Differential expression of voltage-gated calcium channels in identified visual cortical neurons

Neuron,

Vol. 6, 321-332, March, 1991, Copyright

0 1991 by Cell Press

Differential Expression of Voltage-Gated Calcium Channels in Identified Visual Cortical Neurons Kelleen Giffin,* Joel S. Solomon,* Andreas Burkhalter,+ and Jeanne M. Nerbonne* * Department of Pharmacology + Department of Neurosurgery Washington University School of Medicine St. Louis, Missouri 63110

Summary Using the whole-cell patch-clamp technique, Ca*+ channel currents were examined in three distinct types of neurons derived from rat primary visual cortex. Callosal-projecting and superior colliculus-projecting neurons were identified following in vivo retrograde labeling with fluorescent “beads.” A subset of intrinsic CABAergic visual cortical neurons was identified with the monoclonal antibody VCl .l. Although high voltageactivated Caz+ channel currents were measured in all three cell types, clear differences in the densities of these channels were observed. There were also marked variations in the relative amplitudes of the inactivating and noninactivating components of the high voltage-activated currents, suggesting that N- and l-type Caz+ channels are differentially distributed. Although low voltageactivated or T-type currents were measured in subsets of both types of projection neurons, they were not observed in VCl.l-positive cells. These results provide a direct demonstration that voltage-gated Caz+ channels are expressed in neurons of the mammalian visual cortex and reveal that the distributions and densities of different Caz+ channel types in diverseclassesof visual cortical neurons are distinct. Introduction In the peripheral nervous system, it iswidelyaccepted that Ca2+ influx through voltage-gated Ca2+ channels is an important signal in the regulation of a variety of neuronal functions, including the control of membrane excitability and the regulation of neurotransmitter release. Voltage-dependent Ca2+ entry has also been suggested to play a number of other important and diverse roles in the functioning of the central nervous system. It has been proposed, for example, that Ca2+ influx is involved in the generation and the spread of epileptiform activity in the neocortex and hippocampus (Prince, 1986; Pumain et al., 1986) and in the cascade of events leading to excitation-induced cortical cell death (Prince, 1986; Weiss et al., 1990). In addition, in the visual cortex, voltage-dependent Ca2+ influx has been postulated to play a role in control of cortical plasticity during development (Singer, 1985; Geiger and Singer, 1986) and in the induction and maintenance of long-term synaptic potentiation (Artola and Singer, 1990). In spite of their presumed importance and in marked

contrast to other neuronal cell types (for review see McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989), the properties of voltage-gated Ca2+ channels in neurons of the mammalian visual cortex have not been characterized. This is perhaps not surprising, since the cellular heterogeneity of the cortex and the difficulties associated with identifying specific cell types have limited study of the intrinsic membrane properties of cortical neurons in general. Nevertheless, there is evidence to suggest that voltage-gated Ca2+ channels are present in at least some types of neocortical neurons. In dissociated cortical cultures, for example, Ca*+-dependent action potentials have been measured in the presence of tetrodotoxin when outward K+currents were partially suppressed by the addition of extracellular tetraethylammonium of intracellular Cs+ (Dichter et al., 1983; Galvan et al., 1986). Ca2+dependent action potentials have also been demonstrated in analogous experiments in explant cultures (Wolfson et al., 1989) and cortical slices (Connors et al., 1982; Franz et al., 1986; McCormick and Prince, 1987). More direct evidence for the presence of voltage-gated Ca*+ channels in neocortical neurons has been provided by voltage-clamp studies (Franz et al., 1986; Galvan et al., 1986; Sutor and Zieglggnsberger, 1987). In cells loaded with intracellular Cs+, for example, time- and voltage-dependent inward currents that were blocked by the addition of extracellular Cd2+ or Mn2+ were measured during membrane depolarizations to potentials positive to -40 mV (Franz et al., 1986; Galvan et al., 1986). By analogy to other cells (McCleskey et al., 1986; Tisen et al., 1988; Bean, 1989), these results suggest the presence of high voltage-activated (HVA) Ca2+ channels. More recently, Ca2+-dependent inward currents that begin to activate at relatively hyperpolarized potentials have been measured in cortical slices (Sutor and ZieglgPnsberger, 1987), suggesting that low voltage-activated (LVA) Ca2+ channels are also present in some neocortical neurons (Friedman and Gutnick, 1987). This study was undertaken to examine directly the properties of voltage-gated Ca*+ channels in neurons of the mammalian visual cortex and to explore the possibility that the types and densities of voltagegated Ca2+ channels vary in different visual cortical cell types. To achieve these goals, we combined the whole-cell patch-clamp recording technique (Hamill et al., 1981) with methods that allow the in vitro identification of visual cortical cell types and we examined Ca2+ channel currents in isolated, identified neurons derived from the rat primary visual cortex. Callosalprojecting and superior colliculus-projecting (referred to here as colliculus-projecting) cortical neurons were identified in vitro following in vivo retrograde labeling with fluorescent latex microspheres or “beads” (Katz et al., 1984; Huettner and Baughman, 1986,1988; Katz and larovici, 1990). A subset of intrinsic, GABAergic

