Voltage-gated ion channels in the axon initial
segment of human cortical pyramidal cells and
their relationship with chandelier cells
Maria Carmen Inda*†, Javier DeFelipe†‡, and Alberto Mun ˜oz*†
*Departamento de Biologı ´a Celular, Universidad Complutense de Madrid, Jose Antonio Novais 2, 28040 Madrid, Spain; and†Instituto Cajal,
Consejo Superior de Investigaciones Cientificas, Avenida Doctor Arce 37, 28002 Madrid, Spain
Communicated by Edward G. Jones, University of California, Davis, CA, December 28, 2005 (received for review November 24, 2005)
The axon initial segment (AIS) of pyramidal cells is a critical region
for the generation of action potentials and for the control of
pyramidal cell activity. Here we show that Na?and K?voltage-
gated channels, together with other molecules involved in the
localization of ion channels, are distributed asymmetrically in the
AIS of pyramidal cells situated in the human temporal neocortex.
of the AIS together with the associated proteins spectrin ?IV and
ankyrin G. In contrast, Kv1.2 channels are associated with the
adhesion molecule Caspr2, and they are mostly localized to the
distal region of the AIS. In general, the distal region of the AIS is
targeted by the GABAergic axon terminals of chandelier cells,
whereas the proximal region is innervated, mostly by other types
of GABAergic interneurons. We suggest that this molecular seg-
regation and the consequent regional specialization of the
GABAergic input to the AIS of pyramidal cells may have important
functional implications for the control of pyramidal cell activity.
inhibition ? interneurons ? neocortex
zation of the sites where the action potential is generated.
Because of the high concentration of voltage-dependent Na?
and K?channels in the axon initial segment (AIS), it has
commonly been assumed that this region is the site with the
lowest threshold, and, as such, it must be the first site at which
the action potential is generated. However, some studies have
suggested that action potentials initiate beyond the AIS (1, 2).
Indeed, it has been shown recently that action potentials in
cortical layer V pyramidal cells (3) and Purkinje cells of the
cerebellum (4) are first generated at the first node of Ranvier.
Under these circumstances, voltage-dependent Na?channels in
the AIS would serve to back-propagate action potentials into the
resistivity, the diameter, length, and the density of voltage-
dependent Na?channels (5). Furthermore, the different bio-
physical properties of ion channels in the AIS and nodes of
Ranvier might reflect the differences in the distribution of
voltage-dependent Na?and K?channels or in the molecular
isoforms expressed in these structures. Such variations might
determine the threshold for the initiation of the action potential
at each site.
Chandelier cells are a particular type of GABAergic inter-
neuron whose axon terminals [chandelier cell axon terminals
(Ch-terminals)] specifically contact the AIS of cortical pyrami-
dal cells. Alterations in the connectivity of chandelier cells have
been associated with syndromes such as epilepsy (6) and schizo-
phrenia (7), and it has traditionally been presumed that chan-
delier cells are key elements that control the output of pyramidal
cells (1, 8–10). However, if action potentials are initiated in the
first node of Ranvier, they would be propagated in a retrograde
manner to the AIS, where they can be reproduced and back-
o understand how synaptic input is converted into neuronal
output, it is first essential to fully comprehend the organi-
propagated to the soma and dendrites. In this case, GABAergic
synapses established by chandelier cells would not only control
the output of pyramidal cells but also influence the back-
propagation of action potentials, modulating the integration of
information in the soma and dendrites. In fact, recent studies
have shown that rather than simply shunting action potentials in
pyramidal cells, chandelier cells participate in complex activities
such as the synchronization of the firing patterns of large
populations of hippocampal pyramidal cells in different states of
consciousness (11, 12).
Voltage-dependent Na?and K?channels are spatially segre-
gated and symmetrically distributed at the nodes of Ranvier.
