial

sections (Figure 3c–h) and displayed characteristics of AIS.

TH-immunolabeling was mainly associated with dendrites (Figure

3c) and cell bodies (not shown). Ank-G1/TH1 profiles were relatively

sparse, and they varied in size and shape (Figure 3c–g). Ank-G immuno-

gold labeling in these structures was located beneath the membrane

(Figure 3c–g). The submembranous density usually observed on AIS

(Palay et al., 1968; Peters, Palay, & Webster, 1976) was difficult to vis-

ualize in our material due to the peroxidase reaction product revealing

TH immunoreactivity. Serial analysis of 311 ultrathin sections contain-

ing Ank-G1/TH1 double-labeled profiles revealed that Ank-G1/TH1

structures had characteristics of AIS and, in support of this, they were

not myelinated thus indicating that they were not NR.

In order to further ensure that Ank-G1/TH1 profiles corre-

sponded to AIS and therefore had a size and shape consistent with

what we found for AIS at the light microscopy level, we partially recon-

structed and morphologically analyzed three Ank-G1/TH1 profiles (in

a total of 215 ultrathin sections, Figure 3i) using the software Recon-

struct. The lengths of those three partially reconstructed AIS were

�11, 6, and 13 lm, their surface areas were 28, 17, and 11 lm2, and

their average diameters were 0.53, 0.43, and 0.43 lm, respectively.

The values for average diameter, and for partial lengths and surface

FIGURE 1 Axon initial segment (AIS) labeling in neocortex and SNc. (a) Ankyrin-G (Ank-G) immunostaining in visual cortex reveals the uni-form arrangement of cortical AIS (some indicated by large arrows, a1) along with some nodes of Ranvier (NR, small arrows, a1). Pia surface istoward up right corner, according to visual cortex orientation in the mouse at the level of the SNc. Ank-G immunolabeling colocalized withPan-Nav immunostaining as seen in the merged image (a3; large and small arrows for AIS and NR, respectively correspond to those in a1 anda2). Far right AIS and NR columns: higher magnification images showing the colocalization of Ank-G and Pan-Nav in the AIS profile indicatedby a large arrow flanked by arrowheads (first column) in (a1–a3), and for the NR (second column) indicated by the arrowhead on the left ofimages (a1–a3). (b, c) Ank-G and Pan-Nav immunostaining is expressed by TH1 processes. Ank-G immunostaining in the substantia nigrareveals a large number of NRs (small arrows in b1–c1) and a smaller number of AIS (large arrows in b1–c1). NR and AIS co-localize immunolab-eling for the Pan-Nav (small and large arrows in b2–c2). Some Ank-G1/Pan-Nav1 processes also colocalized TH immunoreactivity (largearrows flanked by small arrowheads in b1–b3 and c1–c3). Far right AIS and NR columns: Higher power images showing colocalization ofimmunoreactivity for Ank-G and Pan-Nav in AIS profiles (from large arrow profiles flanked by arrowheads in a1–a3, b1–b3, and c1–c3) and NRprofiles (from small arrow profiles in a1–a3, b1–b3, and c1–c3). Ank-G and Pan-Nav also colocalized with TH in AIS, but not NR profiles. Scalebar in (c3)520 lm for (a), (b), and (c) and510 lm for far right AIS and NR columns

3534 | The Journal ofComparative Neurology

GONZ�ALEZ-CABRERA ET AL.

areas, are consistent with those values obtained for entire AIS in the

light microscopic analysis (Figure 2, Table 3).

In contrast to cortical and hippocampal pyramidal neurons (Howard,

Tamas, & Soltesz, 2005; Somogyi, Freund, & Cowey, 1982), synaptic

innervation of the AIS is not a common feature of central neurons (Iwa-

kura, Uchigashima, Miyazaki, Yamasaki, & Watanabe, 2012; Palay et al.,

1968; Peters et al., 1976; Somogyi & Hamori, 1976; Somogyi et al., 1982).

