Differential Distribution of Three Members of a Gene Family Encoding Low Voltage-Activated (T-Type) Calcium Channels

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Differential Distribution of Three Members of a Gene FamilyEncoding Low Voltage-Activated (T-Type) Calcium Channels

Edmund M. Talley,1 Leanne L. Cribbs,2 Jung-Ha Lee,2 Asif Daud,2 Edward Perez-Reyes,2 and

Douglas A. Bayliss1

1Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, and 2Department of Physiology,Loyola University Medical Center, Maywood, Illinois 60153

Low voltage-activated (T-type) calcium currents are observed in

many central and peripheral neurons and display distinct phys-

iological and functional properties. Using in situ hybridization,

we have localized central and peripheral nervous system ex-

pression 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 ofthese 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 hypothal-

amus, 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 allthree transcripts. Our results are consistent with the hypothesis

that differential gene expression underlies pharmacological and

physiological heterogeneity observed in neuronal T-type cal-

cium 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; anti-

convulsant; rat; T-type calcium channels

Voltage-dependent calcium channels play a dual role in the CNS;they couple electrical activity to calcium influx, thereby triggeringmyriad intracellular biochemical events, and they contribute to

membrane properties that determine the precise nature of excit-ability in different cell types. Low voltage-activated (LVA) cal-cium channels have a hand in both of these roles that is readilydistinguishable from their high voltage-activated (HVA) counter-parts, in part because they activate at potentials near the restingmembrane potential. First, in addition to participating in spike-induced calcium entry (McCobb and Beam, 1991; Scroggs andFox, 1992b; Umemiya and Berger, 1994), they allow calciuminflux at potentials below threshold. This influx can occur whencells are at rest (Magee et al., 1996) or in response to subthresholdsynaptic inputs (Miyakawa et al., 1992; Markram and Sakmann,1994; Magee et al., 1995). Second, LVA calcium channels can bea crucial component in shaping subthreshold membrane fluctua-

tions and thereby contribute to such behaviors as rebound burstfiring (Llinas and Yarom, 1981), rhythmic oscillation (Gutnickand 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 LVAcalcium 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 encodethe subunits forming HVA calcium channels. The central core ofthese channels consists of an 1 subunit that has four internalrepeats, each repeat consisting of six membrane-spanning regionsand a pore-forming loop (for review, see Catterall, 1995; Perez-Reyes and Schneider, 1995). Currently, at least six1 subunitshave been identified that are believed to generate the physiolog-ically and pharmacologically defined H VA calcium channel sub-types (generally designated L, N, P, Q, and R). Although one ofthese 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 bythis gene do not possess all of the properties that are common forT-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 de-activation, and small single-channel conductance (for review, seeHuguenard, 1996).

Recently, a subfamily of genes (designated CaVT) has beendiscovered encoding 1 subunits that are 30% homologous toHVA subunit genes in their putative membrane-spanning regions(Cribbs et al., 1998; Perez-Reyes et al., 1998a,b). When expressedheterologously, two of these proteins, 1G and1H, show all theproperties hallmark of neuronal T-type calcium channels. A third

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.). Wethank Drs. Madaline B. Harrison, Ruth L. Stornetta, Patrice G. Guyenet, and theInformation Technology Services at the University of Virginia for providing imagingequipment and support.

Correspondence should be addressed to Edmund M. Talley, Department ofPharmacology, Box 448, Health Sciences Center, University of Virginia, Charlottes-

ville, VA 22908.

Copyright 1999 Society for Neuroscience 0270-6474/99/191895-17$05.00/0

The Journal of Neuroscience, March 15, 1999, 19(6):18951911


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member of this family, designated 1I, also encodes a calciumchannel that displays a number of T-type properties (Lee et el.,1999).

Given that expression of T-type channels has a unique impacton neuronal properties, it is of great interest to know whichneuronal populations express these subunits. Furthermore,T-type channels are pharmacologically and physiologically heter-ogeneous (Akaike et al., 1991; Huguenard, 1996; Tarasenko et al.,

1997). This heterogeneity may reflect different functions of thesechannels in neurons, and may stem at least in part from differen-tial expression of each of these three genes. Therefore, using in

situ hybridization histochemistry, we have localized the regionaland cellular distribution of gene expression for the three knownmembers of the CaVT family in the rat central and peripheralnervous systems. We find that each gene has a unique expressionpattern and that the location of these three channel types is to alarge ex tent complementary.

MATERIALS AND METHODS

Tissue preparation. Male Sprague Dawley rats (250350 gm; Hilltop)were anestheti zed with ketamine xyla zine and decapitated. Brains, spi-nal cords, and ganglia were removed and frozen on dry ice. Sections (10m) were thaw-mounted onto charged slides (Superfrost Plus; FisherScientific, Houston, TX) and stored at 80C for later use. In prelimi-nary experiments, sagittal and horizontal sections were used from sixanimals. For detailed comparative analysis, coronal sections from threeanimals were taken from the entire brain (200500 m apart). Inaddition, representative sections from three or four animals were takenfrom nodose, superior cervical, and dorsal root ganglia, as well as fromcervical, thoracic, and lumbar spinal cord. Slides were pretreated forhybridization as described previously (Talley et al., 1997). Sections werefixed 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 aceticanhydride (0.25% in 0.1 M triethanolamine, 0.9% saline, pH 8; 10 min)and subsequently dehydrated in a graded series of ethanols and chloro-form. Hybridization was performed overnight at 37C in a buffer of 50%formamide, 4 SSC (1 SSC: 150 mMNaCl and 15 mMsodium citrate,pH 7), 1 Denhardts solution (0.02% each of Ficoll, polyvinylpyrroli-done, and bovine serum albumin), 10% dextran sulfate, 100 m M DTT,250 g/ml yeast tRNA, and 0.5 mg/ml salmon testes DNA. After hy-bridization, slides were washed through four changes of 1 SSC at 55C(15 min each) and once for an hour in 1 SSC at room temperature.

