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The Journal of Neuroscience, March 15, 1999, 19(6):1895-1911
Differential Distribution of Three Members of a Gene Family
Encoding Low Voltage-Activated (T-Type) Calcium Channels
Edmund M.
Talley1,
Leanne L.
Cribbs2,
Jung-Ha
Lee2,
Asif
Daud2,
Edward
Perez-Reyes2, and
Douglas A.
Bayliss1
1 Department of Pharmacology, University of Virginia,
Charlottesville, Virginia 22908, and 2 Department of
Physiology, Loyola University Medical Center, Maywood, Illinois 60153
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ABSTRACT |
Low voltage-activated (T-type) calcium currents are observed in
many central and peripheral neurons and display distinct physiological and functional properties. Using in situ hybridization,
we have localized central and peripheral nervous system expression of three transcripts (
1G, 1H, and 1I) of the T-type calcium
channel family (CaVT). Each mRNA demonstrated a unique
distribution, and expression of the three genes was largely
complementary. We found high levels of expression of these transcripts
in regions associated with prominent T-type currents, including
inferior olivary and thalamic relay neurons (which expressed 1G),
sensory ganglia, pituitary, and dentate gyrus granule neurons (1H),
and thalamic reticular neurons (1I and 1H). Other regions of high
expression included the Purkinje cell layer of the cerebellum, the bed
nucleus of the stria terminalis, the claustrum (1G), the olfactory
tubercles (1H and 1I), and the subthalamic nucleus (1I and
1G). Some neurons expressed high levels of all three genes,
including hippocampal pyramidal neurons and olfactory granule cells.
Many brain regions showed a predominance of labeling for 1G,
including the amygdala, cerebral cortex, rostral hypothalamus,
brainstem, and spinal cord. Exceptions included the basal ganglia,
which showed more prominent labeling for 1H and 1I, and the
olfactory bulb, the hippocampus, and the caudal hypothalamus, which
showed more even levels of all three transcripts. Our results are
consistent with the hypothesis that differential gene expression
underlies pharmacological and physiological heterogeneity observed in
neuronal T-type calcium currents, and they provide a molecular basis
for the study of T-type channels in particular neurons.
Key words:
in situ hybridization; calcium channel; CNS; anticonvulsant; rat; T-type calcium channels
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INTRODUCTION |
Voltage-dependent calcium channels
play a dual role in the CNS; they couple electrical activity to
calcium influx, thereby triggering myriad intracellular biochemical
events, and they contribute to membrane properties that determine the
precise nature of excitability in different cell types. Low
voltage-activated (LVA) calcium channels have a hand in both of these
roles that is readily distinguishable from their high voltage-activated
(HVA) counterparts, in part because they activate at potentials near
the resting membrane potential. First, in addition to participating in
spike-induced calcium entry (McCobb and Beam, 1991
; Scroggs and Fox,
1992b; Umemiya and Berger, 1994), they allow calcium influx at
potentials below threshold. This influx can occur when cells are at
rest (Magee et al., 1996) or in response to subthreshold synaptic
inputs (Miyakawa et al., 1992; Markram and Sakmann, 1994; Magee et al., 1995). Second, LVA calcium channels can be a crucial component in
shaping subthreshold membrane fluctuations and thereby contribute to
such behaviors as rebound burst firing (Llinas and Yarom, 1981), rhythmic oscillation (Gutnick and Yarom, 1989; Bal and McCormick, 1993), and resonance (Hutcheon et al., 1994; Puil et al., 1994). Given
their proposed role in oscillatory behavior, it is perhaps not
surprising that LVA calcium channel dysfunction is implicated in
epileptiform activity (Tsakiridou et al., 1995) and that these channels
are targets for antiepileptic drugs (Coulter et al., 1989b).
Information regarding the structure of calcium channels has been
determined primarily from the cloning of genes that encode the subunits
forming HVA calcium channels. The central core of these channels
consists of an 1 subunit that has four internal repeats, each repeat
consisting of six membrane-spanning regions and a pore-forming loop
(for review, see Catterall, 1995; Perez-Reyes and Schneider, 1995).
Currently, at least six 1 subunits have been identified that are
believed to generate the physiologically and pharmacologically defined
HVA calcium channel subtypes (generally designated L, N, P, Q, and R).
Although one of these subunits (1E) expresses currents that are
transient and whose voltage-dependent properties suggest that it may
encode an LVA channel (Soong et al., 1993), the currents produced by
this gene do not possess all of the properties that are common for
"T-type" LVA calcium channels (e.g., Randall and Tsien, 1997).
T-type properties in neurons include low voltage activation, strongly
voltage-dependent kinetics, rapid inactivation, slow deactivation, and
small single-channel conductance (for review, see Huguenard, 1996).
Recently, a subfamily of genes (designated CaVT) has been
discovered encoding 1 subunits that are ~30% homologous to HVA subunit genes in their putative membrane-spanning regions (Cribbs et
al., 1998; Perez-Reyes et al., 1998a,b). When expressed heterologously, two of these proteins, 1G and 1H, show all the properties
hallmark of neuronal T-type calcium channels. A third member of this
family, designated 1I, also encodes a calcium channel that displays
a number of T-type properties (Lee et el., 1999).
Given that expression of T-type channels has a unique impact on
neuronal properties, it is of great interest to know which neuronal
populations express these subunits. Furthermore, T-type channels are
pharmacologically and physiologically heterogeneous (Akaike et al.,
1991; Huguenard, 1996; Tarasenko et al., 1997). This heterogeneity may
reflect different functions of these channels in neurons, and may stem
at least in part from differential expression of each of these three
genes. Therefore, using in situ hybridization histochemistry, we have localized the regional and cellular
distribution of gene expression for the three known members of the
CaVT family in the rat central and peripheral nervous
systems. We find that each gene has a unique expression pattern and
that the location of these three channel types is to a large extent complementary.