Both in the central and peripheral nervous system, they are
restricted to specific isolated membrane domains, the nodal and
juxtaparanodal regions, respectively, which are separated by the
paranodal region (13, 14). Their restricted and defined distri-
bution is maintained by the expression of key molecules involved
in defining the localization of ion channels. These molecules
include proteins that interact with the cytoskeleton, such as the
spectrin ?IV and ankyrin G, and adhesion molecules such as
Neurofascin, NrCAM, Caspr, and Caspr2. The presence of some
of these molecules has also been reported in the AIS of
cerebellar Purkinje cells and of cortical cells in the rat, together
with other molecules that regulate pyramidal cell excitability
such as 5-HT1Aand GABABreceptors in humans and KCNQ2?3
channels in rats (15–20). However, the precise distribution of
voltage-dependent Na?and K?channels and related molecules
in the pyramidal cell AIS and their spatial relationship with the
GABAergic Ch-terminals has not yet been explored.
In the present study, we have examined the relationship
between Ch-terminals and the regional distribution of both
such as spectrin ?IV, ankyrin G, and Caspr2 in the AIS of
pyramidal cells in the human temporal neocortex. The results
indicate that these molecules are distributed asymmetrically in
the AIS, displaying a partial spatial segregation. Furthermore,
of the AIS, a domain that is characterized by a high density of
Voltage-Gated Na?Channels (VGSC). When we analyzed the distri-
bution of VGSC in layers II–VI of the neocortex, numerous
strongly labeled VGSC-immunoreactive (ir) AISs were observed
(Figs. 1 and 2A), but no somata or dendrites were labeled.
Indeed, they were so numerous that although we did not attempt
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: AIS, axon initial segment; Ch-terminals: chandelier cell axon terminals; ir,
immunoreactive; VGSC, voltage-gated Na?channels.
‡To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
February 21, 2006 ?
vol. 103 ?
to perform a quantitative study, it appeared that the AISs of all
neurons were VGSC-positive. The labeled AISs extended dis-
tally for ?11–40 ?m (mean length ? SD: 23.12 ? 7.05 ?m; n ?
180 in layer III) from their somata of origin.
Spectrin ?IV. Whereas spectrin ?IV immunoreactivity was weak
in the soma of pyramidal cells, the AISs of these cells were
intensely labeled (Fig. 2 B and E). The spectrin ?IV immuno-
staining in the AIS extended distally from the somata of origin
for ?11–47 ?m (mean length ? SD: 25.6 ? 6.8; n ? 212 in layer
III). In double-labeling experiments, nearly all AISs in layer III
that were labeled for VGSC (136 of 138; 98.5%) were also
immunoreactive for spectrin ?IV (Fig. 2 A–C).
Ankyrin G. Ankyrin G immunocytochemistry labeled the AIS of
relatively few cells, although numerous dendritic processes were
labeled, as were the somata of a sparse population of pyramidal
neurons distributed through layers II–VI (Fig. 2D). Only the
AISs in 23 of 163 spectrin ?IV-ir cells (14%) were immuno-
stained for ankyrin-G (Fig. 2 D–F). Thus, ankyrin G immuno-
cytochemistry labels the AIS of a subpopulation of pyramidal
cells that coexpress VGSC and spectrin ?IV.
GAT-1 Labeling of Ch-Terminals. GAT-1-ir Ch-terminals (Figs. 1,
2H, and 3E) ranged in length from 10 to 39 ?m in layer III (mean
length ? SD: 22.2 ? 5.5 ?m; n ? 210), and they were usually
located ?8–19 ?m (mean distance ? SD: 13.4 ? 2.8 ?m; n ? 22)
below the unstained somata of the pyramidal cells from which
the AIS innervated by the GAT-1-ir Ch-terminals originated. In
sections double-labeled for GAT-1 and VGSC, the GAT-1-ir
Ch-terminals surrounded most VGSC-ir pyramidal cell axons
(Fig. 2 G–I). Moreover, it was clear that there was a regional
specialization of the AIS because VGSC immunoreactivity was
the majority of GAT-1-ir Ch-terminals only innervated the distal
region of the VGSC-ir AIS (Fig. 2 G–I).
