Characterization of the axon initial segment of mice ......basis that underlies firing of...
Transcript of Characterization of the axon initial segment of mice ......basis that underlies firing of...
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: [email protected]; [email protected]
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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