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MATERIALS AND METHODS |
Tissue preparation. Male Sprague Dawley rats
(250-350 gm; Hilltop) were anesthetized with ketamine-xylazine and
decapitated. Brains, spinal cords, and ganglia were removed and frozen
on dry ice. Sections (10 µm) were thaw-mounted onto charged slides
(Superfrost Plus; Fisher Scientific, Houston, TX) and stored at
80°C for later use. In preliminary experiments, sagittal and
horizontal sections were used from six animals. For detailed
comparative analysis, coronal sections from three animals were taken
from the entire brain (~200-500 µm apart). In addition,
representative sections from three or four animals were taken from
nodose, superior cervical, and dorsal root ganglia, as well as from
cervical, thoracic, and lumbar spinal cord. Slides were pretreated for
hybridization as described previously (Talley et al., 1997). Sections
were fixed in 4% paraformaldehyde (5 min) and rinsed extensively with
PBS, pH 7.4. They were treated with glycine (0.2% in PBS; 5 min) and acetic anhydride (0.25% in 0.1 M triethanolamine,
0.9% saline, pH 8; 10 min) and subsequently dehydrated in a graded
series of ethanols and chloroform. Hybridization was performed
overnight at 37°C in a buffer of 50% formamide, 4× SSC (1× SSC:
150 mM NaCl and 15 mM sodium citrate, pH 7),
1× Denhardt's solution (0.02% each of Ficoll, polyvinylpyrrolidone,
and bovine serum albumin), 10% dextran sulfate, 100 mM
DTT, 250 µg/ml yeast tRNA, and 0.5 mg/ml salmon testes DNA. After
hybridization, slides were washed through four changes of 1× SSC at
55°C (15 min each) and once for an hour in 1× SSC at room temperature.
Oligonucleotide probes. Sequence analysis of 1G, 1H,
and 1I has revealed a high degree of homology in the putative
transmembrane regions of these three genes (Perez-Reyes et al., 1998b).
In contrast, there is particularly low conservation in the sequences
that connect each of the four repeating domains. Therefore, antisense
oligonucleotide probes (33 bases in length) were designed to hybridize
to the cytoplasmic I-II "linker" region of the rat homolog of each
gene (L. L. Cribbs, J.-H. Lee, and E. Perez-Reyes, unpublished
data). Sequences were chosen that had minimal homology both to other calcium channel genes and to other sequences present in GenBank. Probes
were labeled using terminal deoxyribonucleotidyl transferase (Life
Technologies, Gaithersburg, MD); unincorporated nucleotides were
removed using Sephadex G-50 spin columns (Pharmacia, Piscataway, NJ).
Multiple oligonucleotides were used to probe each gene (three
oligonucleotides for 1G, three for 1H, and two for 1I).
Subsequent to preliminary experiments characterizing the probes, the
oligonucleotides corresponding to each gene were hybridized in a
cocktail. We found that using the probes in combination resulted in an
enhanced signal but had no effect on relative distribution of signal
intensities in different brain regions (see below). The concentration
of each oligonucleotide was ~30 × 106 cpm/ml
(~2 nM). The sequences of the probes were as
follows: 1G: 5'-CCAGCCCGCACGCCTATAGCCCTAGAGACCTGG-3',
5'-TCCGATGGGCATCTGGGAGGGGGTGCCTGGCAA-3', and
5'-TGTCGCCGCCGGCTGTGGGGATCCCGGAGGTCA-3'; 1H:
5'-ATGCCGTACATCCTGGGTAAACTCATAGACTCC-3', 5'-AGCCCCTTGGGTCGTGAGCTGGTGCCACCTTTG-3', and
5'-ATCCCTCCTGGTTGTGAGGCCTTCCGCAGTGGT-3'; 1I:
5'-AGCCACCAGAACCTGAGCCTTCCTGGCCTGAGT-3' and
5'-TCGCGCCACACATCCCCACACAGTCGGGCTGCC-3'.
Control experiments. We performed a number of preliminary
control experiments. First, we hybridized each oligonucleotide
separately to sagittal and horizontal sections. Cognate
oligonucleotides generated an identical tissue distribution, indicating
that these independent probes recognized the same gene product and
ruling out the possibility of spurious cross-reactivity. Higher wash temperatures (60 and 65°C in 1× SSC) resulted in diminished
hybridization, but for each oligonucleotide the distribution of
labeling was unchanged, indicating that the same binding site was
labeled in different brain areas. For each set of probes, specific
binding was eliminated by prior digestion with RNase A (50 µg/ml;
Boehringer Mannheim, Indianapolis, IN). Nonspecific binding was
assessed in competition experiments (examples of which are shown in
Fig. 3), with ~500-fold excess unlabeled oligonucleotide (1 µM) included in the hybridization mixture. In these
experiments, nonspecific binding to tissue was barely distinguishable
from the overall background, demonstrating that specific binding was
saturable and that nonspecific binding was minimal.
Data analysis and presentation. Slides were exposed to film
(Hyperfilm 
-MAX; Amersham, Arlington Heights, IL) for 1 week to
generate autoradiograms (Fig.
1),
which were analyzed with the aid of image analysis software
(MCID; Imaging Research) to determine the relative intensity of
labeling in different brain regions. For resolution of cellular
labeling, slides also were dipped in liquid autoradiographic emulsion
(NTB2; Eastman Kodak, Rochester, NY), exposed for 5-9 weeks, and
examined by dark-field and bright-field microscopy. Images of silver
grains from these slides (see Figs. 2-7) were captured using a Pixera
video camera mounted on a Leitz Diaplan microscope. In addition,
high-power bright-field micrographs (such as those shown in Figs. 4, 6,
7) were made of labeled cells from different brain regions for
side-by-side comparison of the relative numbers of silver grains
overlaying various cell types.
