Neuroscience Letters 407 (2006) 42–47
Localization of L-type calcium channel CaV1.3 in cat lumbar spinal
cord – with emphasis on motoneurons
Mengliang Zhang a,∗ , Natalya Sukiasyan a , Morten Møller b , Ilya Bezprozvanny c ,
Hua Zhang c , Jacob Wienecke a , Hans Hultborn a
a
Department of Medical Physiology, the Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark
Department of Medical Anatomy, the Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark
c Department of Physiology, University of Texas Southwestern Medical Centre at Dallas, Dallas, TX, USA
b
Received 24 May 2006; received in revised form 27 July 2006; accepted 31 July 2006
Abstract
Voltage-dependent persistent inward currents (PICs) which underlie the plateau potentials are an important intrinsic property of spinal motoneurons. Electrophysiological experiments have indicated that a subtype of the low threshold L-type calcium channel, CaV 1.3, mediates this current.
In mouse and turtle lumbar spinal cord it has been shown that these channel proteins are mainly found on motoneuron dendrites. In the present
study we have used immunohistochemistry to locate these channels in lumbar spinal neurons, especially motoneurons, of the cat. The results
indicate that CaV 1.3 immunoreactivity was unevenly distributed among the laminae of the spinal grey matter. The small neurons in superficial
dorsal horn (laminae I–III) were sparsely and weakly labelled, while large neurons in ventral horn were frequently and densely labelled. Groups of
motoneurons in lamina IX that were immunoreactive to choline acetyltransferase also co-expressed CaV 1.3. The immmunoreactivity was mainly
associated with neuronal somata and proximal dendrites. Double staining with antibodies against CaV 1.3 and MAP2 (a dendritic marker) showed
that some fine fibres, which may include distal dendrites, were also labelled. These results in the cat spinal cord show some differences from studies
in mouse and turtle motoneurons where the immunoreactivity against this channel was mainly localized to the dendrites.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Lumbar spinal cord; Motoneuron; Persistent inward currents; Plateau potential; L-type calcium channel; Immunohistochemistry
Voltage-dependent persistent inward currents (PICs) make
important contributions to the intrinsic properties of spinal
motoneurons [7,11,22,23]. These PICs manifest themselves as
“plateau potentials” and may cause self-sustained firing following brief activation of the motoneurons. In a physiological
context these PICs are assumed to provide amplification of the
normal synaptic input to the motoneurons. Several investigations have demonstrated that nifidepine-sensitive L-type calcium
channels are responsible for a major part of the PICs in turtle
[9], mouse [3], and rat motoneurons [15]. Electrophysiological
evidence suggests that these PICs are located mainly at dendritic regions, a strategic position to amplify synaptic inputs
[2,3,5,6,10,12,25].
L-type calcium channels are encoded by CaV 1-genes and
include four different subtypes referred to as CaV 1.1–CaV 1.4
∗
Corresponding author. Tel.: +45 35 327590; fax: +45 35 327499.
E-mail address: [email protected] (M. Zhang).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2006.07.073
[16]. Only CaV 1.2 and CaV 1.3 are expressed in the central
nervous system, including the spinal cord [3,4,8,13,24,27]. Electrophysiological properties of the motoneuron PICs suggest that
they depend on CaV 1.3 channels [7]. Initial immunohistochemical studies in rats using antibodies against the CaV 1.3 channel protein (produced in Catterall’s laboratory) demonstrated
immunoreactivity mainly in the region of the somata and proximal dendrites of spinal motoneurons [27]. However, in recent
studies in mice [3,13] and turtles [24], the distal dendritic regions
of spinal motoneurons have shown strong immunoreactivity for
CaV 1.3 channels. Since many of the studies on the electrophysiological aspects of PICs in spinal motoneurons are performed in
the cat, and since there may be species differences in the neuronal
CaV 1.3 channel distribution, the aim of the present study was to
extend these immunohistochemical studies to cat motoneurons.