Oligonucleotide probes. Sequence analysis of 1G, 1H, and 1I hasrevealed a high degree of homology in the putative transmembraneregions of these three genes (Perez-Reyes et al., 1998b). In contrast, thereis particularly low conservation in the sequences that connect each of thefour repeating domains. T herefore, anti sense oligonucleotide probes (33bases in length) were designed to hybridize to the cytoplasmic I-IIlinker region of the rat homolog of each gene (L. L. Cribbs, J.-H. Lee,and E. Perez-Reyes, unpublished data). Sequences were chosen that hadminimal homology both to other calcium channel genes and to othersequences present in GenBank. Probes were labeled using terminaldeoxyribonucleotidyl transferase (Life Technologies, Gaithersburg,MD); unincorporated nucleotides were removed using Sephadex G-50spin columns (Pharmacia, Piscataway, NJ).

Multiple oligonucleotides were used to probe each gene (three oligo-nucleotides for 1G, three for 1H, and two for 1I). Subsequent topreliminary experiments characterizing the probes, the oligonucleotidescorresponding to each gene were hybridized in a cocktail. We found thatusing the probes in combination resulted in an enhanced signal but hadno effect on relative distribution of signal intensities in different brainregions (see below). T he concentration of each oligonucleotide was30 10 6 cpm/ml (2 nM). The sequences of the probes wereas follows: 1G: 5-CCAGCCCGCACGCCTATAGCCCTAGAGACC-TGG-3, 5-TCCGATGGGCATCTGGGAGGGGGTGCCTGGCAA-3, an d 5-TGTCGCCGCCGGCTGTGGGGATCCCGGAGGTCA-3;1H: 5-ATGCCGTACATCCTGGGTAAACTCATAGACTCC-3 , 5-AGCCCC TTGGGTCGTGAGC TGGTGCCACC TT TG-3, and 5-AT-CCCTCCTGGTTGTGAGGCCTTCCGCAGTGGT-3 ; 1I: 5-AGCC-ACCAGAA CC TGAGCC TTCC TGGCC TGAGT-3 and 5-TCGCG-CCACACATCCCCACACAGTCGGGCTGCC-3.

Control ex periments.We performed a number of preliminary controlexperiments. First, we hybridized each oligonucleotide separately tosagittal and horizontal sections. Cognate oligonucleotides generated anidentical tissue distribution, indicating that these independent probesrecognized the same gene product and ruling out the possibility ofspurious cross-reactivity. Higher wash temperatures (60 and 65C in 1SSC) resulted in diminished hybridization, but for each oligonucleotidethe distribution of labeling was unchanged, indicating that the samebinding site was labeled in different brain areas. For each set of probes,

specific binding was eliminated by prior digestion with RNase A (50g/ml; Boehringer Mannheim, Indianapolis, IN). Nonspecific bindingwas assessed in competition exper iments (examples of which are shownin Fig. 3), with 500-fold excess unlabeled oligonucleotide (1 M)included in the hybridization mixture. In these experiments, nonspecificbinding to ti ssue was barely distingui shable from the overall background,demonstrating that specific binding was saturable and that nonspecificbinding was minimal.

Data anal ysis and presentation. Slides were exposed to film (Hyperfilm-MAX; Amersham, Arlington Heights, IL) for 1 week to generateautoradiograms (Fig. 1), which were analyzed with the aid of imageanalysis software (MCID; Imaging Research) to determine the relativeintensity of labeling in different brain regions. For resolution of cellularlabeling, slides also were dipped in liquid autoradiographic emulsion(NTB2; Eastman Kodak, Rochester, NY), exposed for 59 weeks, andexamined by dark-field and bright-field microscopy. Images of silvergrains from these slides (see Figs. 27) were captured using a Pixeravideo camera mounted on a Leit z 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-sidecomparison of the relative numbers of silver grains overlaying variouscell types.

By combining densitometry of film autoradiograms with informationon the specific cellular localization of hybridization, we determined thecomparative distribution of each of the three transcripts. The results ofthis analysis are presented in Table 1 as a system of pluses, with fivepluses () representing the highest levels of expression. It isimportant to understand that because a number of factors other thantranscript levels (particularly the hybridization efficiency of individualprobes) can affect signal intensity, this scoring system reflects relativeamounts of individual transcripts in different brain regions, rather thancomparisons among the three different CaVT transcripts. However, thefact that the various probes to each gene (when hybridized individually)

generated similar signal intensities suggests that the influence of factorssuch as differences in hybridization efficiency were minor. Therefore,relative levels of the three different mRNA species may be compared, solong as such comparison is viewed with caution. In this regard, it is alsoimportant to note that only two probes were used to detect 1I (asopposed to three each for1G and1H). As stated above, we found thatcombining oligonucleotides in a cocktail had no effect on relative distri-bution but resulted in enhanced signal intensity. Thus, because one fewerprobe was used for 1I, transcript levels for this gene may have beensomewhat under-represented relative to those of the other two genes.

RESULTS

Overview

We performed in situ hybridization to determine the regional

distribution of C aVT (1G,1H, and1I) gene expression in thecentral and peripheral nervous systems. Figure 1 shows represen-tative film autoradiograms of transverse sections that were takenthrough the entire brain. Figures 27 show representative dark-field and bright-field images of silver grains from emulsion-dippedslides. The results of combined analysis from film autoradiogramsand emulsion-dipped slides are summarized in Table 1.

