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

Olfactory system

All three probes gave prominent labeling in the olfactory bulb (MOB; Figs. ​Figs.11A, ​,2).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 α1G, whereas α1I was present at very high levels in scattered members of this cell population (Fig. ​(Fig.22).

Moderate to high levels of message were also found for all three genes in olfactory cortical structures, including the anterior olfactory nucleus (AO; Fig. ​Fig.11A) and piriform cortex (Pir; Fig.​Fig.11B–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 α1G mRNA (Figs. ​(Figs.11B–D, 2). Instead, it contained very high levels of α1H in the dense cellular layer as well as very high levels of α1H and α1I in the cellular islands of Calleja.

Basal forebrain

α1H and α1I transcripts were both present throughout the striatum (CPu; Figs. ​Figs.11C–I, 4) and the accumbens nucleus (Acb; Fig. ​Fig.11B,C). α1H was detected at moderate levels in these structures, whereas α1I 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. α1G 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.​(Fig.11E–H). Another basal ganglia structure, the subthalamic nucleus (STh; Fig. ​Fig.11I) contained high levels of α1I, moderate levels of α1G, and only scattered expression of α1H.

In the septum (Fig. ​(Fig.11C–E), labeling was moderate for α1G 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). α1H labeling was found at low levels in all parts of the lateral and medial septum and absent in the triangular septum (TS). Labeling for α1I was opposite to that of α1H; 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. ​Fig.11C), which had very high levels of α1H as well as low levels of α1G.

The BST (Fig. ​(Fig.11D,E) displayed expression for all three genes, but only in the medial edge of the posterior region of the nucleus. In this restricted area, α1G expression was quite high, α1H was moderate, and α1I was low. In the rest of the nucleus, levels of α1G and α1H expression were low, and α1I expression was not detected. Another basal forebrain structure, the claustrum (Cl; Fig.​Fig.11B–F), also had prominent α1G expression along with low levels of α1I.

Amygdala

In the amygdala, α1G and α1H were predominant, with α1I mRNA expressed at low levels in most nuclei (Fig.​(Fig.11G–K). Highest levels of all three genes were found in the nucleus of the lateral olfactory tract (LOT; Fig.​Fig.11F) and the related bed nucleus of the accessory olfactory tract (BAOT; Fig. ​Fig.11G). In the central nucleus (Ce; Fig. ​Fig.11G,H) and the lateral nucleus (La; Fig. ​Fig.11G–I), α1H expression was uniformly low, but α1G 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.​Fig.11H,I) than in the anterior part (MeA; Fig.​Fig.11G). Expression for α1G and α1H was most evident in the caudal tip of this nucleus, as shown in Figure​Figure11I.

Hippocampal formation

In many CNS regions, the hybridization levels of the α1G probes tended to be higher than those of the other two sets of probes. Such was not the case for the hippocampal formation (Fig.​(Fig.11G–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 Ca_VT expression in the nonpyramidal cell layers of Ammon’s Horn was, for the most part, restricted to α1G. In the granule cell layer of the dentate gyrus, α1H 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 ​Figures11A–L and 4, α1G and α1I 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 α1G was present in the deepest part of layer VI. These intensely labeled cells appeared to be restricted to the insular/perirhinal cortices (Ins, Fig.​Fig.11B–G; PRh, Fig.​Fig.11H–K) and secondary somatosensory areas (e.g., Fig. ​Fig.11E). With respect to particular cell types, probes for α1G and α1I 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 α1H. 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 α1H-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. ​(Fig.11B). The other structure, the Pir (Fig. ​(Fig.11B–I), had mixed expression levels of all three genes. Both α1H and α1I were present in high amounts in the superficial neurons of this cortical region but were not detected in deeper layers. On the other hand, α1G 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.​Fig.11C–J).