All experiments were conducted in accord with the guidelines of EU Directive 86/609/EEC and were approved by
the Danish Council for Animal Experiments. Four adult cats
(2.5–4.5 kg body weight) were used in this study. Three animals
M. Zhang et al. / Neuroscience Letters 407 (2006) 42–47
were anaesthetized with isoflurane (2.0–2.5%) and decerebrated
by either ligating the basilar and both common carotid arteries
(anaemic decerebration) or by a coronal knife lesion between
the cranial and caudal colliculi (intercollicular decerebration
with removal of the rostral part). Electrophysiological recording
was obtained from cervical or lumbar motoneurons for 10–20 h
in each of these animals (for detailed experiment protocol,
see [12]). Subsequently, the animals were perfusion fixed
for immunohistochemical analysis (see below). One cat was
used only for immunohistochemical analysis. Anaesthesia was
induced in this animal by isoflurane followed by Nembutal
(50 mg/kg body weight, i.p.). Perfusion fixation was performed
transcardially with 0.9% saline containing 7500 IU/L heparin
followed by 2 L 0.1 M cold phosphate buffered-saline (PBS)
containing 4% paraformaldehyde, 0.36% l-lysine and 0.05%
sodium m-periodate. The whole spinal cord was then removed
immediately and postfixed in the same fixative solution
overnight at 4 ◦ C. The next day, the spinal cord was dissected
into smaller segments and cryoprotected in PBS with 30%
sucrose up to 48 h at 4 ◦ C. Selected segments from the lumbar
spinal cord (L4–L7) were cut transversely into 40 ␮m-thick
sections using a sliding microtome.
Every fifth transverse section was processed for CaV 1.3
immunohistochemistry. Rabbit anti-CaV 1.3a polyclonal antibody (AM9742) was raised against a segment of C-terminus
of a long-spliced variant of rat neuronal L-type calcium channel
subunit CaV 1.3a, LDC5, and affinity purified on a Sepharoseconjugated LDC6 peptide (for details see [28]). The specificity
of this antibody was verified by Western Blot using cat spinal
tissues (data not shown).
Tissue sections were rinsed in 0.1M PBS for 10 min and
then incubated in 0.3% H2 O2 in PBS for 30 min. To minimize non-specific staining, the sections were preincubated in
PBS containing 0.3% Triton X-100 (PBS-T), 2% bovine serum
albumin (BSA) and 5% normal goat serum (NGS) for 1 h. The
sections were then incubated in the same solution containing
primary rabbit anti-CaV 1.3a antibody (1:500) for 2–3 days at
4 ◦ C. The sections were sequentially incubated with biotinylated
goat anti-rabbit IgG (1:500; Dako, Denmark) in PBS-T with 1%
BSA and 2% NGS and avidin–biotin complex (ABC, 1:100;
Vector Labs, Burlingame, CA) in PBS-T for 1 h each. Finally,
the sections were incubated for 5–10 min in 0.05 M Tris buffer
(pH 7.5) containing 0.04% diaminobenzidine tetrahydrochloride and 0.01% H2 O2 . Following this, they were dried, cleared
and coverslipped.
To identify motoneurons, neuronal dendrites and CaV 1.3
channels in the spinal cord, double-immunostaining was carried
out using goat anti-choline acetyltransferase (ChAT) polyclonal
antibody (1:100) or mouse anti-MAP2 (microtubule-associated
proteins) monoclonal antibody (1:500; both antibodies were
from Chemicon, Temecula, CA) and rabbit CaV 1.3a antibody.
CaV 1.3-immunolabelling was performed first, as described
above, up to the ABC incubation. After washing with PBS-T,
the sections were then incubated in biotinylated tyramide (1:500,
TSA indirect; NEN Life Science Products) in PBS containing
0.005% H2 O2 for 5 min followed by washing with PBS and
incubation in streptavidin-Alexa Fluor 488 (1:100) in PBS-T
43
for 1 h. The sections were then washed and incubated in goat
anti-ChAT or mouse anti-MAP2 antibodies in PBS-T with 2%
BSA and 5% normal donkey serum (NDS) or NGS overnight
at 4 ◦ C. Subsequently the sections were incubated in donkey
anti-goat Alexa Fluor 568 or goat anti-mouse Alexa Fluor 568
(1:100–200) in PBS-T with 1% BSA and 2% NDS or NGS
for 1 h. All fluorescent-labelled secondary antibodies were from
Molecular Probes (Eugene, OR). The sections were mounted
with Fluorescent Mounting Medium (Dako, Denmark).
Transverse sections adjacent to those immunostained for
CaV 1.3 were stained with thionine to reveal the general cytoarchitecture of the spinal cord. Control immunohistochemical
staining was done using the same procedures as described above
except that either the primary antibodies were omitted or the
primary antibodies were absorbed with the antigen sequence
against which the antibody was raised. No specific staining was
present in these control sections.