The distribution of the three transcripts was to a great extentcomplementary, and the expression pattern of each gene wasunique. Only a few regions, including the granule cell layer of theolfactory bulb (Figs. 1A, 2), fields CA1 and CA3 of the hippocam-pus (Figs. 1GK, 3), and the tenia tecta (TT; Fig. 1B), di splayedexpression of all three transcripts in abundance. Cells with the

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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.1GK, 3), and in sensory ganglia (Fig. 7). Labeling for 1I washighest in the olfactory bulb (Figs. 1A, 2), the cell islands ofCalleja (ICj; Figs. 1C, 2), and fields CA1 and CA3 of the hip-pocampus (Figs. 1GK, 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 thisstructure was one of a limited number of brain regions with highexpression of all three channel subtypes. In contrast, the mitralcell layer was not noticeably labeled, and where mitral cells wereidentified by Nissl stain, silver grains were not detected abovebackground (data not shown). Small neurons in the glomerular

Figure 1. C NS distribution of CaVT gene expression. Sections were hybridized with oligonucleotides specific for 1G (left panels),1H (middle panels),and1I (right panels) and exposed to autoradiographic film. Li ne drawings on the far rightare adapted from Paxinos and Watson (1997; reproduced withpermission) and indicate the relevant labeled areas. For abbreviations, refer to Table 2. Figure 1 continues.

Talley et al. T-type Calcium Channel Distribution J. Neurosci., March 15, 1999,19(6):18951911 1897


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layer contained moderate levels of1G, whereas1I was presentat very high levels in scattered members of this cell population(Fig. 2).

Moderate to high levels of message were also found for all threegenes in olfactory cortical structures, including the anterior ol-

factory nucleus (AO; Fig. 1A) and piriform cortex (Pir; Fig. 1BI;see below). The olfactory tubercle, by contrast, had an expressionpattern that was more reminiscent of the striatum (see below)insofar as it contained little expression of 1G mRNA (Figs.1BD, 2). Instead, it contained very high levels of 1H in the

Figure 1 continued.



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dense cellular layer as well as very high levels of1H and 1I inthe cellular islands of Calleja.

Basal forebrain

1H and 1I transcripts were both present throughout the stria-tum (CPu; Figs. 1CI, 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 de-tectable levels in the accumbens and the medial striatum, but itdisplayed moderate levels at the lateral and caudal edges of thestriatum. 1G mRNA was generally undetectable in these struc-

Figure 1 continued.



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tures, although scattered cells were labeled: these were moreevident medially at the edge of the globus pallidus (GP), a nucleusthat was not marked by expression of any of the transcripts (Fig.1EH). Another basal ganglia structure, the subthalamic nucleus(STh; Fig. 1I) contained high levels of 1I, moderate levels of1G, and only scattered expression of1H.

In the septum (Fig. 1CE), labeling was moderate for 1G inthe dorsal and ventral part of the lateral septum (LSD, LSV) andlow in the intermediate part (LSI) and in the medial septum(MS). 1H labeling was found at low levels in all parts of thelateral and medial septum and absent in the triangular septum(TS). Labeling for 1I was opposite to that of1H; it was not

Figure 1 continued.



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detected in the lateral and medial septum, but it was present inlow amounts in the triangular septum. This region is traversed bya cortical structure, the indusium griseum (IG; Fig. 1C), whichhad very high levels of1H as well as low levels of1G.

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 wasmoderate, and 1I was low. In the rest of the nucleus, levels of

1G and 1H expression were low, and 1I expression was notdetected. Another basal forebrain structure, the claustrum (Cl;Fig. 1BF), also had prominent 1G expression along with lowlevels of1I.

Amygdala

In the amygdala, 1G and 1H were predominant, with 1ImRNA expressed at low levels in most nuclei (Fig. 1GK). High-est levels of all three genes were found in the nucleus of the lateralolfactory tract (LOT; Fig. 1F) and the related bed nucleus of theaccessory olfactory tract (BAOT; Fig. 1G). In the central nucleus(Ce; Fig. 1G,H) and the lateral nucleus (La; Fig. 1GI), 1Hexpression 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 con-trast, expression for all three genes was mixed; it was higher in theposterior part (MeP; Fig. 1H,I) than in the anterior part (MeA;Fig. 1G). Expression for 1G and 1H was most evident in thecaudal tip of this nucleus, as shown in Figure 1I.

Hippocampal formation

In many C NS regions, the hybridization levels of the 1G probestended to be higher than those of the other two sets of probes.Such was not the case for the hippocampal formation (Fig. 1 GL,3), where labeling intensities were more equivalent between thethree sets of probes. Pyramidal cells in the hippocampus hadmoderate to high levels of all three transcripts, whereas CaVTexpression in the nonpyramidal cell layers of Ammons Horn was,

for the most part, restricted to 1G. In the granule cell layer ofthe dentate gyrus, 1H expression was predominant and ex-tremely high. In contrast, dentate gyrus polymorph cells hadlabeling that was more closely matched between the three sets ofprobes.

Cerebral cortex

All three transcripts were present in the cerebral cortex, as show nin Figures 1ALand 4, 1G and 1I were present fairly uniformlyin all cell layers, the major exception being layer IV, where bothtranscripts were detectable in more cells. In addition, i n the

ventral part of the cortex just dorsal to the claustrum, a band ofneurons that was intensely labeled with the probes for 1G waspresent in the deepest part of layer VI. These intensely labeled

cells appeared to be restricted to the insular/perirhinal cortices(Ins, Fig. 1BG; PRh, Fig. 1HK) and secondary somatosensoryareas (e.g., Fig. 1E). With respect to particular cell t ypes, probesfor 1G and 1I labeled both large (presumably pyramidal) andsmall (presumably granule) neurons. However, this labeling wasnot uniform, and in some cells expression was not detected. Thus,no clear preference emerged for expression of either one of thesetranscripts by specific t ypes of cortical neurons.