From the analysis of 311 serial ultrathin sections, including those used for

FIGURE 2 Structural features of the AIS of SNc dopaminergic neurons. (a) Ordinal placement of AIS based on length for the 29reconstructed AIS. (b) 3D orthogonal views of a typical AIS (indicated by an asterisk in a). (c, d) Length and surface area for all AIS shown in(a). Length was positively correlated with surface area (top right inset in d), but not with volume (bottom right inset in d). (e) Average (blackline), maximum (top value), and minimum (bottom value) diameters for all AIS shown in (a). There was no significant correlation betweenAIS length and average (open circles, middle regression line), or minimum (black solid circles, bottom regression line) or maximum (graycircles, top regression line) AIS diameter (top right inset in E). Furthermore, there was no correlation between length and degree oftapering, as measured by the maximum to minimum diameter ratio (bottom right inset in E). Scale bar in (b)55 lm

GONZ�ALEZ-CABRERA ET AL. The Journal ofComparative Neurology

| 3535

the three partially reconstructed profiles shown in Figure 3i, we found

three cases of synaptic innervation of Ank-G1/TH1 structures (Figure 3j).

Given that the surface area of the 3D reconstructed AIS mentioned above

(215 AIS profiles) was�55 lm2 (28, 17, and 11 lm2), we extrapolated that

the surface area of 311 AIS profiles would be �80 lm2. This equates to a

density of �0.038 synapses/lm2. Given that the average surface area per

individual AIS found in this study was 29.3 lm2 (Table 3), we would expect

an average of�1 synapse per AIS of mouse dopaminergic neurons.

3.4 | Expression mRNA for Nav subunits in SNc

Finally, in order to gain insight into the Nav subunits that compose the

AIS in SNc dopaminergic neurons, we analyzed the mRNA expression

of the main Nav subunits observed in the central nervous system (Kole

& Stuart, 2012) by in situ hybridization. As shown in Figure 4, we found

Nav1.1 mRNA to be expressed in only a few scattered neurons within

the SNc, but absent in the substantia nigra pars reticulata (SNr) or other

adjacent regions (Figure 4a). This contrasted markedly with the mRNA

for the Nav1.2 subunit, which was expressed throughout the SNc and

in the majority of neurons therein. mRNA for Nav1.2 was also strongly

expressed in the parabrachial pigmented (PBP) subdivision of the ven-

tral tegmental area, and although more scattered, also in the SNr (Fig-

ure 4b). Scattered neurons both in the SNc and SNr also expressed

Nav1.6 mRNA, although sometimes forming clear clusters with no clear

boundaries spanning both the SNc and SNr (Figure 4c). Examples of

labeled cells can be seen in Figure 4d,e. Because we only found sub-

stantial labeling for Nav1.2 and Nav1.6, and not Nav1.1, we estimated

the proportion of Nav1.2 versus Nav1.6 within the SNc. We systemati-

cally sampled and imaged the SNc in both Nav1.2 and Nav1.6 series

and counted the number of profiles we encountered in each series.

From the total number of profiles counted in each animal (n53), we

found that 87% corresponded to Nav 1.2 mRNA expressing cells

(132612, mean6 SEM, respectively), and 13% to Nav 1.6 mRNA

expressing cells (1963, mean6 SEM, respectively).

4 | DISCUSSION

The main findings of the present study are: (a) the AIS of dopaminergic

SNc neurons are diverse in length and diameter, (b) there is a decrease

in diameter along AIS axis, independent of size, (c) Ank-G immunoreac-

tivity is restricted to a sub-membranous localization, consistent with

the role in anchoring Nav channels at the AIS of these neurons, (d)

although in low number and incidence, there are cases of afferent syn-

aptic innervation directly onto the AIS of, at least, some neurons, and

(e) SNc neurons prominently express Nav1.2 mRNA. These features

could help to explain not only some common electrical behavior of

dopaminergic neurons, but also provide a substrate for physiological

variability and vulnerability to degeneration. They could also provide

valuable information which may contribute to the future design of

therapies for disorders of the dopaminergic system.