Kv1.2. Kv1.2 immunocytochemistry revealed the presence of
numerous labeled AISs in all cortical layers (Figs. 1 and 3 A
and D). In layer III, these Kv1.2-ir AISs ranged in length from
7 to 27 ?m (mean length ? SD: 14.82 ? 4.54 ?m; n ? 116).
However, Kv1.2 immunostaining did not extend along the
whole length of the AIS; rather it was confined to the more
distal region (Fig. 3 A–D).
Caspr2. Caspr2 immunocytochemistry also labeled numerous
AISs ranging in length from 11 to 26 ?m (mean length ? SD:
15.85 ? 3.12 ?m; n ? 79 in layer III) with a similar pattern to
that observed for Kv1.2. Indeed, in sections labeled for both
Kv1.2 and Caspr2, the immunostaining for Caspr2 was specific
to the distal AIS in perfect register with the distribution of
Kv1.2 (Fig. 3 A–C). Of the AISs that were labeled by antibodies
against Kv1.2, 92% were also labeled for Caspr2. Indeed,
labeling for Caspr2 failed to coincide with that for Kv1.2 in
only 8 of 99 AISs labeled for Kv1.2. Furthermore, in sections
double-labeled for Kv1.2 and GAT-1, Kv1.2 channels were
restricted to the distal region of the AIS in register with the
region that is innervated by GAT-1-ir Ch-terminals (Fig. 3
D–F). It should be noted that VGSC and Caspr2 also colo-
calized in the AIS of the majority of pyramidal cells in
double-labeled sections. Only 3 of 102 VGSC-ir AISs were not
labeled by antibodies against Caspr2. Therefore, both VGSC
and Kv1.2 overlapped in the distal AIS, whereas the proximal
AISs were characterized by the presence of VGSC and the
absence of Kv1.2 (Figs. 2 and 3) and Ch-terminals.
The basis for the generation and saltatory propagation of action
potentials is the exquisite molecular architecture of nerve fibers,
notably in the segregation of voltage-gated ion channels into
distinct membrane domains (13, 21–24). This segregation is
achieved through specific sorting mechanisms coupled to the
anchoring and clustering of these proteins in the plasma mem-
brane. The ion channels that underlie excitability are compart-
mentalized into sharply defined subcellular regions and form
heteromultimeric complexes that also contain intracellular scaf-
folding, adapter, and cytoskeleton proteins, transmembrane
proteins, and extracellular matrix elements (see below). The
similarities in the membranes of the AIS and the nodes of
Ranvier (25, 26) extend to the presence of an electron-dense
membrane undercoat that is necessary to cluster the voltage-
gated ion channels (27). However, whereas the molecular archi-
tecture of the nodes of Ranvier has been extensively studied in
the rat CNS and peripheral nervous system, much less is known
the distance from the base of the pyramidal cell body to the beginning of the AIS labeled for Kv1.2 (B) or to the GAT-1-ir Ch-terminal (C). The asterisks indicate
the location of pyramidal cell bodies. (Scale bar: 10 ?m.) (C Right) The plots shown are presented as a function of the mean distance from the cell body based
on optical density readings of the immunostaining for Na?and Kv1.2 channels along the AIS (Upper) or for GAT-1 Ch-terminals (Lower). Note that
immunostaining for the VGSC is present along the whole length of the AIS, whereas for Kv1.2, the staining is restricted to the distal region of the AIS that is
contacted by GAT-1-ir Ch-terminals (See Fig. 3 D–F).
Inda et al.
February 21, 2006 ?
vol. 103 ?
no. 8 ?
about the AIS. We present a morphological description of the
distribution of voltage-dependent Na?and K?channels in the
AIS of human pyramidal cells. The results indicate that some
features observed in the AIS are common to the nodes of
Ranvier, particularly regarding the molecules with which ion
channels may associate. However, the results also indicate
fundamental differences in ion channel distribution between the
AIS and the nodes of Ranvier.