By combining densitometry of film autoradiograms with information on
the specific cellular localization of hybridization, we determined the
comparative distribution of each of the three transcripts. The results
of this analysis are presented in Table 1
as a system of pluses, with five pluses (+++++) representing the
highest levels of expression. It is important to understand that
because a number of factors other than transcript levels (particularly
the hybridization efficiency of individual probes) can affect signal
intensity, this scoring system reflects relative amounts of individual
transcripts in different brain regions, rather than comparisons among
the three different CaVT transcripts. However, the fact
that the various probes to each gene (when hybridized individually)
generated similar signal intensities suggests that the influence of
factors such as differences in hybridization efficiency were minor.
Therefore, relative levels of the three different mRNA species may be
compared, so long as such comparison is viewed with caution. In this
regard, it is also important to note that only two probes were used to
detect 1I (as opposed to three each for 1G and 1H). As stated
above, we found that combining oligonucleotides in a cocktail had no
effect on relative distribution but resulted in enhanced signal
intensity. Thus, because one fewer probe was used for 1I, transcript
levels for this gene may have been somewhat under-represented relative
to those of the other two genes.
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RESULTS |
Overview
We performed in situ hybridization to determine the
regional distribution of CaVT (1G, 1H, and 1I)
gene expression in the central and peripheral nervous systems. Figure 1
shows representative film autoradiograms of transverse sections that
were taken through the entire brain. Figures
2-7
show representative dark-field and bright-field images of silver grains
from emulsion-dipped slides. The results of combined analysis from film
autoradiograms and emulsion-dipped slides are summarized in Table
1.
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Figure 2.
Dark-field micrographs demonstrating cellular
labeling of olfactory structures. Slides were exposed to
autoradiographic emulsion; silver grains were imaged using dark-field
microscopy. Left panels show silver grains over cells of
the main olfactory bulb. Note that whereas all three transcripts were
present in the granule cell layer (asterisks), labeling
of small neurons in the glomerular layer (arrowheads)
was limited to 1G and 1I. Right panels show the
olfactory tubercles. Labeling for 1H and 1I was very high in the
cell islands of Calleja (open arrows); labeling was also
high for 1H but only moderate for 1I in the dense cell layer of
the tubercles (closed arrows). Scale bar, 500 µm.
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Figure 3.
Dark-field micrographs of horizontal sections
through the ventral portion of the hippocampus. Left
panels show cross sections in the horizontal plane through the
ventral hippocampus. Note that whereas all three transcripts were
present, each had a different relative distribution through the various
hippocampal fields. Fields CA1 and CA3 are indicated, as are the
dentate gyrus (DG), the subiculum
(S), and the ventral lateral geniculate nucleus
(VLG) of the thalamus. Open arrow points
to 1G-labeled (presumably nonpyramidal) cells in stratum radiatum.
The dentate gyrus is shown at higher magnification in the middle
panels. Note that the granule cell layer (Gr,
arrowheads in all three sets of panels) shows
particularly strong labeling for 1H, whereas cells of the polymorph
layer (Po) show more even levels of the three
transcripts. Right panels show images of the dentate
gyrus from control sections hybridized in the presence of ~500-fold
excess cold oligonucleotide. Note that this nonspecific labeling was
uniformly low. Scale bar, 400 µm (left panels); 100 µm (middle and right panels).
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Figure 4.
Differential hybridization to neurons in the
cerebral cortex. Distribution of 1G, 1H, and 1I mRNA in the
primary somatosensory cortex is shown at increasing levels of
magnification. Top panels demonstrate laminar
distribution of labeling; the relevant cortical layers (layers II-VI)
are indicated to the right of these panels, as are the
external capsule (ec) and the striatum
(CPu). Arrowheads indicate large
(presumably pyramidal) neurons in layer V. The same neurons are
depicted at higher magnification in the middle panels
(using dark-field optics) and at still higher magnification in
bottom panels (using bright-field optics). Note that
1G and 1I were found in all cortical layers, but expression of
1H was for the most part restricted to layer V. Scale bar, 400 µm
(top panels); 100 µm (middle panels);
25 µm (bottom panels).
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Figure 5.
Distribution of CaVT expression in the
thalamus. Left panels show differential labeling of the
thalamic reticular nucleus (Rt) and ventral posterior
thalamic nucleus (VP). Note that neurons of the
reticular nucleus contained 1H and 1I mRNA, whereas 1G
expression was limited to thalamic relay nuclei, including VP.
Right panels show labeling in the habenulae and midline
thalamic nuclei. Neurons of the lateral habenular nucleus
(LHb) expressed 1G and 1I mRNA (see Results for
details). DG, Dentate gyrus; PV,
paraventricular thalamic nucleus. Scale bar, 250 µm.
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Figure 6.
Parasagittal sections demonstrating labeling of
the cerebellum and inferior olivary nucleus. Left panels
show bright-field images of film autoradiograms exposed to sagittal
sections through the brainstem and cerebellum. Lobules 9 and 1 of the
cerebellar vermis are indicated and correspond to higher magnification
dark-field micrographs of emulsion-dipped sections shown on the
right. Note that for 1G, the granule cell layer
(Gr) of the cerebellum displayed a rostrocaudal gradient
of expression, with lobule 9 labeled intensely and lobule 1 showing
very low levels. In contrast, expression of 1I in the granule cell
layer was fairly uniform throughout the cerebellum and showed similar
levels in both lobules. Probes for 1G labeled Purkinje neurons
(P) at extremely high levels. One of these
neurons (arrowhead) is depicted at higher power using
bright-field optics (inset). The inferior olivary
nucleus (InO) also is indicated in the autoradiograms
and corresponds to dark-field images in the bottom
panels. Labeling of this structure also was heterogeneous:
1G was uniformly high; labeling for 1I was limited to the caudal
part of the nucleus. Scale bar, ~4.8 mm (left panels);
250 µm (right panels); 50 µm (inset);
400 µm (bottom panels).