4.1 | Structural characteristics of the AIS

The average length of the AIS of dopaminergic neurons in mice is �25

lm. While on average this length is smaller than that for mouse baso-

lateral amygdala pyramidal cells (�60 lm) (Veres, Nagy, Vereczki,

Andrasi, & Hajos, 2014), rat somatosensory cortex pyramidal cells

(�40–60 lm) (Baranauskas, David, & Fleidervish, 2013; Palmer &

Stuart, 2006), similar to mouse spinal motoneurons (�30 lm) (Duflocq,

Chareyre, Giovannini, Couraud, & Davenne, 2011) and rat dentate

granule cells (�19 lm) (Evans et al., 2015), and larger than avian audi-

tory magnocellular neurons (�12 lm) (Kuba et al., 2010), we found

considerable variability in AIS length (�12–60 lm) for dopamine neu-

rons. Longer AIS are associated with higher spontaneous firing rates in

both avian auditory magnocellular neurons and rat dentate granule cells

(Evans et al., 2015; Kuba et al., 2010), suggesting that AIS length may

relate to the differences in in vivo firing rate observed in dopaminergic

neurons (Brown, Henny, Bolam, & Magill, 2009; Henny et al., 2012;

Neuhoff, Neu, Liss, & Roeper, 2002). As expected, length positively

correlated with surface area. However, differences in length did not

correlate with differences in average diameter, thus demonstrating that

length is the major factor contributing to changes in AIS surface area.

This is relevant insofar as larger AIS may allow a larger number of Nav

at its surface, and thus a lower threshold for firing, which could then

translate into higher firing rates (Mainen, Joerges, Huguenard, & Sej-

nowski, 1995). The diameter of AIS of dopaminergic neurons was also

variable (0.2–0.8 lm), with an average value of <0.4 lm, which is in

line with previous data reported for the axons intracellularly labeled

dopaminergic neurons (Tepper et al., 1987). We also established that,

on average, and independently of AIS length, the diameter of individual

AIS decreased (tapered) by �50% along its length. This is relevant in

view of the fact that, as shown in pyramidal neurons, the thinnest and

most distal region of the AIS is where action potentials are initiated,

implying that AIS tapering in dopaminergic may define a sub-region of

the AIS where action potentials are initiated (Mainen et al., 1995;

Palmer & Stuart, 2006).

4.2 | Ultrastructural characterization and synaptic

innervation of the AIS

At the ultrastructural level, Ank-G was restricted to a submembranous

position, both at the NR and AIS, which is consistent with its function

TABLE 3 Structural parameters of axon initial segment SNc dopa-minergic neurons (n529, from three animals), as extracted fromvector-based 3D reconstructions following double immunolabelingfor ankyrin-G and TH

Mean SEM Range

Length (lm) 25.8 1.7 12.9–62.1

Surface area (lm2) 29.3 2.2 10.4–64.2

Volume (lm3) 3.06 0.33 0.42–7.83

Average diameter (lm) 0.37 0.019 0.2–0.6

Minimum diameter (lm) 0.27 0.019 0.2–0.6

Maximum diameter (lm) 0.53 0.033 0.2–0.8

Max/min diameter ratio 2.07 0.14 1.0–2.67

3536 | The Journal ofComparative Neurology

GONZ�ALEZ-CABRERA ET AL.

as a scaffolding protein anchoring Nav and other voltage-gated chan-

nels to the membrane (Hill et al., 2008; Jones et al., 2014; King et al.,

2014; Zhou et al., 1998). At the AIS, the submembranous region is

characterized by a dense undercoating (Palay et al., 1968) which in our

hands was difficult to observe due to the diffuse and intense labeling

produced by the peroxidase reaction product that we used to label TH-

FIGURE 3 Ultrastructural features of AIS of SNc dopaminergic neurons. Ank-G immunostaining was present in both TH1 and TH2 struc-