Neuron 322

Figure 1. Bead-Labeled Colliculus-Projecting and Callosal-Projecting Neurons of the Visual Cortex Coronal section through the primary visual cortex of a P8 animal following rhodamine bead injection into the ipsilateral superior colliculus (A) and fluorescein bead injection into the contralateral area 17 (B). Both pictures are of the same section photo graphed under rhodamine (A) or fluorescein (B) optics. Cortical layers were assigned from Nissl staining of adjacent sections. Bar, 200 urn.

Figure 2. Isolated, jetting and VCl.l+ Cortex

Identified Neurons

Callosal-Pro of the Visual

(A and B) Dissociated bead-labeled callosal-projecting cortical neuron isolated at P8 and visualized approximately 12 hr after plating under epifluorescence (A) and phase-contrast (B) illumination. (C and D) VCl.l+ cortical neuron isolated at P8 and visualized approximately 12 hr after dissociation under epifluorescence (C) and phase-contrast (D) illumination. This culture was fixed prior to exposure to VC1.l, and antibody labeling was completed as described in Experimental Procedures. Bars, 50 pm.

Ca2+ Channels 3 !3

in Identified

Cortical

Neurons

visual cortical neuronswas identified usingthe monoclonal antibody VC1.l (Arimatsu et al., 1987; Naegele et al., 1988). The results reveal that HVA Ca2+ channels are present in all threetypes of visual cortical neurons, although the densities of these channels are significantly lower in VCl.l-positive (VCl.l+) cells than in both types of projection neurons. Currents through LVAor T-type Ca*+ channels, in contrast, were measured cnly in subsets of callosal- and colliculus-projecting cells and were never observed in VCl.l+ intrinsic neurons. These results provide a direct demonstration that voltage-gated Ca*+ channels are present in visual cortical neurons and reveal that the distributions and densities of different types of voltage-gated Ca2+ chanreels in anatomically and functionally diverse classes of visual cortical neurons are distinct.

Kesults Identification of Cortical Cell Types :n cortical sections, callosal-projecting and colliculusprojecting cellswere identified following in vivo retrograde labeling with fluorescent latex microspheres or beads (Katz et al., 1984; Katz and larovici, 1990). As shown in Figure IA, colliculus-projecting neurons \,vere confined entirely to layer 5 (Sefton et al., 1981). :n contrast, callosal-projecting cells (Figure IB) were distributed throughout layers 2-6 (Olavarria and Van ‘iluyters, 1985). Although there are layer 5 cells pro:ecting to both targets, we have never seen doubleabeled cells either in cortical sections or in dissocisited cultures, indicating that colliculus-projecting ctnd callosal-projecting cells are distinct classes of corical projection neurons. Bead-labeled callosal- and :olliculus-projecting cells were also readily identified n dissociated cell cultures (Figure 2). Typically, bead,abeled cells represented 2%-3% of the neurons dis‘iociated from postnatal days 8 to 14 (PB-P14) animals. Because beads do not fade or diffuse from isolated cells n vitro, labeled neurons can be identified in freshly