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Figure 7.
Differential accumulation of CaVT
transcripts in the spinal cord and sensory ganglia. Left
panels show low-power dark-field images of transverse sections
through the lumbar spinal cord. All three transcripts were present in
the dorsal horn (asterisks), with 1H mRNA limited to
neurons of the external lamina. Note also that 1G and 1H were
expressed in motor neurons in the ventral horn (open
arrows). Middle panels show high-power
bright-field images of dorsal root ganglia (DRG)
neurons. Probes for 1H and 1I labeled small- and medium-sized
neurons (arrowheads); in contrast, large neurons
(asterisks) were unlabeled. In the nodose ganglia
(right panels) expression was for the most part limited
to 1H (arrowhead). Scale bar, 400 µm (left
panels); 25 µm (middle and right
panels).
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The distribution of the three transcripts was to a great extent
complementary, and the expression pattern of each gene was unique. Only
a few regions, including the granule cell layer of the olfactory bulb
(Figs. 1A, 2), fields CA1 and CA3 of the hippocampus (Figs. 1G-K, 3), and the tenia tecta (TT; Fig.
1B), displayed expression of all three transcripts in
abundance. Cells with the highest 1G mRNA levels included
cerebellar Purkinje cells (Fig. 6), thalamic relay neurons (Fig. 5),
inferior olivary cells (InO; Fig. 6), and neurons of the bed nucleus of
the stria terminalis (BST; Fig. 1E). Labeling for
1H was highest in the olfactory tubercle (Tu; Figs.
1C,D, 2), granule cells of the dentate gyrus (Figs. 1G-K, 3), and in sensory ganglia (Fig.
7). Labeling for 1I was highest in the olfactory bulb (Figs.
1A, 2), the cell islands of Calleja (ICj; Figs.
1C, 2), and fields CA1 and CA3 of the hippocampus (Figs.
1G-K, 3).
Olfactory system
All three probes gave prominent labeling in the
olfactory bulb (MOB; Figs. 1A, 2). As noted above,
the granule cell layer of this structure was one of a limited number of
brain regions with high expression of all three channel subtypes. In
contrast, the mitral cell layer was not noticeably labeled, and where
mitral cells were identified by Nissl stain, silver grains were not
detected above background (data not shown). Small neurons in the
glomerular layer contained moderate levels of 1G, whereas 1I was
present at very high levels in scattered members of this cell
population (Fig. 2).
Moderate to high levels of message were also found for all three genes
in olfactory cortical structures, including the anterior olfactory
nucleus (AO; Fig. 1A) and piriform cortex (Pir; Fig. 1B-I; see below). The olfactory tubercle,
by contrast, had an expression pattern that was more reminiscent of the
striatum (see below) insofar as it contained little expression of 1G
mRNA (Figs. 1B-D, 2). Instead, it
contained very high levels of 1H in the dense cellular layer as well
as very high levels of 1H and 1I in the cellular islands of Calleja.
Basal forebrain
1H and 1I transcripts were both present throughout
the striatum (CPu; Figs. 1C-I, 4) and the
accumbens nucleus (Acb; Fig. 1B,C).
1H was detected at moderate levels in these structures, whereas
1I distribution was mixed: it was found at barely detectable levels
in the accumbens and the medial striatum, but it displayed moderate
levels at the lateral and caudal edges of the striatum. 1G mRNA was
generally undetectable in these structures, although scattered cells
were labeled: these were more evident medially at the edge of the
globus pallidus (GP), a nucleus that was not marked by expression of
any of the transcripts (Fig.
1E-H). Another basal ganglia
structure, the subthalamic nucleus (STh; Fig. 1I) contained high levels of 1I, moderate levels of 1G, and only scattered expression of 1H.
In the septum (Fig. 1C-E), labeling was moderate
for 1G in the dorsal and ventral part of the lateral septum (LSD,
LSV) and low in the intermediate part (LSI) and in the medial septum
(MS). 1H labeling was found at low levels in all
parts of the lateral and medial septum and absent in the triangular
septum (TS). Labeling for 1I was opposite to that of 1H; it was
not detected in the lateral and medial septum, but it was present in
low amounts in the triangular septum. This region is traversed by a
cortical structure, the indusium griseum (IG; Fig. 1C),
which had very high levels of 1H as well as low levels of 1G.
The BST (Fig. 1D,E)
displayed expression for all three genes, but only in the medial edge
of the posterior region of the nucleus. In this restricted area, 1G
expression was quite high, 1H was moderate, and 1I was low. In
the rest of the nucleus, levels of 1G and 1H expression were low,
and 1I expression was not detected. Another basal forebrain
structure, the claustrum (Cl; Fig.
1B-F), also had prominent 1G
expression along with low levels of 1I.
Amygdala
In the amygdala, 1G and 1H were predominant, with 1I mRNA
expressed at low levels in most nuclei (Fig.
1G-K). Highest levels of all three genes
were found in the nucleus of the lateral olfactory tract (LOT; Fig.
1F) and the related bed nucleus of the accessory olfactory tract (BAOT; Fig. 1G). In the central nucleus (Ce;
Fig. 1G,H) and the lateral nucleus (La;
Fig. 1G-I), 1H expression was
uniformly low, but 1G expression was varied; it was higher in medial
portions of the central nucleus and in lateral portions of the lateral
nucleus. In the medial amygdala, by contrast, expression for all three
genes was mixed; it was higher in the posterior part (MeP; Fig.
1H,I) than in the anterior part (MeA; Fig.
1G). Expression for 1G and 1H was most evident in the
caudal tip of this nucleus, as shown in Figure
1I.