Such a general distribution in the neocortex was not the casefor 1H. It was seen at very high levels in a subset of layer Vpyramidal neurons; otherwise it was only present at very lowlevels in the externalmost cellular layer (layer II) and was notdetected in layers IV and VI. The 1H-expressing pyramidal

Table 1. Distribution of1G/1H/1I mRNA

Brain region 1G 1H 1I

Olfactory systemOlfactory bulb

Glomerular layer bt Granule cell layer

Anterior olfactory nucleus Olfactory tubercle Islands of Calleja

Basal forebrainStriatum bt /Accumbens bt Globus pallidus bt bt btSubthalamic nucleus bt/ Septum/diagonal band / bt/ bt/Indusium griseum btBed nucleus of the stria terminalis / / bt/Claustrum bt

AmygdalaCentral nucleus / btMedial nucleus / / bt/Lateral nucleus / Basolateral/basomedial nuclei Cortical amygdaloid nuclei / Nucleus of the lateral olfactory tract Amygdalohippocampal area Amygdalopiriform transition area

Cerebral cortexNeocortex

Layers II/III bt/ Layer IV bt Layer V Layer V I / bt

Piriform cortex / /bt /btTenia tecta

HippocampusPyramidal cell layers

Field CA1 Field CA2 Field CA3

Granule cell layer of the dentate gyrus Polymorph layer of the dentate gyrus

ThalamusPrincipal relay nuclei bt bt / Intralaminar nuclei / bt bt / Geniculate nuclei bt bt / Re ticular thalamic nucl eus bt Lateral habenular nucleus / bt /

HypothalamusPreoptic nuclei / Suprachiasmatic nucleus / Supraoptic nucleus bt btLateral hypothalamic area btParaventricular nucleus bt

Arcuate nucleus Dorsomedial/ventromedial nuclei / / Mammillary nuclei / / /

Mibrain and ponsSuperior/inferior colliculus / Periaqueductal gray btTegmental nuclei / bt bt / Raphe nuclei bt/ btSubstantia nigra bt/ bt/ btInterpeduncular nucleus Parabrachial nuclei / bt

Cerebellum and inferior oliveGranule cell layer

Anterior bt Posterior bt

Purkinje cell layer bt btMolecular layer bt btDeep cerebellar nuclei bt btInferior olive bt bt /

Medulla and spinal cordSpinal trigeminal nuclei / bt/ bt/Nucleus of the solitary tract bt

Cochlear nuclei

/

bt/

bt/

Gracile/cuneate nuclei / btSomatic motor neurons btArea postrema Reticular fields bt / bt bt /

OtherPituitary Pineal gland btSensory ganglia /Superior cervical ganglia bt bt

In situ hybridization signals were determined based on relative optical density of film autoradio-

grams, as well as silver grain density over cells from emulsion-dipped sections.

, Highest levels of labeling; , very high; , high; , moderate; , low levels;

bt, below the threshold limit for detection.

Note the heterogeneity of labeling within a number of regions; see corresponding sections in

Results for details.



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Table 2. Abbreviations used throughout text and in figures

2 through 10 C erebellar lobules

X II Hypoglossal nucleus7n Facial nerveAcb Accumbens nucleusACo Anterior cortical amygdaloid nucleusAD Anterodorsal thalamic nucleusAH Anterior hypothalamic areaAHi Amygdalohippocampal areaAO Anterior olfactory nucleusAP Area postremaAPi r Amygdalopir iform transition areaAP T Anterior pretectal nucleusArc Arcuate nucleusAu Auditory cortexBAOT Bed nucleus of the ac cessor y ol factor y tractBL Basolateral amygdaloid nucleusBLA Basolateral amygdaloid nucleus, anterior partBLP Basolateral amygdaloid nucleus, posterior partBM Basomedial amygdaloid nucleusBMP Basomedial amygdaloid nucleus, posterior partBST Bed nucleus of the stria terminalisCA1 Field CA1 of hippocampus

CA2 Field CA2 of hippocampusCA3 Field CA3 of hippocampusC e Central amygdaloid nucleusC g Cingulate cortex C l ClaustrumCLi C audal linear nucleus of the rapheC M C entral medial thalamic nucleusC nF Cuneiform nucleusC Pu C audate putamen (striatum)C tx Cerebral cortex Cu Cuneate nucleusDEn Dorsal endopiriform nucleusDG Dentate gyrusDLG Dorsal lateral geniculate nucleusDM Dorsomedial hypothalamic nucleus

DR Dorsal raphe nucleusDTg Dorsal tegmental nucleusEct Ectorhinal cortex ECu E xternal cuneate nucleusEnt Entorhinal cortex FrA Frontal association cortex Gi Gigantocellular reticular nucleusGP Globus pallidusGr Gracile nucleusic Internal capsuleIC Inferior colliculusICj Islands of CallejaIG Indusium griseumInO Inferior oliveIns Insular cortex I P Interpeduncular nucleus

La Lateral amygdaloid nucleusL A L ateroanterior hypothalamic nucleusLC Locus coeruleusLD Laterodorsal thalamic nucleusLDTg L aterodorsal tegmental nucleusL Ent Lateral entorhinal cortex L H Lateral hypothalamic areaL Hb Lateral habenular nucleusL L Nuclei of the lateral lemniscusL M Lateral mammillary nucleusL OT Nucleus of the lateral ol factor y tractL PB Lateral parabrachial nucleusL PO Lateral preoptic area

LRt Lateral reticular nucleusLS Lateral septal nucleusL SD L ateral septal nucleus, dorsal partL SI L ateral septal nucleus, intermediate partL SV L ateral septal nucleus, ventral partM Motor cortex MD Mediodorsal thalamic nucleusMeA Medial amygdaloid nucleus, anterior partMeP Medial amygdaloid nucleus, posterior partMG Medial geniculate nucleusML Medial mammillar y nucleus, lateral partM M Medial mammillar y nucleus, medial partMnPO Median preoptic nucleusMnR Median raphe nucleusMo5 Motor trigeminal nucleusMOB Main olfactory bulbM PA Medial preoptic areaM PO Medial preoptic nucleusM PT Medial pretectal nucleusMS Medial septal nucleusM Ve Medial vestibular nucleusOrb Orbital cortex