dissociated preparations, as well as in long-term cultures. Also, as discussed previously by Huettner and Baughman (1986), we found no evidence that the presence of beads in dissociated callosal-projecting or colliculus-projecting cells influences subsequent survival in vitro: cell counts completed hours after dissociation and following several days in vitro reveal no measurable differences in the percentage of beadlabeled cells (J. P. Doyle and J. M. Nerbonne, unpublished data). The monoclonal antibody VC1.l (Arimatsu et al., 1987; Naegele et al., 1988), generously provided by Dr. Janice Naegele, has been shown to identify a subset of nonpyramidal interneurons in the cat visual cortex that express GABAergic properties (Naegele et al., 1988; Naegele and Barnstable, 1989). In rat area 17, we find that VCl.l+ neurons (Figure 3) also show nonpyramidal morphology, suggesting that this antibody recognizes a similar subset of intrinsic cortical neurons in the rat. In dissociated cultures, VCl.l+ cells were visualized following antibody labeling under epifluorescence illumination (Figure 2C). Approximately 15% of the cells in cultures prepared from P8PI2 animals were VCl.l+. In addition, in separate experiments,wefound thatallVC1.1+cellswerestained following exposure to a monoclonal antibody against the GABA-synthesizing enzyme glutamic acid decarboxylase (Gottlieb et al., 1986), whereas only approximately 50% of the cells positive for glutamic acid decarboxylase are also VC1.1-C (J. M. Nerbonne and J. F. Doyle, unpublished data). Thus, we conclude that, similar to the results obtained in the cat primary visual cortex (Naegele et al., 1988), VC1.l identifies a subset of intrinsic GABAergic neurons in rat primary visual cortex. Periodic examinations of the cortical injection and tissue removal sites were made (see Experimental Procedures) to ensure that the injections and pieces of cortex removed for the preparation of dissociated cultures were confined exclusively to area 17. If histological examination of the tissue revealed that either the

Figure 3. VC1.l Identifies a Subset of Nonpyramidal Neurons in Rat Primary Visual Cortex Coronal section through area 17 photographed under bright-field illumination followingVC1.l stainingasdescribed inExperimental Procedures. There are nonpyramidal VCl.l+ cells in layers 213, 4, and 5 in this photograph. Cortical layers were assigned from Nissl staining of adjacent sections. Bar, 50 urn.

Neuron 324

injection or the tissue removal site extended beyond the borders of area 17, electrophysiological data obtained from labeled cells in that preparation were not subsequently analyzed. Ca*+ Channel Currents in Isolated, identified Neurons of the Visual Cortex In the experiments here, Ba2+ was used (rather than Ca*+) as the charge carrier through voltage-gated Ca2+ channels primarily because, in other cells, inward current amplitudes are larger and the rates of current activation and inactivation are reduced (Hagiwara and Byerly, 1981; Eckert and Chad, 1984; Bean, 1989). Because isolated cortical neurons extend neurites over time in culture, all recordings were obtained from cells within the first 16 hr after isolation to ensure adequate spatial control of the membrane voltage: In addition, cells that were largely devoid of processes were selected for recordings to ensure that space clamp was achieved in freshly isolated cells. Because preliminary experiments revealed that inward Ba*+ current (lea) amplitudes through voltage-gated Ca*+ channels in isolated visual cortical neurons were small relative to the amplitudes of voltage-activated Na+and K+ currents, experiments were conducted using a bath solution containing 140 mM tetraethylammonium-Cl (substituted for NaCI) to provide maximal suppression of inward Na+ and outward K+ currents. Theefficacyof K+and Na+current blockadewasexamined following blockade of Isa by the addition of 5 mM CoCI, to the bath. Under these conditions, no timedependent inward or outward currents were evident during depolarizing voltage steps to potentials between -60 and +50 mV from a holding potential of -80 mV. Thus, we conclude that the lea waveforms measured and analyzed here reflect the currents through voltage-gated Ca2+ channels, uncontaminated by inward Na+ and outward K+ currents. le. Waveforms in Isolated Cortical Projection Neurons Isa waveforms through voltage-gated Ca2+ channels measured in a typical isolated callosal-projecting neuron of the visual cortex (see also below) are shown in Figure 4. During depolarizations from a holding potential of -80 mV, lea begins to activate between -30 and -20 mV, peaks at +I0 mV, and appears to reverse at potentials positive to +50 mV (Figure 4A). In addition, at all test potentials, le, activates rapidly to a peak and subsequently decays to an apparent plateau level that is sustained for the duration of the (125 ms) voltage steps. Current waveforms recorded during prolonged (>250 ms) depolarizations are similar to those displayed, and little or no attenuation of the plateau current amplitude is evident during depolarizations lasting up to 1.5 s (data not shown). Thus, the measured current waveforms reflect the presence of inactivating and noninactivating (i.e., the plateau) current components. The current-voltage relations of the peak and plateau components of leaare indistinguish-