Hippocampal formation
In many CNS regions, the hybridization levels of the 1G probes
tended to be higher than those of the other two sets of probes. Such
was not the case for the hippocampal formation (Fig.
1G-L, 3), where labeling intensities were more
equivalent between the three sets of probes. Pyramidal cells in the
hippocampus had moderate to high levels of all three transcripts,
whereas CaVT expression in the nonpyramidal cell layers of
Ammon's Horn was, for the most part, restricted to 1G. In the
granule cell layer of the dentate gyrus, 1H expression was
predominant and extremely high. In contrast, dentate gyrus polymorph
cells had labeling that was more closely matched between the three sets
of probes.
Cerebral cortex
All three transcripts were present in the cerebral cortex, as
shown in Figures 1A-L and 4, 1G and
1I were present fairly uniformly in all cell layers, the major
exception being layer IV, where both transcripts were detectable in
more cells. In addition, in the ventral part of the cortex just dorsal
to the claustrum, a band of neurons that was intensely labeled with the
probes for 1G was present in the deepest part of layer VI. These
intensely labeled cells appeared to be restricted to the
insular/perirhinal cortices (Ins, Fig.
1B-G; PRh, Fig.
1H-K) and secondary somatosensory areas (e.g., Fig. 1E). With respect to particular
cell types, probes for 1G and 1I labeled both large (presumably
pyramidal) and small (presumably granule) neurons. However, this
labeling was not uniform, and in some cells expression was not
detected. Thus, no clear preference emerged for expression of either
one of these transcripts by specific types of cortical neurons.
Such a general distribution in the neocortex was not the case for
1H. It was seen at very high levels in a subset of layer V pyramidal
neurons; otherwise it was only present at very low levels in the
externalmost cellular layer (layer II) and was not detected in layers
IV and VI. The 1H-expressing pyramidal neurons were predominantly found in the deeper part of layer V, although not
every large neuron at this level was labeled.
Two rostral cortical structures expressed all three transcripts at high
levels. One was the TT (Fig. 1B). The other
structure, the Pir (Fig. 1B-I),
had mixed expression levels of all three genes. Both 1H and 1I
were present in high amounts in the superficial neurons of this
cortical region but were not detected in deeper layers. On the other
hand, 1G mRNA was expressed in moderate amounts in the external
layer but at much higher levels in the deeper layers, especially the
dorsal endopiriform nucleus (DEn; Fig.
1C-J).
Thalamus
The thalamus (Figs. 1E-K, 5) was
characterized by high levels of expression of 1G in thalamocortical
relay nuclei. In many of these nuclei, including the medial dorsal
(MD), the ventral anterior (VA), the ventral and posterior relay nuclei
(Po, VM, and VP), and the medial and dorsal lateral geniculate nuclei
(MG and DLG), detectable expression was limited to 1G. Low levels of
1I were seen in the anterior dorsal nucleus (AD; Fig.
1F) and the rostral part of the lateral dorsal
nucleus (LD; Fig. 1G); low to moderate levels were seen in
the ventral lateral geniculate nucleus (VLG; Fig.
1J). Expression of 1H in relay nuclei was limited
to some of the large neurons of AD that expressed this transcript at
low levels. The intralaminar nuclei, including the central medial
nucleus (CM; Fig. 1G,H), paraventricular
thalamic nucleus (PV; Fig. 1F-I),
the rhomboid (Rh; Fig. 1G), and reuniens (Re; Fig.
1G,H) nuclei had even higher levels of
1G expression. Low levels of 1I expression also characterized a
number of these nuclei (CM, PV, and Rh).
In contrast to the thalamic relay and intralaminar nuclei, the thalamic
reticular nucleus, which is composed of GABAergic interneurons that
modulate and synchronize thalamic output (for review, see Steriade et
al., 1993), had no detectable 1G expression (Fig. 5, left
panels). Instead, these neurons expressed high levels of
1I and moderate levels of 1H. The lateral habenula (Fig. 5,
right panels) had an uneven distribution of 1G and
1I; both transcripts were detected at high levels in the rostral
portion of the nucleus. Caudally, 1I expression was moderate, and
1G expression was low (Fig. 1, compare H,
I). CaVT expression was not detected in
neurons of the medial habenula, a region characterized by high levels
of mRNA encoding 1E (Soong et al., 1993; Williams et al., 1994).
Hypothalamus
In the hypothalamus, 1G was predominant rostrally. In the
preoptic region (Fig. 1D-F), 1G
mRNA levels were higher in medial structures such as the medial
preoptic nucleus (MPO) than in their lateral counterparts (e.g., LPO).
This medial bias also included the rostral portion of the
suprachiasmatic nucleus (SCh; Fig. 1E,F). More caudally,
expression levels were mixed. Expression of all three genes was found
in both the dorsomedial (DM) and the ventromedial (VMH) nuclei, with
high levels of 1G mRNA in the ventrolateral portion of the VMH (Fig.
1H,I). Expression patterns in the mammillary bodies were somewhat complex, with each transcript showing a unique expression pattern (Fig. 1J). In the
medial mammillary nucleus, the medial part (MM) showed a predominance
of 1H expression, whereas the lateral part (ML) displayed 1G and
1I. In the lateral mammillary nucleus (LM), 1G was abundant, with
the other two transcripts present only at low levels.
Midbrain and pons
All three transcripts were expressed in the tectum (SupC and IC;
Fig. 1K-M), with 1H and 1I
at low levels and 1G at low to moderate levels, except in the
external gray area of the superior colliculus, where 1G labeling was
high (Fig. 1K). All three transcripts also were found
in the pretectal nuclei (MPT and APT; Fig. 1J), which
were notable as a result of high 1H labeling in scattered cells of
the anterior pretectal nucleus (APT). The periaqueductal gray (PAG;
Fig. 1J-L) had moderate levels of
labeling for 1G and low levels of 1H. More caudally, the
tegmental nuclei ventral to the fourth ventricle (Fig.