tures. (a) A TH2 myelinated axon cut longitudinally, showing Ank-G immunolabeling (gold particles, small arrows) at a NR. Ank-G staining islocalized beneath the cytoplasmic membrane. Note the myelin sheath covering adjacent portions of the axon (large arrows) but not the NRitself. (b) A TH2 myelinated axon cut transversally showing Ank-G immunolabeling (gold particles, small arrows) at a NR. Ank-G1 immuno-gold particles, located beneath the cytoplasmic membrane, are present at the bottom half of the axonal profile. The upper half of the axonalprofile is myelin coated (large arrow) and is not Ank-G labeled. (c) A large TH1 profile (asterisk) revealed by the peroxidase reaction productwhich also contains Ank-G1 staining (gold particles, arrows), identifying them as the AIS of dopaminergic neurons. TH1 immunostaining isrevealed by the amorphous precipitate within the cytoplasm, typical of the peroxidase reaction product. This contrasts with the electrondense, particulate immunolabeling for Ank-G (small solid arrowheads) with a restricted distribution beneath the membrane. A multi-vesicularbody within the AIS is also visible (small solid arrowhead). Note in (c) the presence of adjacent TH1 profiles devoid of Ank-G1 staining(double solid arrowheads), most likely thin dendritic processes. Note also adjacent TH2 profiles, including an unlabeled thin dendrite (dou-ble open arrowhead), an unlabeled axon terminal filled with synaptic vesicles (open arrowhead) and unlabeled myelinated axons (small openarrowhead). (d–g) Various TH1 profiles (asterisks) revealed by the peroxidase reaction product, showing Ank-G1 staining (gold particles,arrows), indicative of AIS of dopaminergic neurons. Note difference in size between (d), (e), and (g) (small) and (f) (large) AIS. (h) A long TH2

profile also immunopositive for Ank-G (small arrows), indicative of an AIS of a non-dopaminergic neuron within the substantial nigra. (i) Par-tial 3D reconstructions of three Ank-G1/TH1 profiles. The reconstructed profiles show a typical tubular shape and their diameters are sim-ilar to those obtained for AIS from light microscopy reconstructions (�0.5 lm, see Figure 2). (j) Micrograph of a synaptic contact onto theAIS of a dopaminergic neuron. As described above, TH1 staining is cytoplasmatic (asterisk) and Ank-G1 staining is peripheral and submem-branous (arrows). The presynaptic terminal is indicated by a black arrowhead and the postsynaptic density is indicated by white arrowheads.Synaptic contacts were followed through serial ultrathin sections and confirmed from tilted views (not shown). Scale bar in (h)5500 nm for(a–h), 1 lm for (i) and 500 nm for (j). [Color figure can be viewed at wileyonlinelibrary.com]

GONZ�ALEZ-CABRERA ET AL. The Journal ofComparative Neurology

| 3537

immunoreactive structures. We on the other hand, detected multive-

sicular bodies, structures that are also typically observed in AIS (Palay

et al., 1968; Peters et al., 1976). In the serial-section analysis and

reconstructions, we did not observe the colocalization of Ank-G and

TH immunoreactivity in myelinated structures nor did we observe

Ank1/TH1 profiles that may have corresponded to NR thus confirm-

ing the unmyelinated nature of dopaminergic axons (Nirenberg,

Vaughan, Uhl, Kuhar, & Pickel, 1996).

Serial-section analysis and reconstructions of double-labeled mate-

rial showed only few cases of afferent innervation of the AIS of dopami-

nergic neurons. This is in agreement with the generally low incidence of

synapses onto the AIS of central neurons (Iwakura et al., 2012; Palay

et al., 1968; Peters et al., 1976; Somogyi & Hamori, 1976), with the

important exception of AIS of pallial pyramidal cells (Somogyi et al.,

1982; Veres et al., 2014). Indeed, the number of synapses per AIS (�1

on average) is very low in contrast to 50–60 synapses received by AIS

of mouse basolateral amygdala pyramidal neurons (Veres et al., 2014).

The density of innervation (0.038 synapses/lm2) is also an order of

magnitude lower than overall synaptic density of the somatodendritic

domain of rat SNc dopaminergic neurons (0.53 synapses/lm2) (Henny

et al., 2012). Notwithstanding, the results support the contention that,

although to a lesser extent, the electrical activity of dopaminergic neu-

rons could be modulated by classical synaptic neurotransmission at the

very place where it is initiated (Blythe et al., 2009). They also highlight

the existence of afferent neurons able to target the AIS of dopaminergic

neurons.