Figure 4. IBaWaveforms in an Identified ron of the Visual Cortex

CallosaCProjecting

Neu-

Currents, evoked during 125 ms voltage steps to potentials between -60 and +50 mV from holding potentials of -80 (A) and -40 (C) mV, were measured as described in Experimental Procedures. Voltage steps were presented in 10 mV increments at 5 s intervals; the voltage-clamp paradigms are illustrated below the current records. The numbers above the individual current traces correspond to the test potentials (see paradigm) at which the currents were evoked. On depolarization from -80 mV (A), 1~~begins to activate between -30 and -20 mV, peaks at +I0 mV, and appears to reverse positive to +50 mV. As is evident, IRa activates rapidly during depolarizations to all test potentials and subsequently decays to an apparent plateau level. (B) The (single-cell) current-voltage relation for the peak (closed circles) and the plateau (open circles) currents evoked during depolarizations from -80 mV (shown in [A]) are indistinguishable and are consistent with both components reflecting currents through HVA Ca*+ channels (see text). During depolarizations from a holding potential of -40 mV (C), current amplitudes at all test potentials are reduced. In addition, the currents do not decay measurably during 125 ms depolarizations. Similar IBawaveforms were recorded in 36 (of 44) callosal-projecting neurons. The numbers above the current records are cell identifiers and are included for our reference purposes only.

able (Figure 4B) and are consistent with both components reflecting currents through HVA Ca*+ channels (Carbone and Lux, 1987). In addition, because the peak IBa reflects the sum of the inactivating and the noninactivating current components, these results also suggest that the voltage dependences of activation of these two (HVA) current components are similar. By analogy to other cells, therefore, we speculated that the inactivating and the noninactivating current components likely reflected Ba*+ currents through N- and L-type Ca2+ channels, respectively (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989). In other cells, N-and L-type channels have been separated based on

Ca” Channels 3 !5

in Identified

0

l-10

Cortical

Neurons

1 l-20

Percent Inactivation

21-30

3

mV are similar to those evoked from -80 mV (Figure 4A), except that current amplitudes at all test potentials are reduced and the currents do not decay measurably during the voltage steps. The absence of the inactivating current component is consistent with the suggestion that it reflects the activation of N-type Ca2+ channels, which are inactivated at a holding potential of -40 mV (Tsien et al., 1988). Although Is,waveformssimilartothosedisplayed in Figure 4 were measured in 36 (of 44) callosal-projecting cortical neurons isolated at P8-PI4 (see also below), there were substantial variations in the relative amplitudes of the peak and plateau currents measured in different cells, suggesting that the relative contributions of the inactivating and nonina.ctivating current components to the peak IBawere variable. To quantify these differences, the percent contribution of the inactivating component to the peak current in individual cells was determined by dividing the amplitude of the decaying current component evoked at +I0 mV from a holding potential of -80 mV by the peak current evoked during the same voltage step. The procedure is illustrated in Figure 5A. These analyses revealed that the inactivating component contributed between 4% and 39% to the peak (Figure 5B), with a mean (*SD) of 22.3% + 8.0% (n = 36) (Table 1). If the interpretation concerning the origin of the inactivating and noninactivating components of the HVA current (see above) is correct, these results suggest that N- and L-type Cal+ channels coex.ist in all P&P14 callosal-projecting neurons, although the relative distribution of these channel types varies among cells. The variations in the percent contribution of the inactivating component to the peak current did not correlate with the age of the animal frorn which the cells were isolated, the absolute sizes of the cells, the peak current amplitudes, or the peak c:urrent densities, suggesting that it reflects differences in the distribution of N- and L-type Ca2+ channels in callosalprojecting neurons. It is important to note, however, that there is an alternative interpretation of the presence of inactivating and noninactivating components of the HVA Ba2+ currents. It is possible, for example, that the two current components reflect alternate modes of gating of a single type of HVA Ca2+ channel (SwanduIlaandArmstrong,1988). lfthisinterpretation