1M,N) were a little more
heterogeneous, with 1G mRNA at high levels in the posterodorsal
tegmental nucleus (PDTg; Fig. 1N) and 1I present
in the laterodorsal nucleus (LDTg; Fig. 1M).
Labeling for 1G and 1H were found at low levels in the raphe
nuclei (Fig. 1K,L). These two
transcripts were also found in the substantia nigra (SN; Fig.
1J,K); in the compact part
1G labeling was moderate and 1H low, whereas the reticular part was for the most part unlabeled, except for some cells that contained 1H (data not shown). All three transcripts were found in the interpeduncular nucleus (IP; Fig. 1K) with 1G and
1H at moderate levels and 1I at low levels. The lateral
parabrachial nucleus (LPB; Fig.
1M,N) had low levels of
1G and 1H, except for the external portion, which had moderate to
high levels of 1G (Fig. 1M). One other area of
note was the pontine nuclei (not shown), which had low to moderate
levels of both 1G and 1I.
Cerebellum and inferior olive
Expression of CaVT transcripts in the cerebellum is
illustrated in Figures 1M-P and 6. 1G
expression was strikingly high in every Purkinje cell examined, and
granule cells were labeled with 1G probes in a rostral-to-caudal
gradient of increasing levels of expression (in both the hemispheres
and the vermis), as has been seen for a variety of cerebellar markers
(Herrup and Kuemerle, 1997). 1I mRNA also was present in the granule
cell layer at moderate levels, although it lacked the graded expression seen for 1G. The InO (Figs. 1O,P, 6) also
contained 1G and 1I mRNA; in this case 1G was expressed at
very high levels throughout the nucleus, whereas 1I was expressed at
moderate levels, but only in caudal olivary neurons.
Medulla and spinal cord
In the medulla and in the spinal cord, probes for 1G mRNA once
again generated a higher signal than did those for 1H and 1I.
Labeling for all three transcripts was sparse or absent in brainstem
reticular areas, including those of the midbrain and pons (Fig.
1L-N). In the medullary reticular
fields, 1G was present at low levels in the lateral but not the
medial areas (Fig. 1O, compare PCRt, Gi); 1I
was found in the lateral reticular nucleus (Fig.
1P).
All three transcripts were detected in sensory areas. In the spinal
trigeminal nucleus (Sp5), the three mRNAs were found at higher levels
caudally than rostrally (Fig. 1, compare O, P). All three also were seen in the dorsal cochlear nucleus (data not
shown), but only 1G was present (and at lower levels) in the ventral
cochlear nucleus (VC; Fig.
1M,N). Similarly, all three transcripts were present in the dorsal horn of the spinal cord (Fig.
7). 1G was present at moderate levels; 1H was for the most part
restricted to the outermost layers (layers 1-2). 1I was somewhat
more evenly distributed at low levels, but it was more prominent in
layers 3-4. Only 1G and 1H were detected in the nucleus of the
solitary tract (Sol; Fig. 1O,P).
Somatic motor neurons in the brainstem and spinal cord contained 1G
and 1H mRNA, at moderate and low levels, respectively (Figs.
1M,P, 7). Other labeled medullary
regions included the inferior olive (discussed above) and the area
postrema (AP; Fig. 1P); in the AP all three
transcripts were found, with 1G at high levels.
Other areas
In addition to surveying the CNS, we also hybridized the
CaVT probes to sections from the sensory and sympathetic
ganglia, specifically the nodose, dorsal root ganglia (DRG) and
superior cervical ganglia. High-power bright-field micrographs of
sensory neurons of the DRG and nodose ganglia are shown in Figure 7
(middle and left panels). In the DRG, high
levels of 1H and moderate levels of 1I mRNA were found in
scattered medium-sized neurons, whereas the extremely large neurons
were not labeled. The nodose ganglia also contained high levels of
1H mRNA in many neurons, although in contrast to the DRG, there did
not appear to be a bias in the size of the labeled neurons. Neurons of
the superior cervical ganglia (not shown) only expressed very low
levels of 1G. We also examined cells of the pituitary and pineal
glands. Both of these structures showed high levels of expression of
1H mRNA (data not shown).
| |
DISCUSSION |
We used in situ hybridization to show that
three members of a novel family of calcium channels with T-type
properties are expressed widely in the CNS and in peripheral neurons,
and that each of these three transcripts has a unique pattern of
distribution. Our results reveal that expression of these genes is
prominent in a number of brain areas where T-type currents have been
recorded and absent in specific regions believed to be devoid of these currents. Furthermore, our data are in accord with the hypothesis that
differential expression of specific CaVT subtypes may
account for at least some of the heterogeneity observed in CNS T-type calcium currents.
CaVT transcripts are expressed in cells with
prominent T-type currents
As anticipated, we detected robust expression of CaVT
mRNA in areas where prominent T-type calcium currents have been
observed. For example, we found expression of all three transcripts in
the thalamus, where the role of T-type calcium channels has been
studied extensively (for review, see Steriade et al., 1993). We found 1G mRNA to be predominant in thalamocortical relay areas but absent
in the thalamic reticular nucleus, which instead expressed high and
moderate levels of 1I and 1H mRNA, respectively. It is believed
that the differences in the characteristics of T-type currents in these
two cell types (discussed below) are important for the generation of
synchronized thalamocortical rhythms (McCormick and Bal, 1997).