VGSC. VGSC are responsible for the rapid inward sodium cur-
rents and the consequent depolarization required for the induc-
tion of the action potential and saltatory conduction (28, 29).
The anti-pan-sodium channel antibody used in the present study
recognizes all of the known vertebrate Na?channel isoforms
(30), and, therefore, it is thought to reflect the complete
repertoire of VGSC in the AIS, where they are distributed along
its whole length. At the nodal region of the nodes of Ranvier,
VGSC are clustered at a high density. In these structures, they
are associated with the underlying actin-spectrin cytoskeleton
through the adaptor protein ankyrin G and to NrCAM and
neurofascin 186, nodal cell adhesion molecules of the Ig super-
family (13, 14). The same molecular interactions seem to occur
in the AIS in different neuronal populations of the rat CNS,
where NrCAM and neurofascin 186 have been shown to be
concentrated (18, 31–35). In addition, spectrin ?IV has been
seen to concentrate at the AISs of both neocortical and hip-
pocampal cells, where it might be involved in the localization of
VGSC and ankyrin G (19, 36, 37). In keeping with these studies,
the AISs of human neocortical pyramidal cells express VGSC
and spectrin ?IV, which colocalize along the length of the AIS
in virtually all pyramidal cells. However, we observed little
colocalization with ankyrin G. The antibody used in the present
study to recognize ankyrin G identifies a single protein of
between 200 and 300 kDa that might correspond to only one of
the established nodal isoforms (270 kDa). Hence, it may not
recognize the 480-kDa (33, 34, 38) or the 97-kDa (39, 40)
trating double labeling for VGSC (Na?Ch) and spectrin ?IV (A–C), ankyrin G
and spectrin ?IV (D–F), and VGSC and GAT-1 (G–I) in the AIS of pyramidal cells
from the human temporal neocortex. C, F, and I were obtained after combin-
of VGSC and spectrin ?IV (filled arrows in A and B) and of ankyrin G and
along the length of the AIS from the somata, whereas the GAT-1-ir Ch-
A–C represent stacks of 15 optical sections obtained at a distance of 1.5 ?m in
the z axis (total: 22 ?m). Images D–F represent stacks of seven optical sections
7 ?m). (Scale bar: A–C, 25 ?m; D–I, 15 ?m.)
Confocal images from the same section and microscopic field illus-
VGSC and Caspr2 (G–I) in the human temporal neocortex. The asterisks indi-
cate the location of the pyramidal cell bodies. C, F, and I were obtained after
combining the images A and B, D and E, and G and H, respectively. Note that
is also expressed (filled arrows in A and B). This distal AIS specifically receives
GABAergic innervation from the GAT-1-ir Ch-terminals (filled arrows in D and
the proximal AIS is characterized by the presence of VGSC and the absence of
Kv1.2 (D–I). Images A–C represent stacks of nine optical sections obtained at
a distance of 1.1 ?m in the z axis (total: 9 ?m). Images D–F and G–I represent
stacks of four optical sections obtained at a distance of 1.1 ?m in the z axis
(total: 3 ?m). (Scale bar: 11 ?m.)
Confocal images from the same section and microscopic field illus-
www.pnas.org?cgi?doi?10.1073?pnas.0511197103 Inda et al.
isoforms previously identified as nodal components in the CNS.
Therefore, because the 270-kDa isoform of ankyrin G seems to
be present in only ?15% of the human neocortical pyramidal
cells (this study), different ankyrin G polypeptides or other
anchoring proteins might participate in the clustering of VGSC
in the AIS of specific pyramidal cell populations.