I

of the Peak

-igure 5. The Contribution of the Inactivating lent to the Peak HVA Current Varies among

Current Cells

Compo-

A) Protocol used to calculate the percent inactivation of the leak IBi during 125 ms depolarizations to +I0 mV from a holding lotentral of -80 mV. (B) Percent inactivation of the peak HVA 3a2‘ current, determined using the method illustrated in (A), n cailosal-projecting (open bars), colliculus-projecting (closed Jars), and VCl.l+ (slashed bars) cortical neurons. For the projecion neurons, only those cells without measurable LVA currents ire included in the histogram. The mean (f SD) percent inactivaion of the peak current was 22.3% + 8.0% for callosal-projecting :ells (t-1 = 36), 8.7% t 7.1% for colliculus-projecting cells (n = 15), and 12.5% + 8.8% for VCl.l+ cells (n = 12) (see text and Table ‘I).

differing voltage dependence of steady-state inactiva.ion (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989). Therefore, lea waveforms in callosal-projecting :ells were also measured during voltage steps from nore depolarized holding potentials (which should nactivate N-type Ca*+ channels). As shown in Figure K, currents evoked from a holding potential of -40

Table ‘I. BaF Currents

in Isolated,

Identified

Neurons

of the Visual

Cortex

Peak IBa Range

Peak IBa

Peak 1~~Density

Cell Type

(PAP

(pA)a”

CpNpW

Percent Inactivatior+

n

Callosal-projecting

128-2053 128-2053 298-850 128-1787 120-618

691 662 510 520 270

42.6 42.0 47.7 44.7 27.0

22.3 22.6 8.7 14.5 12.5

36’ 44d 15’ 32d 12

Colliculus-projecting vc1.1+ il Measured b Values are c Only those d All P8-PI4

+ + * + f

425 406 292 361 158

during depolarizations to +I0 mV from a holding potential mean f SD. cells without measurable LVA currents are included. cells examined are included.

f i f f f

23.2 22.6 23.0 24.7 14.5

f f + f f

8.0 7.9 7.1 10.2 8.8 -

of -80 mV.

--

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-300v Figure 6. LVA Ca2+ Channels Projecting Cortical Neurons

Are Present in a Subset of Callosal-

Currents were measured as described in Figure 4. (A) In this cell, IBa begins to activate between -50 and -40 mV, and the waveformsofthecurrentsatthemorehyperpolarizedtestpotentials are transient; this is clearly evident in (B), where the currents evoked during depolarizations to -60 to -10 mV are shown at higher gain. The numbers above the current records are cell identifiers and are included for our reference purposes only. (C) Single-cell current-voltage relation for the peak (closed circles) and plateau (open circles) components of the current. The peak current begins to activate at a potential approximately 20 mV more hyperpolarized than the plateau current, consistent with the presence of LVA Ca2+ channels in this cell. Similar current waveforms were recorded in 8 (of 44) callosal-projecting cells.

is correct, our results suggest a cell-to-cell variability in the distribution of these two gating pathways. Resolution of this issuewill require pharmacological analyses of the currents and examination of the properties of single HVA Ca*+ channels (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989). In addition to differences in the relative amplitude of the inactivating and noninactivating components, there were also substantial variations in the absolute peak HVA current amplitudes measured in callosalprojecting cells. Peak 1~~ amplitudes evoked at +I0 mV from a holding potential of -80 mV, for example, varied over the range of 128-2053 pA, with a mean (+ SD) value of 691 & 425 pA (n = 36) (Table 1). Because there were also differences in cell sizes, the peak IBa amplitude in each cell was normalized to the measured whole-cell membrane capacitance (see Experimental Procedures) to allow comparison of peak Isa densities among cells. For all P8-PI4 callosal-projetting cells, peak le, densities varied over the range of 16-109 pA/pF, with a mean (f SD) of 42.6 + 23.2 pA/ pF (n = 36) (Table 1, see also below).