Pa Paraventricular hypothalamic nucleusPAG Periaqueductal grayPaS ParasubiculumPCRt Par vicellular reticular nucleusPDTg Posterodorsal tegmental nucleusPFl ParaflocculusPir Piriform cortex PLCo Posterolateral cortical amygdaloid nucleusPMCo Posteromedial cortical amygdaloid nucleusPo Posterior thalamic nuclear groupPost PostsubiculumPr Prepositus nucleusPr5 Principal sensory trigeminal nucleusPRh Perirhinal cortex PrL Prelimbic cortex

PV Paraventricular thalamic nucleusR Red nucleusRe Reuniens thalamic nucleusRh Rhomboid thalamic nucleusRMg Raphe magnus nucleusROb Raphe obscurus nucleusRS Retrosplenial cortex Rt Reticular thalamic nucleusS SubiculumS1 Primary somatosensory cortex S2 Secondary somatosensory cortex SCh Suprachiasmatic nucleusSN Substantia nigraSol Nucleus of the solitary tractSp5 Spinal trigeminal nucleusst Stria terminalis

STh Subthalamic nucleusSuM Supramammillary nucleusSupC Superior colliculusTS Triangular septal nucleusTT Tenia tectaTu Olfactory tubercleV Visual cortex VC Ventral cochlear nucleusV LG Ventral lateral geniculate nucleusV M Ventromedial thalamic nucleusV M H Ventromedial hypothalamic nucleusV P Ventral posterior thalamic nucleusV TA Ventral tegmental area



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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 athigh levels. One was the TT (Fig. 1B). The other structure, the Pir(Fig. 1BI), had mixed expression levels of all three genes. Both1H and 1I were present in high amounts in the superficialneurons of this cortical region but were not detected in deeperlayers. On the other hand, 1G mRNA was expressed in moder-

ate amounts in the external layer but at much higher levels in thedeeper layers, especially the dorsal endopiriform nucleus (DEn;Fig. 1CJ).

Thalamus

The thalamus (Figs. 1EK, 5) was characterized by high levels ofexpression of 1G in thalamocortical relay nuclei. In many ofthese nuclei, including the medial dorsal (MD), the ventral ante-rior (VA), the ventral and posterior relay nuclei (Po, VM, andVP), and the medial and dorsal lateral geniculate nuclei (MG andDLG), detectable expression was limited to 1G. Low levels of1I were seen in the anterior dorsal nucleus (AD; Fig. 1F) andthe 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 of1H in relay nuclei was limited tosome of the large neurons of AD that expressed this transcript atlow levels. The intralaminar nuclei, including the central medialnucleus (CM; Fig. 1G,H), paraventricular thalamic nucleus (PV;Fig. 1FI), the rhomboid (Rh; Fig. 1G), and reuniens (Re; Fig.1G,H) nuclei had even higher levels of 1G expression. Lowlevels of 1I expression also characterized a number of thesenuclei (CM, PV, and Rh).

In contrast to the thalamic relay and intralaminar nuclei, thethalamic reticular nucleus, which is composed of GABAergicinterneurons that modulate and synchronize thalamic output (forreview, see Steriade et al., 1993), had no detectable 1G expres-sion (Fig. 5, left panels). Instead, these neurons expressed high

levels of1I and moderate levels of1H. The lateral habenula(Fig. 5, right panels) had an uneven distribution of1G and 1I;both transcripts were detected at high levels in the rostral portionof the nucleus. Caudally, 1I expression was moderate, and 1Gexpression was low (Fig. 1, compareH, I). CaVT expression wasnot detected in neurons of the medial habenula, a region charac-terized by high levels of mRNA encoding 1E (Soong et al., 1993;Williams et al., 1994).

Hypothalamus

In the hy pothalamus, 1G was predominant rostrally. In thepreoptic region (Fig. 1DF), 1G mRNA levels were higher inmedial structures such as the medial preoptic nucleus (MPO)

than in their lateral counterparts (e.g., LPO). This medial biasalso 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 of1G mRNA in the ventrolateral portion of the VMH (Fig. 1H,I).Expression patterns in the mammillary bodies were somewhatcomplex, with each transcript showing a unique expression pat-tern (Fig. 1J). In the medial mammillary nucleus, the medial part(MM) showed a predominance of1H expression, whereas thelateral part (ML) displayed1G and1I. In the lateral mammil-lary nucleus (LM), 1G was abundant, with the other two tran-scripts present only at low levels.

Midbrain and pons

All three transcripts were ex pressed in the tectum (SupC and IC;Fig. 1KM), with 1H and 1I at low levels and 1G at low tomoderate levels, except in the external gray area of the superiorcolliculus, where 1G labeling was high (Fig. 1K). All threetranscripts also were found in the pretectal nuclei (MPT and

AP T; Fig. 1J), which were notable as a result of high 1H labelingin scattered cells of the anterior pretectal nucleus (APT). Theperiaqueductal gray (PAG; Fig. 1JL) had moderate levels oflabeling for 1G and low levels of 1H. More caudally, thetegmental nuclei ventral to the fourth ventricle (Fig. 1M,N) werea little more heterogeneous, with 1G mRNA at high levels in theposterodorsal tegmental nucleus (PDTg; Fig. 1N) and 1I presentin the laterodorsal nucleus (LDTg; Fig. 1M).

Figure 2. Dark-field micrographs demonstrating cellular labeling of ol-factory structures. Slides were exposed to autoradiographic emulsion;silver grains were imaged using dark-field microscopy. Left panelsshowsilver grains over cells of the main olfactory bulb. Note that whereas allthree transcripts were present in the granule cell layer (asterisks), labelingof small neurons in the glomerular layer (arrowheads) was limited to 1Gand 1I.Right panelsshow the olfactory tubercles. Labeling for 1H and1I was very high in the cell islands of Calleja (open arrows); labeling wasalso high for1H but only moderate for 1I in the dense cell layer of thetubercles (closed ar rows). Scale bar, 500 m.