Thalamus

The thalamus (Figs. ​(Figs.11E–K, 5) was characterized by high levels of expression of α1G 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 α1G. Low levels of α1I were seen in the anterior dorsal nucleus (AD; Fig.​Fig.11F) and the rostral part of the lateral dorsal nucleus (LD; Fig. ​Fig.11G); low to moderate levels were seen in the ventral lateral geniculate nucleus (VLG; Fig.​Fig.11J). Expression of α1H 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. ​Fig.11G,H), paraventricular thalamic nucleus (PV; Fig. ​Fig.11F–I), the rhomboid (Rh; Fig. ​Fig.11G), and reuniens (Re; Fig.​Fig.11G,H) nuclei had even higher levels of α1G expression. Low levels of α1I 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 α1G expression (Fig. ​(Fig.5,5, left panels). Instead, these neurons expressed high levels of α1I and moderate levels of α1H. The lateral habenula (Fig. ​(Fig.55,right panels) had an uneven distribution of α1G and α1I; both transcripts were detected at high levels in the rostral portion of the nucleus. Caudally, α1I expression was moderate, and α1G expression was low (Fig. ​(Fig.1,1, compare H,I). Ca_VT expression was not detected in neurons of the medial habenula, a region characterized by high levels of mRNA encoding α1E (Soong et al., 1993; Williams et al., 1994).

Hypothalamus

In the hypothalamus, α1G was predominant rostrally. In the preoptic region (Fig. ​(Fig.11D–F), α1G 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.​Fig.11E,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 α1G mRNA in the ventrolateral portion of the VMH (Fig.​(Fig.11H,I). Expression patterns in the mammillary bodies were somewhat complex, with each transcript showing a unique expression pattern (Fig. ​(Fig.11J). In the medial mammillary nucleus, the medial part (MM) showed a predominance of α1H expression, whereas the lateral part (ML) displayed α1G and α1I. In the lateral mammillary nucleus (LM), α1G 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. ​Fig.11K–M), with α1H and α1I at low levels and α1G at low to moderate levels, except in the external gray area of the superior colliculus, where α1G labeling was high (Fig. ​(Fig.11K). All three transcripts also were found in the pretectal nuclei (MPT and APT; Fig. ​Fig.11J), which were notable as a result of high α1H labeling in scattered cells of the anterior pretectal nucleus (APT). The periaqueductal gray (PAG; Fig. ​Fig.11J–L) had moderate levels of labeling for α1G and low levels of α1H. More caudally, the tegmental nuclei ventral to the fourth ventricle (Fig.​(Fig.11M,N) were a little more heterogeneous, with α1G mRNA at high levels in the posterodorsal tegmental nucleus (PDTg; Fig. ​Fig.11N) and α1I present in the laterodorsal nucleus (LDTg; Fig. ​Fig.11M).

Labeling for α1G and α1H were found at low levels in the raphe nuclei (Fig. ​(Fig.11K,L). These two transcripts were also found in the substantia nigra (SN; Fig.​Fig.11J,K); in the compact part α1G labeling was moderate and α1H low, whereas the reticular part was for the most part unlabeled, except for some cells that contained α1H (data not shown). All three transcripts were found in the interpeduncular nucleus (IP; Fig. ​Fig.11K) with α1G and α1H at moderate levels and α1I at low levels. The lateral parabrachial nucleus (LPB; Fig.​Fig.11M,N) had low levels of α1G and α1H, except for the external portion, which had moderate to high levels of α1G (Fig. ​(Fig.11M). One other area of note was the pontine nuclei (not shown), which had low to moderate levels of both α1G and α1I.

Cerebellum and inferior olive

Expression of Ca_VT transcripts in the cerebellum is illustrated in Figures ​Figures11M–P and 6. α1G expression was strikingly high in every Purkinje cell examined, and granule cells were labeled with α1G 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). α1I mRNA also was present in the granule cell layer at moderate levels, although it lacked the graded expression seen for α1G. The InO (Figs. ​(Figs.11O,P, ​,6)6) also contained α1G and α1I mRNA; in this case α1G was expressed at very high levels throughout the nucleus, whereas α1I 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 α1G mRNA once again generated a higher signal than did those for α1H and α1I. Labeling for all three transcripts was sparse or absent in brainstem reticular areas, including those of the midbrain and pons (Fig.​(Fig.11L–N). In the medullary reticular fields, α1G was present at low levels in the lateral but not the medial areas (Fig. ​(Fig.11O, compare PCRt, Gi); α1I was found in the lateral reticular nucleus (Fig.​(Fig.11P).