In contrast to the results above, inward BaL’ current that begin to activate between -50 and -40 mV (Figure 6) were measured in 8 (of the 44) P8-PI4 callosal-projetting cells examined. In addition, the currents evoked at the more hyperpolarized test potentials (i.e., -40 and -30 mV) in these 8 cells were distinct from those evoked at more positive test potentials (Figure 6B). In particular, the currents evoked at hyperpolarized test potentials activate and inactivate rapidly and are transient (Figure 6B). During depolarizations from a holding potential of -40 mV, 1~~waveforms measured in these 8 cells were similar to those displayed in Figure 4C (data not shown). The peak current begins to activate between -50 and -40 mV (Figure 6C), consistent with the presence of LVA or T-type Ca2+ channels (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989). The amplitude of the LVA current is small relative to the amplitude of the HVA current for this cell, and similar results were obtained in all 8 callosal-projetting cells in which LVA currents were measured. On average, the peak amplitude of the LVA current at -30 mV was 5% of the amplitude of the peak inward current at +I0 mV. Nevertheless, these results do indicate that LVA Ca*+ channels coexist with HVA Ca*+ channels in a subset (8 of 44, or 18%) of P8-PI4 callosal-projecting cortical neurons. Interestingly, the presence of a measurable LVA current did not correlate with the absolute amplitude of the HVA current or the peak HVA current density measured in the same cell. In addition, when these 8 cells were included in the analyses of peak HVA current amplitudes and densities, the mean values for both parameters were not significantly (p > 0.05) different from those obtained for cells in which only HVA currents were measured (Table 1). In parallel experiments, le. waveforms in colliculusprojecting cortical neurons were examined. In one group of cells (n = 17), depolarizations from a holding potential of -80 mV revealed inward Ba*+currents that begin to activate between -50 and -40 mV, whereas in the other group of cells (n = 15), inward currents that begin to activate between -30 and -20 mV were measured. By analogy to the results obtained in callosal-projecting cells, these findings suggest that LVA or T-type Ca*+ channels are present in a subset (17of 32, or 53%) of colliculus-projecting cells. As observed for callosal-projecting cells, however, the amplitudes of the LVA currents in colliculus-projecting cells (n = 17) were small relative to the amplitudes of the HVA currents measured in the same cell. The relative amplitudes of the peak and plateau componentsoftheHVAcurrentsalsovariedmarkedly amongcolliculus-projectingcells.Thecontributionof the inactivating component to the peak Isa for colliculus-projecting cells in which only HVAcurrents were measured varied over the range of 0%-24% (Figure 5B), with a mean (+ SD) of 8.7% + 7.1% (n = 15) (Table 1). These variations did not correlate with the peak current amplitude, the age of the animal from which the cells were isolated, or with the absolute cell size.

Za*’ Channels

in Identified

Cortical

Neurons

327

Figure7. Neurons

Only

HVA

Currents

Are Present

in VCl.l+

Cortical

(A and C) I& waveforms in an isolated VCl.l+ cell identified following electrophysiological recording were recorded as described in Figure 4 during voltage steps from holding potentials of -80 (A) and -40 (C) mV. (B) Mean (+ SEM) normalized currentvoltage relation for the peak IB. measured in VCl.l+ cells (n = 12). The numbers above the current records are cell identifiers

and are included for our reference purposes only.