Another region classically associated with prominent T-type calcium
current is the InO, where we found 1G mRNA in great abundance. LVA
currents in these neurons contribute to oscillations that support the
coordination of synchronous rhythmic firing (Manor et al., 1997). These
neurons make monosynaptic contacts with Purkinje cells of the
cerebellum, and their synchronous activity is believed to play a
prominent role in the organization of cerebellar output (Welsh et al.,
1995). It is worth pointing out that we found a heterogeneous
distribution of CaVT transcripts in the InO. Expression in
the rostral part of the nucleus was restricted to 1G, whereas caudal
expression consisted of both 1G and 1I (Figs.
1O,P, 6). It remains to be determined how
this differential distribution of CaVT expression in the
inferior olive might contribute to the functional organization of
olivocerebellar processing.
In addition to 1G and 1I, we also found abundant 1H mRNA in
regions commonly associated with prominent T-type calcium currents. For
example, we found high levels of this transcript in granule cells of
the dentate gyrus and in sensory ganglion neurons. In both of these
cell types, T-type channels have been shown to produce sizable spike
afterdepolarizing potentials (White et al., 1989; Zhang et al., 1993)
that in sensory neurons can trigger bursts of action potentials. It is
notable that in sensory neurons of the nodose ganglia, CaVT
gene expression has been directly implicated in the production of
T-type calcium current; transfection with an antisense oligonucleotide
targeting a sequence shared by the three CaVT genes
specifically and markedly diminished the LVA calcium current in those
cells (Lambert et al., 1998).
In sensory neurons of DRG, we saw high expression of 1H and moderate
levels of 1I mRNA. Expression of both transcripts was restricted to
small- and medium-sized neurons; CaVT transcripts were not
found in the extremely large DRG neurons. These data are in accord with
studies of calcium currents of DRG neurons acutely isolated from adult
rats, where large T-type currents were present in medium-diameter
neurons but were absent in large-diameter neurons (Scroggs and Fox,
1992a). Thus, our results support the view that T-type currents are
expressed specifically in smaller sensory neurons that convey thermal
and nociceptive information and not in larger neurons that subserve
proprioceptive and tactile pathways (Scroggs and Fox, 1992a).
In addition to finding abundant CaVT mRNA in areas where
prominent T-type calcium currents have been observed, there were a
number of regions where we saw little or no detectable CaVT expression and where other investigators have failed to find LVA calcium current. For example, we saw no CaVT expression in
the globus pallidus and saw only very low expression of one of the transcripts (1G) in sympathetic ganglia. In neurons of both of these
areas, T-type channels were not evident in whole-cell calcium current
recordings (Schofield and Ikeda, 1988; Plummer et al., 1989; Surmeier
et al., 1994).
Regions of inconsistency between CaVT expression and
recordings of T-type calcium currents
As noted above, we saw expression of CaVT transcripts
in regions that display prominent T-type currents and failed to detect these transcripts in regions where T-type currents are absent. However,
there were also a number of regions reportedly devoid of these currents
where CaVT genes were expressed (e.g., granule cells in the
cerebellum and cerebral cortex) and conversely, regions where T-type
currents have been recorded but where we found no evidence for
CaVT expression (e.g., olfactory mitral cells).
It is important to point out that comparative characterization of
T-type currents in neurons is problematic because a variety of
experimental factors can affect these recordings (discussed in
Huguenard, 1996). A particular concern for comparing T-type currents is
the converging evidence suggesting that a substantial fraction of these
channels are localized to relatively distal dendrites (Karst et al.,
1993; Markram and Sakmann, 1994; Magee and Johnston, 1995; Kavalali et
al., 1997; Mouginot et al., 1997). As a result of this subcellular
distribution, recordings from intact neurons are subject to voltage-
and space-clamp errors that can have a dramatic impact on the apparent
voltage- and time-dependent properties of T-type calcium currents
generated at distal sites (Destexhe et al., 1996, 1998). However, in
dissociated preparations where electrical recordings only sample
currents from the soma and proximal dendrites, the complement of
calcium channels of a cell may be misrepresented. Therefore, in
addition to some of the more obvious caveats to comparison (e.g.,
species or age differences in the animals being compared), it is
important to note that definitive identification and characterization
of T-type currents in neurons is difficult and may even be subject to
"false negatives."
One region where there were discrepancies between CaVT
expression and T-type current recordings was the cerebellum. For
example, Purkinje cells are most often associated with their prominent P-type HVA calcium currents (Mintz et al., 1992), and it has been suggested that T-type currents in these neurons, which are robust in
neonatal animals (Regan, 1991), might only be transiently expressed early during postnatal development (Usowicz et al., 1992, but see
Kaneda et al., 1990). However the data presented here, which show
extremely high levels of 1G mRNA in adult Purkinje cells (Fig. 6),
do not support this possibility. Moreover, our preliminary results
indicate that expression of 1G mRNA is present in the neonate and
actually increases postnatally (E. M. Talley and D. A. Bayliss, unpublished observations).
Similar to Purkinje neurons, cerebellar granule cells also have been
studied extensively for the characterization of their HVA current
(Slesinger and Lansman, 1991; Pearson et al., 1995; Randall and Tsien,
1995), and T-type currents are reported to be absent in these neurons
(Rossi et al., 1994). However, we found a prominent distribution of
CaVT expression in these cells. It is noteworthy that this
distribution was heterogeneous. Although moderate levels of 1I were
found evenly distributed in granule cells throughout the cerebellar
cortex, 1G was found at high levels only in the caudal lobules of
both the vermis and the hemispheres (Fig.
6A,B). This type of gradient also
has been observed for expression of KV4.2 and
KV4.3 (Serodio and Rudy, 1998), two genes believed to
contribute to transient subthreshold potassium currents. Thus, based on
the expression of CaVT- and KV4-family genes,
we would expect to find differences in subthreshold membrane properties of rostral and caudal cerebellar granule neurons.