Voltage-Dependent K?Channels. Voltage-dependent K?channels
of the Kv1 subfamily of Shaker-type delayed-rectifying K?
channels are present at the nodes of Ranvier where they are
found in the juxtaparanodal axonal membrane beneath the
overlying compact myelin (41–44). Kv1 channels generate low-
threshold voltage-dependent outward currents that seem to
regulate the action potential threshold. They contribute to the
repolarization of single-action potentials, they modulate action
potential duration and frequency, and they maintain the inter-
nodal resting potential (29, 34, 45, 46). Juxtaparanodal Kv
channels are composed of various heteromultimeric combina-
tions of pore-forming Kv1 ? (Kv 1.1, 1.2, 1.4, and 1.6) and the
associated cytoplasmic Kv?2 subunits (42, 44, 47, 48). In the
juxtaparanodal region, Kv1.1 and Kv1.2 have been shown to
colocalize with, and to interact and?or cluster with, Caspr2, a
interacts with other proteins necessary for the accumulation of
Kv channels at the juxtaparanodal region and for their interac-
tion with the actin–spectrin cytoskeleton (14, 51–56). The
present results show that in human neocortical pyramidal cells,
Kv1.2 channels are clustered at and colocalize with the adhesion
molecule Caspr2 exclusively in the distal domain of the AIS. This
region of the AIS is innervated by Ch-terminals, and it is
therefore devoid of myelin. In contrast, Kv1.2 is located beneath
the compact myelin sheath at the juxtaparanodes. Whether
Caspr2 or other adhesion molecules that might be present at the
distal region of the AIS specifically interact with the adhesion
molecules of Ch-terminals is a possibility that should be further
Differential Distribution of Voltage-Dependent Na?and K?Channels
at the AIS and the Node of Ranvier.At the nodes of Ranvier, VGSC
Indeed, VGSC and Kv1.2 are confined to the nodal and jux-
taparanodal regions, respectively, separated by the paranodal
region (14, 24). The present results show that in contrast to the
nodes of Ranvier, voltage-dependent Na?and K?channels are
only partially segregated in the AIS and that there is a certain
degree of overlap in the distal portion of the AIS. Whether this
difference might be relevant to define the physiological at-
tributes of action potentials generated in the AIS when com-
pared to those in the nodes of Ranvier should be further studied.
The spatial distribution of ion channels is another relevant
molecular difference between the nodes of Ranvier and the AIS.
At the nodes of Ranvier, ion channels are symetrically distrib-
uted, and VGSC in the nodal region are flanked by K?channels
expressed in the juxtaparanodal regions. In contrast, in the AIS,
the Kv1.2 channels are exclusively found in the distal domain
where VGSC are expressed. It is important to note that over the
first 3 weeks of postnatal development in the rat peripheral
nervous system, Kv1 channel clustering is often asymmetrical
(50%), and channels may be found exclusively in the distal
juxtaparanode (46). However, this asymmetrical distribution is
transient, lasting only until the end of the third postnatal week,
and it may lead to instabilities in the propagation of the action
potential (46). Thus, it is of interest to determine what the
functional consequences of the asymmetrical distribution of
voltage-gated Na?and K?channels are in the AIS of human
the generation of the action potential and for the directionality
of action potential transmission.
Modulation of Action Potential Firing at the AIS by Chandelier Cells.
In different neuronal types, including Purkinje cells of the
cerebellum (4) and pyramidal cells of the subiculum (1) and
neocortex (3), it has been shown that action potentials are first
generated beyond the AIS. Indeed, this event occurs at the first
node of Ranvier, which shows a lower threshold for the initiation
of the action potential. Action potentials generated at distal
axonal locations then invade the soma and dendrites, a process
that depends on the presence of VGSC at the AIS that serve to
back-propagate action potentials into the soma and dendrites
(1–5). Inhibition of the AIS by GABAergic inputs from chan-
delier cells or through other neurotransmitters would dissociate
the soma from the site of action potential initiation. As a result,
it would not only be suited for orthodromic inhibition that would
simply suppress the initiation of the action potential but also for
antidromic inhibition. This latter type of inhibition might have
important consequences in controlling the back-propagation of
action potentials, influencing the level of neuronal output to the
soma and the dendritic tree that is necessary for the integration
of information (57). In fact, recent studies indicate that chan-
delier cells, together with other interneurons, are involved in
complex activities. These actions contribute to ensuring well
timed hyperpolarization that regulates the firing of pyramidal
cells and shapes the network output and the rhythms generated
in different states of consciousness (11, 12, 58–61).
region of the AIS where voltage-gated K?channels are located.