Similar results were obtained when cells containing both LVAand HVAcurrentswere included in theanalyses (Table 1). Thus, we suggest that the difference in the relative peak-to-plateau lea current amplitudes reflects the variable distribution of L- and N-type (HVA) Ca2+ channels in colliculus-projecting cells. In addition, the finding that in 4 cells there was no measurable decay of the peak HVA current (Figure 56) suggests that in a subset (4 of 32, or 25%) of colliculusprojecting cells only L-type channels are present, i.e., no N- or T-type channel currents were evident. lea Waveforms in Isolated VCl.l+ Cortical Neurons To compare the properties of the currents through voltage-gated Ca*+ channels in cortical projection neurons with those in intrinsic cortical neurons, lea waveforms in a subset of GABAergic interneurons, identified with the monoclonal antibody VC1.l (Arimatsu et al., 1987; Naegele et al., 1988), were measured and analyzed. In initial experiments, VCl.l+ cells were identified prior to electrophysiological recordings (see Experimental Procedures), and isa waveforms consistent with the presence of HVA Ca*+ channels were measured. Peak lea amplitudes measured in these cells, however, were much smaller than those

measured in both types of projection neurons. In addition, the pattern of VC1.l labeling was markedly different from that seen on cells in fixed cultures. Specifically, the labeling was punctate in appearance, suggesting “capping” and possibly internalization of the antigen-antibody complexes. As a result, we were concerned that the labeling procedure itself may have altered the membrane properties of VCl.l+ cells. To examine this possibility directly, dissociated cortical neurons were plated on gridded coverslips (see Experimental Procedures), and electrophysiological recordings were obtained from cells selected at random. The locations on the grids of the cells from which recordings were obtained were noted, and following recordings, the cultures were fixed and stained for VC1.l. Representative 1~~waveforms measured in a VCl.l+ cell in one such experiment are displayed in Figure 7. The mean normalized current-voltage relation for the currents evoked from a holding potential of -80 mV in VCl.l+ cells, identified beforeand after electrophysiological recordings (n = 12), is displayed in Figure 7B. The properties of the measured currents are consistent with the presence of HVA Ca2+ channels, and in none of the VCl.l+ cells examined (n = 12) were LVA Ca2+ channel currents measured. In addition, although complete current-voltage curves were not obtained, similar results were obtained in 7 other VCl.l+ cells. Thus, in contrast to the results on cortical projection neurons, we found no evidence that LVA Ca2+ channels coexist with HVA channels in VCl.l+, CABAergic cortical interneurons (n = 19). The experiments completed on VCl.l+ cells identified after recordings confirmed the finding for prelabeled cells that peak leaamplitudes in VCl.l+ cells are lower than those in projection neurons. For all VCl.l+ cells, peak inward currents varied over the range of 120-618 pA, with a mean of 270 f 158 pA (n = 12) (Table 1). When current amplitudes were normalized for differences in cell size, the difference between projection neurons and VCl.l& cells was also evident. The mean (+ SD) peak lea density in VCl.l+ cells was 27.0 f 14.5 pA/pF (n = 12), a value that is significantly lower (p < 0.01) than that observed in callosalor colliculus-projecting cells (Table 1). The relative contribution of the inactivating component to the peak lea amplitude also varied markedly among VCl.l+ cells (Figure 5). For all cells, the percentage of the inactivating current component varied over the range of 0%-24%, with a mean of 12.5% f 8.8% (n = 12) (Table 1). Thus, on average, there was significantly (p < 0.001) less decay of the peak current in VCI .I+ than in callosal-projecting cells. In addition, in 2 of the 12 VCl.l+ cells, there was no measurable decay of the peak current during 125 ms voltage steps, suggesting that on/y L-type Ca2+ channels are present in a subset (2 of 12, or 16%) of VCl.l+ cells. Developmental Changes in HVA Ca2+ Channel Densities in Visual Cortical Neurons As noted above, there were large variations

in the

Neuron 328

Table 2. Developmental

Changes

in Ca If Channel

Postnatal

Age

Density

in Cortical

Projection

Neurons

Peak IBa

Ct.