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. ​(Fig.1,1, compare O, P). All three also were seen in the dorsal cochlear nucleus (data not shown), but only α1G was present (and at lower levels) in the ventral cochlear nucleus (VC; Fig.​Fig.11M,N). Similarly, all three transcripts were present in the dorsal horn of the spinal cord (Fig.​(Fig.7).7). α1G was present at moderate levels; α1H was for the most part restricted to the outermost layers (layers 1–2). α1I was somewhat more evenly distributed at low levels, but it was more prominent in layers 3–4. Only α1G and α1H were detected in the nucleus of the solitary tract (Sol; Fig. ​Fig.11O,P).

Somatic motor neurons in the brainstem and spinal cord contained α1G and α1H mRNA, at moderate and low levels, respectively (Figs.​(Figs.11M,P, ​,7).7). Other labeled medullary regions included the inferior olive (discussed above) and the area postrema (AP; Fig. ​Fig.11P); in the AP all three transcripts were found, with α1G at high levels.

Ca_VT transcripts are expressed in cells with prominent T-type currents

As anticipated, we detected robust expression of Ca_VT 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 α1G mRNA to be predominant in thalamocortical relay areas but absent in the thalamic reticular nucleus, which instead expressed high and moderate levels of α1I and α1H 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 α1G 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 Ca_VT transcripts in the InO. Expression in the rostral part of the nucleus was restricted to α1G, whereas caudal expression consisted of both α1G and α1I (Figs.​(Figs.11O,P, ​,6).6). It remains to be determined how this differential distribution of Ca_VT expression in the inferior olive might contribute to the functional organization of olivocerebellar processing.

In addition to α1G and α1I, we also found abundant α1H 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, Ca_VT 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 Ca_VT 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 α1H and moderate levels of α1I mRNA. Expression of both transcripts was restricted to small- and medium-sized neurons; Ca_VT 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 Ca_VT 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 Ca_VT expression and where other investigators have failed to find LVA calcium current. For example, we saw no Ca_VT expression in the globus pallidus and saw only very low expression of one of the transcripts (α1G) 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 Ca_VT expression and recordings of T-type calcium currents

As noted above, we saw expression of Ca_VT 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 Ca_VT 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 Ca_VT 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 inHuguenard, 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 Ca_VT 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 seeKaneda et al., 1990). However the data presented here, which show extremely high levels of α1G mRNA in adult Purkinje cells (Fig. ​(Fig.6),6), do not support this possibility. Moreover, our preliminary results indicate that expression of α1G 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 Ca_VT expression in these cells. It is noteworthy that this distribution was heterogeneous. Although moderate levels of α1I were found evenly distributed in granule cells throughout the cerebellar cortex, α1G was found at high levels only in the caudal lobules of both the vermis and the hemispheres (Fig.​(Fig.66A,B). This type of gradient also has been observed for expression of K_V4.2 and K_V4.3 (Serodio and Rudy, 1998), two genes believed to contribute to transient subthreshold potassium currents. Thus, based on the expression of Ca_VT- and K_V4-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 Ca_VT 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 α1H expression is largely limited to layer V pyramidal neurons. However, we also found substantial expression of α1G and α1I in all cortical layers, and this expression did not appear to be limited to pyramidal neurons. Given the generalized expression patterns of α1G and α1I 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 Ca_VT expression in neurons thought to be devoid of T-type currents, there were also cells in which we failed to find Ca_VT 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 Ca_VT gene-family members, these cells are well stained by an antibody to the α1E (HVA family) calcium channel (Yokoyama et al., 1995). It is possible that LVA currents in these neurons receive a contribution from the α1E 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).