The sections were observed with a conventional light, epifluorescence microscope (Axioplan2, Zeiss, Germany) or a
laser scanning confocal microscope (Leica TCS SP2 system)
equipped with argon and helium-neon lasers. Images were captured digitally (AxioCam camera, Zeiss, Germany) and processed with Adobe Photoshop CS2 (version 9.0).
The CaV 1.3a staining pattern was the same for all four cats;
no differences were seen between the three cats that were perfused after electrophysiological studies and the cat that was
perfused directly. CaV 1.3a immunoreactive-like (CaV 1.3a-IR)
neurons were seen throughout the lumbar spinal grey matter
(Fig. 1A). No axon-like profiles were labelled in the grey matter. No dendrites, axons or glial cells were labelled in the white
matter except for those associated with an occasionally labelled
neuron.
The frequency and intensity of labelled neurons varied
depending on the Rexed lamina of the grey matter. The frequency increased gradually from the superficial dorsal horn to
the ventral horn (Fig. 1). When compared with adjacent Nissl
stained sections, only a small fraction of neurons in laminae I–III
were labelled (Fig. 1A–C). Whereas many neurons were labelled
in laminae IV–VI and X, nearly all neurons were labelled in
laminae VII–IX (Fig. 1A and B). The CaV 1.3a labelled neurons
may include projection neurons, interneurons and motoneurons.
Some neurons in lamina I and certain large neurons in laminae
IV–VI might be projection neurons (Fig. 1C and D). Most ChATnegative neurons in laminae I–VII are likely to be interneurons,
while ChAT-positive neurons in laminae VIII and IX are probably motoneurons (Fig. 2A1–A3). The intensity of CaV 1.3a-IR
also varied among neurons located in different laminae. The
large neurons in lamina IX, probably motoneurons, were the
most intensely labelled neurons. Some relatively large neurons
in the deep dorsal horn (laminae IV–VI) and in the region surrounding the central canal were also strongly labelled.
In the large (moto)neurons the immunoreactivity of the dendrites could be traced up to 100–200 ␮m along the proximal
dendrites. As the labelling pattern was punctate it was difficult to follow the dendrites distally. However, there were
indeed quite a few small CaV 1.3a-IR dots scattered within
motoneuronal area in lamina IX (Fig. 1E, 2B1, 3A1). Previous
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M. Zhang et al. / Neuroscience Letters 407 (2006) 42–47
Fig. 1. Photomicrographs of CaV 1.3a immunolabelling and Nissl staining in lumbar spinal cord. (A) Low power photomicrograph of CaV 1.3a reactivity in different
laminae (I–X) of spinal grey matter stained by the immunoperoxidase method. Labelling of neurons is sparse in the superficial dorsal horn, increases gradually
towards the ventral horn and becomes frequent and intense especially in lamina IX. (B) An adjacent section stained with thionine to compare the general neuron
distribution with neurons labelled by CaV 1.3a antibody in (A). Stars in (A) and (B) indicate the same blood vessel notch. (C–E) Enlargements of the parts in (A)
delimited by rectangles showing the detailed CaV 1.3a labelling pattern in the superficial dorsal horn (laminae I–III (C)), deep dorsal horn (laminae IV–VI (D)) and
lateral ventral horn (lamina IX (E)). Note that the labelling intensity is low for labelled neurons in the superficial dorsal horn (arrows in (C)), whereas it is high for
large neurons in the deep dorsal and lateral ventral horns. The initial parts of proximal dendrites can be seen clearly labelled in some neurons especially in lamina
IX. Scale bar in (A), valid for (A) and (B), 300 ␮m; in (C), valid for (C–E), 100 ␮m.
immunohistochemical studies on mouse and turtle motoneurons
have shown that CaV 1.3 channels are located primarily in dendrites (even distal dendrites) of the cells [3,13,24], so an important issue in this study was to establish whether this same pattern
of labelling exists in the cat. Double-immunostaining patterns of
CaV 1.3a and ChAT or MAP2 were therefore compared in large
motoneurons in lamina IX. As seen in Fig. 2B1–B3, CaV 1.3a
and ChAT double-labelling demonstrates that neurons in lamina IX labelled by ChAT were also positive for CaV 1.3a. ChAT
antibody not only labelled cell bodies and proximal dendrites
but also many fine fibres which may include distal dendrites.