Given the lack of a strong synaptic innervation at the AIS itself,

synaptic regulation of dopaminergic neurons may be facilitated by

innervation of regions near the AIS, specifically those of the axon bear-

ing dendrite, which in both dopaminergic and hippocampal neurons

(Blythe et al., 2009; Hamada, Goethals, de Vries, Brette, & Kole, 2016)

has been shown to be particularly important for action potential gener-

ation and neuronal excitability.

4.3 | Nav subunit expression in the SNc

Of the nine types of Nav subunits, Nav1.1, 1.2, and 1.6 have been

shown to be expressed in the AIS of central neurons (Kole & Stuart,

FIGURE 3 (Continued)

3538 | The Journal ofComparative Neurology

GONZ�ALEZ-CABRERA ET AL.

2012). According to our in situ hybridization data, the majority of

neurons within the SNc express the Nav1.2 subunit, with many fewer

expressing the Nav1.6 subunit and very few expressing the Nav1.1

subunit. Assuming that following translation, Ank-G anchors Nav

subunits to the AIS, the results indicate that Nav1.2 would be the

main channel subunit contributing to PanNav immunolabeling in the

AIS of dopaminergic neurons (Figure 1). Nav1.6 is the most

abundantly expressed subunit in the adult brain, and has also been

shown to endow neurons with a lower threshold for action potential

triggering and high repetitive firing rate (Kole & Stuart, 2012; Royeck

et al., 2008; Rush, Dib-Hajj, & Waxman, 2005; Van Wart & Mat-

thews, 2006). Nav1.2, in contrast, has a more restricted expression in

the adult brain (Kole & Stuart, 2012), activates at more depolarized

voltages, and appears to inactivate faster than Nav1.6 channel

FIGURE 4 Expression of mRNA for Nav subunits in SNc. (a) In situ hybridization for Nav1.1 in the SNc. Low (a1) and high (a2)magnification micrographs of ventral midbrain showing scant expression of Nav1.1 in the SNc, and not in the underlying SNr nor otheradjacent regions. This contrasted with Nav1.1 expression by neurons of the piriform cortex (Pir; a3). (b) Low (b1) and high (b2)magnification micrographs of the SNc, SNr, and adjacent parabrachial pigmented (PBP) depicting labeling of Nav1.2 in most neurons in theSNc, with Nav1.21 neurons also observed in the underlying SNr. In situ hybridization signal for Nav1.2 is also widely expressed in thepiriform cortex (b3). (c) Low (c1) and high (c2) magnification images of Nav1.6 expression in the SNc, SNr, and PBP. Scattered cells areobserved in the SNc, (c1, c2), sometimes forming relatively denser groups of cells spanning the SNc and SNr (c2, right). Expression ofNav1.6 in the underlying SNr appears denser than for Nav1.2. Nav1.6 expression in piriform cortex is shown in (c3). (d, e) Highmagnification images showing examples of Nav1.21 (d1, d2) and Nav1.61 (e1, e2) cells (arrowheads) in the SNc. Scale bar in (c1)5500 lmfor (a1), (b1), and (c1); (c3)5100 lm for rest of micrographs in (a), (b), and (c); (e2)520 lm for (d) and (e) micrographs. PBP5 parabrachialpigmented area of the ventral tegmental area; Pir5 piriform cortex; RN5 red nucleus; SNc5 substantia nigra pars compacta;SNr5 substantia nigra pars reticulata [Color figure can be viewed at wileyonlinelibrary.com]