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present at low levels in the lateral but not the medial areas (Fig.1O, compare PCRt, Gi); 1I was found in the lateral reticularnucleus (Fig. 1P).

All three transcripts were detected in sensory areas. In thespinal trigeminal nucleus (Sp5), the three mRNAs were found athigher levels caudally than rostrally (Fig. 1, compare O, P). Allthree also were seen in the dorsal cochlear nucleus (data notshown), but only 1G was present (and at lower levels) in the

ventral cochlear nucleus (VC; Fig. 1M,N). Similarly, all threetranscripts were present in the dorsal horn of the spinal cord (Fig.7). 1G was present at moderate levels; 1H was for the most partrestricted to the outermost layers (layers 12). 1I was somewhatmore evenly distributed at low levels, but it was more prominentin layers 34. Only 1G and1H were detected in the nucleus ofthe solitary tract (Sol; Fig. 1O,P).

Somatic motor neurons in the brainstem and spinal cord con-tained 1G and1H mRNA, at moderate and low levels, respec-tively (Figs. 1M,P, 7). Other labeled medullary regions includedthe inferior olive (discussed above) and the area postrema (AP;Fig. 1P); in the AP all three transcripts were found, with 1G athigh levels.

Other areas

In addition to surveying the CNS, we also hybridized the CaVTprobes to sections from the sensory and sympathetic ganglia,specifically the nodose, dorsal root ganglia (DRG) and superiorcervical ganglia. High-power bright-field micrographs of sensoryneurons of the DRG and nodose ganglia are shown in Figure 7(middle and left panels). In the DRG, high levels of 1H andmoderate levels of1I mRNA were found in scattered medium-

sized neurons, whereas the extremely large neurons were notlabeled. The nodose ganglia also contained high levels of 1HmRNA in many neurons, although in contrast to the DRG, theredid not appear to be a bias in the size of the labeled neurons.Neurons of the superior cervical ganglia (not shown) only ex-pressed very low levels of 1G. We also examined cells of thepituitary and pineal glands. Both of these structures showed highlevels of expression of1H mRNA (data not shown).

DISCUSSION

We used in situ hybridization to show that three members of anovel family of calcium channels with T-type properties areexpressed widely in the CNS and in peripheral neurons, and that

Figure 4. Differential hybridization toneurons in the cerebral cortex. Distribu-tion of1G, 1H, and 1I mRNA in theprimary somatosensory cortex is shownat increasing levels of magnification.Top panels demonstrate laminar distri-bution of labeling; the relevant corticallayers (layers II-VI) are indicated to the

rightof these panels, as are the externalcapsule (ec) and the striatum (CPu).Ar-

rowheads indicate large (presumably py-ramidal) neurons in layer V. The sameneurons are depicted at higher magnifi-cation in the middle panels (using dark-field optics) and at still higher magnifi-cation in bottom panels (using bright-

field optics). Note that 1G and 1Iwere found i n all cortical layers, but ex-pression of1H was for the most partrestricted to layer V. Scale bar, 400 m(top panels); 100m (middle panels); 25m (bottom panels).



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each of these three transcripts has a unique pattern of distribu-tion. Our results reveal that expression of these genes is promi-nent in a number of brain areas where T-type currents have beenrecorded and absent in specific regions believed to be devoid ofthese currents. Furthermore, our data are in accord with thehypothesis that differential ex pression of specific C aVT subtypesmay account for at least some of the heterogeneity observed inCNS T-type calcium currents.

CaVT transcripts are expressed in cells with prominentT-type currents

As anticipated, we detected robust expression of C aVT mRNA inareas where prominent T-type calcium currents have been ob-served. For example, we found ex pression of all three transcriptsin the thalamus, where the role of T-type calcium channels hasbeen studied extensively (for review, see Steriade et al., 1993). Wefound 1G mRNA to be predominant in thalamocortical relayareas but absent in the thalamic reticular nucleus, which insteadexpressed high and moderate levels of 1I and 1H mRNA,respectively. It is believed that the differences in the characteris-tics of T-type currents in these two cell types (discussed below)

are important for the generation of synchronized thalamocorticalrhythms (McCormick and Bal, 1997).

Another region classically associated with prominent T-typecalcium current is the InO, where we found 1G mRNA in greatabundance. LVA currents in these neurons contribute to oscilla-tions that support the coordination of synchronous rhythmicfiring (Manor et al., 1997). These neurons make monosynaptic

contacts with Purkinje cells of the cerebellum, and their synchro-nous activity is believed to play a prominent role in the organi-zation of cerebellar output (Welsh et al., 1995). It is worthpointing out that we found a heterogeneous distribution of CaVTtranscripts in the InO. E xpression in the rostral part of thenucleus was restricted to 1G, whereas caudal expression con-sisted of both 1G and 1I (Figs. 1O,P, 6). It remains to bedetermined how this differential distribution of CaVT expressionin the inferior olive might contribute to the functional organiza-tion of olivocerebellar processing.

In addition to 1G and 1I, we also found abundant 1HmRNA in regions commonly associated with prominent T-typecalcium currents. For example, we found high levels of this

Figure 5. Distribution of CaVT ex pression in the thalamus.Left panelsshow differential labeling of the thalamic reticu-lar 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 tothalamic relay nuclei, including V P.Right panelsshow label-ing in the habenulae and midline thalamic nuclei. Neuronsof the lateral habenular nucleus (LHb) expressed 1G and1I mRNA (see Results for details).DG, Dentate gyrus;PV,paraventricular thalamic nucleus. Scale bar, 250 m.