On the other hand, the proximal region of the AIS shows a high
density of VGSC, which seem to be less accessible to the
inhibitory influence of Ch-terminals. This proximal region is
likely to be innervated by other types of interneurons that
occasionally form synapses with the AIS (62, 63). The precise
point at which the action potential is initiated in human cortical
cells is not yet known. Nevertheless, it seems that inhibition by
the selective innervation of the distal AIS by Ch-terminals in
human pyramidal cells might be directionally selective (60),
being more effective from either the soma to the axon or vice
versa. This directional selectivity, and the consequent pattern of
action potential firing and back-propagation in pyramidal cells,
might vary depending on the regulation of K?conductances at
the AIS by different neurotransmitter systems such as the
serotonergic and cholinergic ascending systems (17, 39, 40, 64).
These regulatory systems, together with chandelier cells and
other types of GABAergic interneurons, might modify the
physiological requirements for the generation of action poten-
tials and, possibly, the location at which the action potential is
initiated in human pyramidal cells. This regulation may occur
through their direct action on the AIS, an intriguing possibility
that has to be evaluated in further studies.
Materials and Methods
Human neocortical tissue from the anterolateral temporal cor-
tex was obtained by surgical resection from one male and four
female patients diagnosed with intractable temporal lobe epi-
lepsy with a mesial origin (age range ? 21–54 years). All patients
were evaluated presurgically by scalp electroencephalography
(EEG), interictal single-photon emission computer tomography,
magnetic resonance imaging (MRI) 1.5 T, and videoelectroen-
according to the international 10–20 system and foramen-ovale
electrodes. Informed consent was obtained individually for all
patients, having been previously approved by ethical committee
of the ‘‘Hospital de la Princesa’’ (Madrid). During surgery,
electrocorticography was performed with a grid of 4 ? 5
electrodes embedded in Sylastic. The electrodes were placed
directly over the exposed lateral temporal cortex, and they were
of 1.2 mm in diameter with a 1 cm center-to-center interelec-
trode distance (Add-Tech, Racine, WI). Recordings were sam-
Inda et al.
February 21, 2006 ?
vol. 103 ?
no. 8 ?
pled at 400 Hz with a bandwidth of 1–70 Hz and over a minimum
period of 20 min, by using a 32-channel Easy EEG II system
(Cadwell, Kennewick, WA). Spiking areas were identified as
electrodes showing spikes (?80 ms) or sharp waves (80–200 ms)
with a mean frequency ?1 spike?min. Nonspiking areas were
defined as electrodes where no spikes, sharp waves, or slow
activity was observed. Photographs of the placement of the
electrodes were taken before grid removal, and the anatomical
location of the spiking and nonspiking areas was defined before
In all cases, tailored temporal lobectomy and amygdalohip-
pocampectomy was performed under electrocorticography guid-
ance. After surgery, the lateral neocortex and mesial structures
were subjected to standard neuropathological assessment. All of
the lateral neocortical biopsies were histologically normal,
whereas the hippocampal formation displayed neuronal loss and
gliosis (hippocampal sclerosis). In the present study, only normal
nonspiking areas of the lateral neocortex were used. Biopsy
samples were fixed in cold 4% paraformaldehyde in 0.1 M
phosphate buffer at pH 7.4 (PB) for 4–5 h at 4°C. Vibratome
sections (100 ?m thick) were immunolabeled by using the
following antibodies: mouse anti-Na?channels (1:100; Sigma),
mouse anti-K?channel Kv1.2 (1:50; Upstate Biotechnology,
Lake Placid, NY), mouse anti-ankyrin G (1:150; Zymed),
chicken anti-spectrin ?IV (1:100, gift from M. Komada, Tokyo
Institute of Technology, Yokohama, Japan; see ref. 19), rabbit
anti-GAT-1 (1:500; Chemicon), and rabbit anti-Caspr2 (1:250;
United States Biological).