GONZ�ALEZ-CABRERA ET AL. The Journal ofComparative Neurology

| 3539

subunits (Rush et al., 2005). This implies that dopaminergic neurons

should be less excitable than neurons expressing Nav1.6, a sugges-

tion that is consistent with previously reported electrophysiological

features of dopaminergic neurons having high threshold for firing

(Seutin & Engel, 2010). The preferential expression of Nav1.2 may

also explain the relatively low frequency at which dopaminergic neu-

rons fire. On the other hand, some SNc neurons did express Nav1.6,

which suggests the presence of a subpopulation of dopaminergic

neurons within the SNc selectively expressing Nav1.6, or co-

expressing Nav1.2 and Nav1.6, as do cortical neurons (Hu et al.,

2009). Alternatively, this population may represent non-

dopaminergic, GABAergic neurons within the boundaries of SNc

(Nair-Roberts et al., 2008) that express Nav1.6. In conclusion, it is

likely that most dopaminergic SNc neurons only express Nav1.2

channel subunit at their AIS. Future studies looking at the specific

localization of the Nav1.2 (and Nav1.6) protein in SNc dopaminergic

neurons may confirm this suggestion, while also assessing whether

its distribution across the AIS itself is homogeneous (Hu et al., 2009).

These studies could also examine its presence in other non-AIS sub-

cellular compartments (Martinez-Hernandez et al., 2013).

4.4 | Functional aspects and conclusion

As mentioned above, it has been previously shown that neurons with

larger AIS are associated with faster firing and excitability than small-

AIS neurons (Evans et al., 2015; Kuba et al., 2010). On the other hand,

it has been proposed that increased excitability and firing rate may be

a characteristic of more vulnerable SNc dopaminergic neurons (Brown

et al., 2009; Neuhoff et al., 2002). It is possible therefore to propose

that heterogeneity of AIS length in dopaminergic neurons may also

relate to differences in vulnerability, on the contention that neurons

with larger AIS fire faster and would be more vulnerable.

In addition to reflect structural and possibly physiological variabili-

ty in SNc dopaminergic neurons, AIS variability may also reflect

ongoing plastic changes which have been shown possible to occur in

the AIS of other neuronal types (Evans et al., 2015; Kuba et al., 2010).

Finally, the apparent exclusive expression of Nav1.2 in this popula-

tion opens the interesting possibility of regulation of the activity of the

dopaminergic system by either reducing or enhancing excitability

through direct action on Nav1.2, once specific blockers (or openers) are

available, as it is the case for Nav1.1 subunits (Osteen et al., 2016), or

by modulation of post-transcriptional silencing of Nav1.2 through the

use of siRNA, as also shown for Nav1.1 subunits (Mishra et al., 2015).

In summary, our data indicate that the AIS of SNc dopaminergic

neurons share common characteristics with the AIS of other types of

neurons but also display less common features such as the likely

expression of Nav1.2 and, although of low density, afferent synaptic

innervation. The results also show an important degree of variability in

terms of size and shape of the AIS. All these characteristics may under-

lie some typical features of midbrain dopaminergic neurons including a

low excitability and firing rate, but also differences observed between

individual neurons regarding in vivo firing pattern, frequency and

response to aversive stimulation (Brischoux et al., 2009; Henny et al.,

2012; Roeper, 2013).

ACKNOWLEDGMENTS

We thank the Centro de Investigaciones M�edicas (CIM) and

Direcci�on de Investigaci�on (DIDEMUC) for the use of the Micros-

copy Unit, funded by MECESUP PUC0815 grant for scientific equip-

ment, and the Unidad de Microscopia Avanzada de Ciencias

Biol�ogicas facility at the Pontificia Universidad Cat�olica de Chile.

CONFLICT OF INTEREST

The authors declare no competing financial interests.

ORCID

Cristian Gonz�alez-Cabrera http://orcid.org/0000-0003-0515-0254

Rodrigo Meza http://orcid.org/0000-0003-0770-6535

Pablo Henny http://orcid.org/0000-0001-8470-8222

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How to cite this article: Gonz�alez-Cabrera C, Meza R, Ulloa L,

et al. Characterization of the axon initial segment of mice sub-

stantia nigra dopaminergic neurons. J Comp Neurol.

2017;525:3529–3542. https://doi.org/10.1002/cne.24288

3542 | The Journal ofComparative Neurology

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