However, very few fine fibres labelled by ChAT were CaV 1.3apositive. Nevertheless, when comparing CaV 1.3a with MAP2
staining some fine punctate structures were seen to be doublelabelled (Fig. 3A1–A3). While this indicates that some distal
dendrites may be CaV 1.3a-positive, most double-labelled punctate or patch structures were found in cell bodies and proximal
dendrites connecting to the somata (Fig. 3B1–B3). Exactly how
far distally the dendrites can be labelled by CaV 1.3a antibody is
still a pending question and needs further studies that combine
intracellular labelling of individual motoneurons with CaV 1.3
immunostaining.
M. Zhang et al. / Neuroscience Letters 407 (2006) 42–47
45
Fig. 2. Epifluorescent photomicrographs of lumbar spinal cord co-stained for CaV 1.3a and choline acetyltransferase (ChAT). (A1–A3) Low power photomicrographs
show that all the neurons positive for ChAT in the lateral ventral horn and a region close to the central canal were also CaV 1.3a-positive. Many neurons in the medial
and intermediate parts of the ventral horn, which are probably interneurons, were labelled by CaV 1.3a antibody but not ChAT. (B1–B3) Enlargements of the parts
demarcated in (A1–A3) show the detailed labelling pattern of individual motoneurons in the lateral part of the ventral horn. Cell bodies and proximal dendrites
(arrows) of motoneurons were clearly labelled by CaV 1.3a whereas most fine fibres, which most likely include distal dendrites were well stained by ChAT but not
by CaV 1.3a. Scale bar in (A1), valid for (A1–A3), 400 ␮m; in (B1), valid for (B1–B3), 100 ␮m.
As observed from CaV 1.3a and MAP2 double-staining,
CaV 1.3a-IR products were mainly found in neuronal cell bodies
and dendrites, localized to the cell membrane and the cytoplasm, but not in the nuclei (Fig. 1C–E, 2B1, 3A1 and B1).
In cell bodies, CaV 1.3a-IR products were relatively evenly distributed throughout the cytoplasm, while in dendrites labelling
was punctate and mainly associated with the cell membrane
(Fig. 3B1–B3). Whether there is CaV 1.3a labelling on the membrane of the cell body needs to be studied further by electron
microscopy.
The present study provides the first morphological evidence
for the presence of CaV 1.3 channels in the cat spinal cord.
These CaV 1.3 channels are common in ventral horn motoneurons, but sparse in the superficial dorsal horn. In the ventral horn
motoneurons, the CaV 1.3-IR products were primarily located in
cell somata and proximal dendrites, results that are consistent
with previous studies on rat spinal cord [27]. Similar subcellular
localization occurs in some brain regions such as the “dorsal
cerebral cortex”, hippocampus and cerebellum [8]. The results
are, however, to a certain extent in contrast to the existing data
from mice and turtles, which have shown that CaV 1.3 channels
are mainly located on the dendrites of the spinal motoneurons
[3,13,24].
There may be several reasons for these differences. First,
there are some differences in experimental procedures used
in the previous and present studies [13,24]. Although the
present study employed both conventional ABC and fluorescent immunohistochemical procedures to avoid inconsistency
induced by different staining techniques, the sensitivity of different methods may vary. Second, the CaV 1.3 antibody used here
is different from those antibodies used by others. The antibody
we used was raised against a peptide sequence of the C-terminus
from the long-spliced variant of rat CaV 1.3 (LDC5). Although
the cat CaV 1.3 channel has not been cloned, it has been shown
that the sequence of this segment is the same for rats, humans and
chickens and thus seems to be well conserved [28]. Most investigations on rat, mouse and turtle use CaV 1.3 antibodies produced
against a sequence between domain II and III which may recognize both short and long splice variants of CaV 1.3 [3,24,27]. In
pilot studies we tried antibodies of this type from different commercial sources (e.g., Alomone, Chemicon, Sigma), but were
not able to achieve distinct specific labelling in cat spinal tissue.