The mouse anti-Na?channel antibody used was raised against
a synthetic peptide derived from the intracellular III-IV loop of
Na?channel, which is identical in all known vertebrate Na?
channels. Mouse Kv1.2 antibodies were generated against a
Kv1.2, and they recognize the Shaker-related ?-subunit Kv1.2 in
different species, including humans. Chicken anti-?IV spectrin
antibodies were raised against the variable region (amino acids
2,171–2,345 of ?IV e1-spectrin) expressed in Escherichia coli as
a glutathione S-transferase fusion protein (19). Mouse anti-
ankyrin G antibodies were raised against a synthetic peptide
derived from the spectrin-binding domain of the human ankyrin
G protein. These antibodies identify a single protein of between
200 and 300 kDa on Western blots, react with all splice forms of
ankyrin G containing a spectrin-binding domain, and do not
cross-react with related ankyrin isoforms. Rabbit anti-Caspr2
was raised against a C-terminus peptide corresponding to intra-
cellular amino acid residues 1,315–1,331 of human Caspr2.
Rabbit anti-GAT-1 was generated against a C-terminus peptide
(amino acids 588–599) of the rat GAT-1.
Sections were single-labeled or double-labeled by using the
following combinations of primary antibodies: Na?channels
with spectrin ?IV, ankyrin G, Caspr2, and GAT-1 or Kv1.2 with
Caspr2 and GAT-1. The sections were then rinsed and incubated
for 2 h at room temperature in biotinylated secondary antibodies
directed against one of the primary antibodies used in each
combination (rabbit anti-chicken, goat anti-rabbit, horse anti-
mouse, or rabbit anti-goat as appropriate). After rinsing in PB,
the sections were incubated for 2 h at room temperature in
streptavidin coupled to Alexa Fluor 488 (1:1,000; Molecular
594 (1:1,000, Molecular Probes) directed against the other
primary antibody used. The sections were then washed, mounted
in 50% glycerol in PB, and examined with a Leica (Cambridge,
U.K.) TCS 4D confocal laser scanning microscope. Z sections
were recorded at 1–2 ?m intervals through separate channels
(Scanware; Leica). Subsequently, Micrografx PICTURE PUB-
LISHER (Dallas) and PHOTOSHOP (Adobe Systems, San Jose, CA)
software were used to construct composite images from each
series by combining the images recorded through both channels
and to generate the figures.
Identification and Quantitative Analysis of the AIS and Ch-Terminals.
The AIS was readily identified as a short, thin, and smooth process
with a characteristic ‘‘eyelash-like’’ appearance (Fig. 1 A and B). In
contrast, GAT-1-ir Ch-terminals were identified as short, vertical
rows of buttons (65) that could be clearly distinguished from other
labeled elements (Fig. 1C). The length of the labeled AIS and the
Ch-terminals was measured on microphotographs at a final mag-
nification of ?1,000 (Fig. 1). The somata from which the Na?
channel-ir AIS originated were identified because the labeled AIS
cells that gave origin to a Kv1.2-ir AIS were considered to be those
unlabeled somata located just above the labeled AIS and in the
AISs were innervated by GAT-1-ir Ch-terminals were considered
to be those located immediately above the labeled Ch-terminal
Fluorescence microscopy was used to measure the intensity of
staining for the different markers along the AIS. The intensity
of immunostaining for Na?and Kv1.2 channels along the AIS
18) was quantified by densitometry by using a 3- to 4-?m-width
rectangular sampling tool (IMAGEJ software; Sun Microsystems,
Santa Clara, CA), extending from the base of soma to the distal
portion of the labeled AIS or Ch-terminal. The means were
plotted as a function of the distance from the cell body (Fig. 1).
We thank Dr. Komada for providing the spectrin ?IV antibody. This
work was supported by ‘‘Ministerio de Educacio ´n y Ciencia’’ Grants BFI
2003-01018 (to A.M.) and BFI 2003-02745 (to J.D.).
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