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transcript in granule cells of the dentate gyrus and in sensoryganglion neurons. In both of these cell types, T-type channelshave been shown to produce sizable spike afterdepolarizing po-tentials (White et al., 1989; Zhang et al., 1993) that in sensoryneurons can trigger bursts of action potentials. It is notable that insensory neurons of the nodose ganglia, CaVT gene expression hasbeen directly implicated in the production of T-type calciumcurrent; transfection with an antisense oligonucleotide targeting asequence shared by the three CaVT genes specifically and mark-edly diminished the LVA calcium current in those cells (Lambertet al., 1998).

In sensory neurons of DRG, we saw high expression of 1H

and moderate levels of1I mRNA. Expression of both transcriptswas restricted to small- and medium-sized neurons; CaVT tran-scripts were not found in the extremely large DRG neurons.These data are in accord with studies of calcium currents of DRGneurons acutely isolated from adult rats, where large T-typecurrents were present in medium-diameter neurons but wereabsent in large-diameter neurons (Scroggs and Fox, 1992a). Thus,our results support the view that T-type currents are expressedspecifically in smaller sensory neurons that convey thermal andnociceptive information and not in larger neurons that subserveproprioceptive and tactile pathways (Scroggs and Fox, 1992a).

In addition to finding abundant CaVT mRNA in areas where

Figure 6. Parasagittal sections demonstrating labeling of the c erebellum and inferior olivary nucleus. Left panels show bright-field images of filmautoradiograms exposed to sagittal sections through the brainstem and cerebellum. Lobules 9 and 1 of the cerebellar vermis are indicated and correspondto higher magnification dark-field micrographs of emulsion-dipped sections shown on the right. Note that for 1G, the granule cell layer (Gr) of thecerebellum displayed a rostrocaudal gradient of expression, with lobule 9 labeled intensely and lobule 1 showing very low levels. In contrast, expressionof1I in the granule cell layer was fairly uniform throughout the cerebellum and showed similar levels in both lobules. Probes for1G labeled Purkinjeneurons (P) at extremely high levels. One of these neurons (arrowhead) i s depicted at higher power using bright-field optics (inset). The inferior olivarynucleus (InO) also is indicated in the autoradiograms and corresponds to dark-field images in the bottom panels. Labeling of this structure also washeterogeneous: 1G was uniformly high; labeling for 1I was limited to the caudal part of the nucleus. Scale bar, 4.8 mm (left panels); 250 m (right

panels); 50 m (inset); 400 m (bottom panels).



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prominent T-type calcium currents have been observed, therewere a number of regions where we saw little or no detectableC aVT expression and where other investigators have failed to findLVA calcium current. For example, we saw no CaVT expressionin the globus pallidus and saw only very low expression of one ofthe transcripts (1G) in sympathetic ganglia. In neurons of bothof these areas, T-type channels were not evident in whole-cellcalcium current recordings (Schofield and Ikeda, 1988; Plummeret al., 1989; Surmeier et al., 1994).

Regions of inconsistency between CaVT expressionand recordings of T-type calcium currents

As noted above, we saw ex pression of C aVT transcripts in regionsthat display prominent T-type currents and failed to detect thesetranscripts in regions where T-type currents are absent. However,there were also a number of regions reportedly devoid of these

currents where C aVT genes were expressed (e.g., granule cells inthe cerebellum and cerebral cortex) and conversely, regions

where T-ty pe currents have been recorded but where we found noevidence for C aVT expression (e.g., olfactory mitral cells).

It is important to point out that comparative characterizationof T-type currents in neurons is problematic because a variety ofexperimental factors can affect these recordings (discussed inHuguenard, 1996). A particular concern for comparing T-typecurrents is the converging evidence suggesting that a substantialfraction of these channels are localized to relatively distal den-drites (Karst et al., 1993; Markram and Sakmann, 1994; Mageeand Johnston, 1995; Kavalali et al., 1997; Mouginot et al., 1997).

As a result of this subcellular di stribution, recordings from intactneurons are subject to voltage- and space-clamp errors that canhave a dramatic impact on the apparent voltage- and time-

Figure 7. Differential accumulation of C aVT transcripts in the spinal cord and sensory ganglia. Left panels show low-power dark-field images oftransverse sections through the lumbar spinal cord. All three transcripts were present in the dorsal horn (asterisks), with1H mRNA limited to neuronsof the ex ternal lamina. Note also that1G and 1H were ex pressed in motor neurons in the ventral horn (open arrows).Middle panelsshow high-powerbright-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 to1H (arrowhead). Scale bar, 400m (left panels); 25 m (middleand right panels).



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dependent properties of T-type calcium currents generated atdistal sites (Destexhe et al., 1996, 1998). However, in dissociatedpreparations where electrical recordings only sample currentsfrom the soma and proximal dendrites, the complement of cal-cium channels of a cell may be misrepresented. Therefore, inaddition to some of the more obvious caveats to comparison (e.g.,species or age differences in the animals being compared), it isimportant to note that definitive identification and characteriza-

tion of T-type currents in neurons is difficult and may even besubject to false negatives.

One region where there were discrepancies between CaVTexpression and T-type current recordings was the cerebellum. Forexample, Purkinje cells are most often associated with theirprominent P-type HVA calcium currents (Mintz et al., 1992), andit has been suggested that T-type currents in these neurons, whichare robust in neonatal animals (Regan, 1991), might only betransiently expressed early during postnatal development (Usow-icz et al., 1992, but see Kaneda et al., 1990). However the datapresented here, which show ex tremely high levels of1G mRNAin adult Purkinje cells (Fig. 6), do not support this possibility.Moreover, our preliminary results indicate that expression of1GmRNA is present in the neonate and actually increases postna-tally (E. M. Talley and D. A. Bayliss, unpublished observations).