There is a possibility that different CaV 1.3 splice variants are differentially targeted within the dendritic tree of the motoneurons
with the long-spliced CaV 1.3 subunit restricted to the proximal
part and the short-spliced CaV 1.3 subunits to the distal part. As
antibodies specific for short-splice variants are not available, we
do not know much about the subcellular localization of these
variants. However, it has been demonstrated that the same antibody as in the present study was able to label very distal dendrites
46
M. Zhang et al. / Neuroscience Letters 407 (2006) 42–47
Fig. 3. Confocal fluorescent photomicrographs from lateral part of the ventral horn co-stained with CaV 1.3a and MAP2. (A1–A3) show that CaV 1.3a-labelled profiles
are well co-localized with MAP2 labelling. In addition to the large patchy structures which are connected to or in close vicinity of the cell bodies (large arrows in
(A1) and (A3)), some fine punctate structures (small arrows in (A1) and (A3)) also show co-localization. Arrowheads in (A1–A3) indicate the same neuronal cell
bodies. (B1–B3) show subcellular distribution of CaV 1.3a labelling of a large neuron from the lateral ventral horn scanned through its nucleus (n). From the merged
image (B3) it is apparent that the CaV 1.3a labelling is mainly located in the cytoplasm of the cell soma but associated with the surface of the proximal dendrites
(arrows). Scale bar in (A1), valid for (A1–A3), 150 ␮m; in (B1), valid for (B1–B3), 50 ␮m.
in rat hippocampal and striatal neurons and that mRNA levels for
the long CaV 1.3 C-terminal splice variant was ∼50-fold higher
than for the short CaV 1.3 variant in rat hippocampal neurons
[18,28]. Whether the same relative molecular levels of the two
CaV 1.3 variants apply to spinal motoneurons is unknown. Third,
there may be species differences in the distribution of CaV 1.3
channels in motoneurons, with mainly dendritic localizations
in turtle, distal dendritic localizations in mouse and primarily
somatic and proximal dendritic localizations in cat. These apparent differences have to be resolved by intracellular labelling and
subsequent CaV 1.3 immunohistochemistry using the same antibody for motoneurons from the different species.
Recent simulation studies using a realistic motoneuron model
could reproduce and predict experimental electrophysiological
results on PICs [5,6]. However, the “perfect” match with the
experimental results was only seen in the model when the PICs
were located in a wide intermediate band 300–850 ␮m from
the soma. This conclusion is thus in contrast to the immunohistochemical results on the distribution of CaV 1.3 channels,
which either place them more distally (turtle and mouse) or
more proximally (rat and cat). There are no electrophysiological results or simulations that would deny the existence of PICs
originating from all parts of the soma-dendritic membrane; the
conclusions only refer to preferential distribution. Thus, future
immunohistochemical studies must be quantitative, rather than
qualitative.
The generation of PICs not only depends on the somadendritic location of the related channels but also is regulated
by a number of neuromodulators [7]. Although CaV 1.3 channels mediate the main portion of the PICs their activity is
contingent on facilitation by monoamines from nerve fibres
descending from the brainstem, including serotonin (5HT) and
noradrenaline (NA) [14,19], which act via a variety of different
receptor subtypes. So far 5TH2 receptors (possibly 2A or 2C) and
NA ␣1 receptors appear to be very important [14,19,20]. Studies
on cat lumbar ␣-motoneurons have shown that 5HT terminals
mainly make contact onto neuronal cell bodies and dendrites
within a distance of 1000 ␮m from the soma [1]. At the same
time, it has been shown that in rat spinal cord 5HT2A receptors
are mainly located on somata and their proximal dendrites [17].
Furthermore, in vitro studies on dissociated deep dorsal horn
neurons from the rat containing only somata and proximal dendrites showed that they have the ability to produce plateau potentials [26], implying that intact distal dendrites are not necessary
for producing plateau potentials. Thus, this study suggests that
CaV 1.3 channels in motoneuron soma and proximal dendrites,
M. Zhang et al. / Neuroscience Letters 407 (2006) 42–47
especially the latter where CaV 1.3 immunoreactivity has been
demonstrated to locate on their surface, are more important than
distal dendrites for mediating PICs. It may also suggest that the
high densities of CaV 1.3 channels on the cell soma and proximal
dendrites serve to mediate Ca2+ entry in response to summed
excitatory input to initiate intracellular regulatory events. The
comparison of the distribution of PICs on one hand and CaV 1.3
channels on the other is further complicated by the fact that a
significant proportion of PICs are mediated by sodium channels
[15,21]. The spatial distribution of these sodium channels on
motoneuron soma and dendrites is still unknown.
Acknowledgements
We are grateful to Lillian Grøndahl for her unfailing assistance in all phases of this investigation. We thank Drs Christine
K. Thomas and Kirsten Thomsen for critical reading of the
manuscript. This project was supported by the Ludvig & Sara
Elsass’ Foundation, the Danish MRC to H.H and NINDS grant
to I.B.
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