Another area of the CNS where there is curious inconsistency between
our findings with regard to CaVT expression and experiments examining the characteristics of neuronal calcium currents is the
cerebral cortex. T-type calcium currents are thought to be more
prominent in pyramidal neurons of the neocortex (Giffin et al., 1991;
Hamill et al., 1991) when compared with nonpyramidal cells.
Furthermore, cells with measurable T-type current were predominantly
found in deeper layers, cortical layer V in the visual cortex (Giffin
et al., 1991) or layers V and VI in the medial frontal cortex (de la
Pena and Geijo-Barrientos, 1996). In accord with those studies, we
found that 1H expression is largely limited to layer V pyramidal
neurons. However, we also found substantial expression of 1G and
1I in all cortical layers, and this expression did not appear to be
limited to pyramidal neurons. Given the generalized expression patterns
of 1G and 1I in the neocortex, it is not clear why only a
restricted population of neurons in this region have been found to
display T-type currents.
In addition to observing CaVT expression in neurons thought
to be devoid of T-type currents, there were also cells in which we
failed to find CaVT expression but which appear to have
T-type calcium currents. This was the case for olfactory mitral cells, which have been shown to have LVA currents of modest amplitude (Wang et
al., 1996). However, although they appear to lack expression of known
CaVT gene-family members, these cells are well stained by
an antibody to the 1E (HVA family) calcium channel (Yokoyama et al.,
1995). It is possible that LVA currents in these neurons receive a
contribution from the 1E channel, which can generate currents with
some T-type properties (Soong et al., 1993; Meir and Dolphin, 1998) and
may account for some of the LVA current in atrial myocytes
(Piedras-Renteria et al., 1997). An alternative possibility is that an
as yet unidentified gene accounts for the LVA currents in mitral cells.
It is worth mentioning at this juncture that in contrast to mitral
cells, we found high levels of expression of all three transcripts in
granule cells of the olfactory bulb. Calcium currents in these neurons
apparently have not been extensively characterized (Bhalla and Bower,
1993).
Heterogeneity of CNS T-type calcium currents
CNS T-type calcium channels are pharmacologically and
physiologically heterogeneous, leading to the hypothesis that at least some of their variability is generated by differences in genes encoding
these channels. With respect to pharmacology, T-type currents have
shown different sensitivities to block by a number of cations and
organic compounds (for review, see Akaike, 1991; Huguenard, 1996). Any
discussion of the contribution of the three CaVT gene
products to this differential sensitivity is preliminary because the
pharmacological profiles of these clones have not been extensively
characterized. It can be pointed out, however, that we do not see any
correlation between the expression profiles of any of the three
CaVT transcripts and regions where particular pharmacological attributes have been observed. For example,
differential sensitivities to both nickel and amiloride have been noted
between DRG neurons and pituitary cells (Todorovic and Lingle, 1998), two regions where we see predominant expression of 1H mRNA.
Conversely, thalamic reticular and relay neurons, which express
different CaVT transcripts, have similar sensitivities to
both nickel and amiloride (Huguenard and Prince, 1992).
Physiological characterization of the three CaVT genes has
been performed, and there appears to be some correlation between expression of these genes and the properties of T-type currents in
different CNS neurons. When expressed in the same cell type (HEK 293 cells), 1G and 1H generate currents with similar kinetic and
voltage-dependent properties. In contrast, 1I currents have slower
kinetics and depolarized voltage dependence of activation, when
compared with the other two channels (Lee et el., 1999).
The contrast in properties between the 1I channels and those of
1G and 1H corresponds to a difference in T-type currents in
different cells of the thalamus, a region where these currents have
been extensively characterized both in dissociated and intact preparations (Destexhe et al., 1996, 1998). When calcium currents were
compared directly in thalamic reticular and thalamic relay neurons
(Coulter et al., 1989a; Huguenard and Prince, 1992), it was found that
the LVA component was distinctly different in the two cell types.
Thalamic reticular neurons, which express high levels of 1I mRNA
along with moderate levels of 1H, had slower kinetics of activation
and inactivation when compared with thalamic relay neurons of the
ventral basal complex, a region that appears to exclusively express
1G. Moreover, in reticular neurons the voltage range of activation
was depolarized relative to that of relay neurons. Given data from the
expressed channels, it appears that the slower kinetics and depolarized
voltage dependence of activation in reticular neurons may result at
least in part from their expression of 1I. Consistent with this
hypothesis, neurons of the lateral habenula, which express both 1G
and 1I (Figs. 4H,I, 5), were
found to have T-current with physiological properties intermediate
between those of reticular and relay neurons (Huguenard et al.,
1993).
It should be pointed out that in addition to CaVT (1I
and 1H) expression, thalamic reticular neurons also apparently
express somewhat elevated levels of 1E (Soong et al., 1993; Williams et al., 1994; but see Yokoyama et al., 1995). As noted above, this gene
is more rapidly inactivating and has a more hyperpolarized threshold
for activation than other genes of the HVA family (Soong et al., 1993).
Therefore, it may be that a combination of 1E, 1H, and 1I
contribute to the LVA current in thalamic reticular neurons. In
addition, we cannot rule out the possibility that there may be as yet
uncloned genes that generate and/or modify LVA currents in these and
other CNS neurons.
| |
FOOTNOTES |
Received Sept. 23, 1998; revised Oct. 28, 1998; accepted Oct. 30, 1998.
This work was supported by National Institutes of Health Grants HL57828
(E.P-R.), NS33583 (D.A.B.), and MH12091 (predoctoral fellowship for
E.M.T.). We thank Drs. Madaline B. Harrison, Ruth L. Stornetta, Patrice
G. Guyenet, and the Information Technology Services at the University
of Virginia for providing imaging equipment and support.
Correspondence should be addressed to Edmund M. Talley, Department of
Pharmacology, Box 448, Health Sciences Center, University of Virginia,
Charlottesville, VA 22908.
| |
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Top
Abstract
Introduction