Similar to Purkinje neurons, cerebellar granule cells also havebeen studied extensively for the characterization of their HVAcurrent (Slesinger and Lansman, 1991; Pearson et al., 1995; Ran-dall and Tsien, 1995), and T-type currents are reported to beabsent in these neurons (Rossi et al., 1994). However, we found aprominent distribution of CaVT expression in these cells. It isnoteworthy that this distribution was heterogeneous. Althoughmoderate levels of1I were found evenly distributed in granulecells throughout the cerebellar cortex, 1G was found at highlevels only in the caudal lobules of both the vermis and thehemispheres (Fig. 6A,B). This type of gradient also has beenobserved for expression of KV4.2 and KV4.3 (Serodio and Rudy,

1998), two genes believed to contribute to transient subthresholdpotassium currents. Thus, based on the expression of CaVT- andKV4-family genes, we would expect to find differences in sub-threshold membrane properties of rostral and caudal cerebellargranule neurons.

Another area of the CNS where there i s curious inconsistencybetween our findings with regard to CaVT ex pression and exper-iments examining the characteristics of neuronal calcium currentsis the cerebral cortex. T-type calcium currents are thought to bemore prominent in pyramidal neurons of the neocortex (Giffin etal., 1991; Hamill et al., 1991) when compared with nonpyramidalcells. Furthermore, cells with measurable T-type current werepredominantly 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 accordwith those studies, we found that 1H expression is largelylimited to layer V pyramidal neurons. However, we also foundsubstantial expression of1G and 1I in all cortical layers, andthis expression did not appear to be limited to pyramidal neurons.Given the generalized expression patterns of1G and 1I in theneocortex, it is not clear why only a restricted population ofneurons in this region have been found to display T-type currents.

In addition to observing CaVT expression in neurons thoughtto be devoid of T-ty pe currents, there were also cells in which wefailed to find CaVT expression but which appear to have T-typecalcium currents. This was the case for olfactory mitral cells,

which have been shown to have LVA currents of modest ampli-

tude (Wang et al., 1996). However, although they appear to lackexpression of known CaVT gene-family members, these cells are

well stained by an antibody to the 1E (HVA family) calciumchannel (Yokoyama et al., 1995). It is possible that LVA currentsin these neurons receive a contribution from the 1E channel,

which can generate currents w ith some T-type properties (Soonget al., 1993; Meir and Dolphin, 1998) and may account for someof the LVA current in atrial myocytes (Piedras-Renteria et al.,

1997). An alternative possibility is that an as yet unidentified geneaccounts for the LVA currents in mitral cells. It is worth men-tioning at this juncture that in contrast to mitral cells, we foundhigh levels of expression of all three transcripts in granule cells ofthe olfactory bulb. C alcium currents in these neurons apparentlyhave not been extensively characterized (Bhalla and Bower,1993).

Heterogeneity of CNS T-type calcium currents

CNS T-type calcium channels are pharmacologically and physio-logically heterogeneous, leading to the hypothesis that at leastsome of their variability is generated by differences in genesencoding these channels. With respect to pharmacology, T-typecurrents have shown different sensitivities to block by a number ofcations and organic compounds (for review, see Akaike, 1991;Huguenard, 1996). Any discussion of the contribution of the threeCaVT gene products to this differential sensitivity is preliminarybecause the pharmacological profiles of these clones have notbeen extensively characterized. It can be pointed out, however,that we do not see any correlation between the expression profilesof any of the three CaVT transcripts and regions where particularpharmacological attributes have been observed. For example,differential sensitivities to both nickel and amiloride have beennoted between DRG neurons and pituitary cells (Todorovic andLingle, 1998), two regions where we see predominant ex pressionof1H mRNA. Conversely, thalamic reticular and relay neurons,

which express different CaVT transcripts, have similar sensitivi-

ties to both nickel and amiloride (Huguenard and Prince, 1992).Physiological characterization of the three CaVT genes hasbeen performed, and there appears to be some correlation be-tween expression of these genes and the properties of T-typecurrents in different CNS neurons. When expressed in the samecell type (HEK 293 cells), 1G and 1H generate currents withsimilar kinetic and voltage-dependent properties. In contrast, 1Icurrents have slower kinetics and depolarized voltage depen-dence of activation, when compared with the other two channels(Lee et el., 1999).

The contrast in properties between the1I channels and thoseof1G and1H corresponds to a difference in T-type currents indifferent cells of the thalamus, a region where these currents havebeen extensively characterized both in dissociated and intact

preparations (Destexhe et al., 1996, 1998). When calcium currentswere compared directly in thalamic reticular and thalamic relayneurons (Coulter et al., 1989a; Huguenard and Prince, 1992), it

was found that the LVA component was di stinctly different in thetwo cell types. Thalamic reticular neurons, which express highlevels of 1I mRNA along with moderate levels of 1H, hadslower kinetics of activation and inactivation when compared withthalamic relay neurons of the ventral basal complex, a region thatappears to exclusively express 1G. Moreover, in reticular neu-rons the voltage range of activation was depolarized relative tothat of relay neurons. Given data from the expressed channels, itappears that the slower kinetics and depolarized voltage depen-dence of activation in reticular neurons may result at least in part



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from their expression of 1I. Consistent with this hypothesis,neurons of the lateral habenula, which express both 1G and1I(Figs. 4H,I, 5), were found to have T-current with physiologicalproperties intermediate between those of reticular and relayneurons (Huguenard et al., 1993).

It should be pointed out that in addition to CaVT (1I and1H) expression, thalamic reticular neurons also apparently ex-press somewhat elevated levels of1E (Soong et al., 1993; Wil-

liams et al., 1994; but see Yokoyama et al., 1995). As noted above,this gene is more rapidly inactivating and has a more hyperpo-larized threshold for activation than other genes of the HVAfamily (Soong et al., 1993). Therefore, it may be that a combina-tion of 1E, 1H, and 1I contribute to the LVA current inthalamic reticular neurons. In addition, we cannot rule out thepossibility that there may be as yet uncloned genes that generateand/or modify LVA currents in these and other CNS neurons.

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Differential Distribution of Three Members of a Gene Family Encoding Low Voltage-Activated (T-Type) Calcium Channels