Peak IB1 Density

Cell Type

(days)

(pAPb

(pFP

(pA/pFP

n

Callosal-projecting

8-9 IO-12 13-14 8-9 IO-12 13-14

341 676 1079 438 424 652

32.3 42.1 55.3 36.8 44.8 49.4

15 18 11 8 11 13

Colliculus-projecting

+ f f + f f

174 274 423 143 274 464

10.8 18.4 24.2 13.2 9.0 13.1

+ + k f + ?r

3.5 8.1 13.8 6.9 2.7 5.6

d Measured during depolarizations to +I0 mV from a holding potential of -80 mV. b Values are mean f SD. c Determined from integration of the capacitative transients evoked during 55 mV voltage

peak amplitudes of the HVA IBa waveforms in P8-PI4 cortical projection neurons (Table 1). Because the ceils were isolated at different ages, variations in peak HVA current densities could reflect developmental changes in Ca2+ channel densities. As shown in Table 2, mean peak current amplitudes in callosal-projecting cells increased during development from P8 to P14. In addition, cell membrane capacitances increased during this period, consistent with the visual observation that cells isolated from older animals were larger. Nevertheless, when peak HVA 1~~amplitudes were normalized for differences in cell size, peak lea densities increased significantly from P8 to PI4 (Table 2). Similar, although somewhat smaller, increases in peak HVA current densities were measured in colliculus-projetting cells (Table 2). Because of the small number of VCl.l+ cells examined, the possibility that there are also developmental variations in HVA current densities in these cells was not explored. Discussion The results presented here reveal that voltage-gated Ca*+ channels with properties similar to those of HVA and LVA Ca2+ channels in other cells (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989) are present in neurons of the rat visual cortex and that these channels are differentially distributed in different visual cortical cell types (Table 3). Currents through LVA or T-type Ca*+ channels, for example, were measured only in subsets of callosal-projecting (8 of 44, or 18%) and colliculus-projecting (17 of 32, or 53%) cells, and we found no evidence that LVA Ca2+ channels coexist with HVA channels in VCl.l+ cells (n = 19) (Table 3). In addition, although HVA channels were evident in all cells, the densities of these channels were significantly (p < 0.001) higher in the projection neurons

Table 3. Differential

Distribution

of Different

Ca2+ Channel

?r. 13.4 f 21.0 f 27.6 + 13.6 f 25.7 f 27.8

steps from a holding

potential

of -60 mV

than in the intrinsic neurons (Table 1). There were also marked differences in the relative contribution of the inactivating and the noninactivating HVA current components to the peak HVA currents measured in allthreecelltypes.Basedonthefindingthattheinactivatingcurrent component is not measured during depolarizations from a holding potential of -40 mV and by analogy to results obtained in other cells (McCleskey et al., 1986; Tsien et al., 1988; Bean, 1989), we suggest that these differences likely reflect the variable distribution of N-and L-type Ca*+ channels in visual cortical neurons. If this interpretation iscorrect, the absence of an inactivating component of the HVA current in subsets of colliculus-projecting (4 of 32, or 25%) and VCl.l+ (2 of 12, or 16%) cortical neurons suggests that, in these cells, on/y L-type Ca*+ channels are expressed (Table 3). Because the experiments here were conducted on cells within hours of dissociation, we suggest that the measured differences in Ca2+ channel distributions and densities reflect differences in the in vivo membrane properties of the cells at the time of isolation. We recognize, however, that it might be suggested that the isolation procedure altered cell membrane properties, Although we cannot exclude this possibility directly, we feel it is unlikely to account for our results for several reasons. First, all the experiments here were conducted on cells isolated using the same methodology. If the dissociation procedure preferentially affected some types of channels, variations in the densities of L-, N-, and T-type Ca*+ channels in different cell types would not be expected. Second, L-, N-, and T-type channel densities varied among cells in the same preparation, as well as among cells isolated at different developmental ages. Third, although it was not possible to demonstrate adequate voltageclamp control for quantitative studies, Ca*+ channel

Types

Cell Type

Percentage of Cells with LVA or T-type Channels

Percentage of Cells with L-type HVA Channels

Percentage of Cells with N-type HVA Channels

Total Number of Cells Examined

Callosal-projecting Colliculus-projecting VCl.l+ (intrinsic)

18 53 0

100 100 100

100 a0 84

44 32 19

:a*+ Channels I29

in Identified

Cortical

Neurons

currents were also measured in cells maintained for 24-72 hr in vitro, and the properties of the currents werequalitativelysimilartothose measured in freshly rsolated cells. Thus, we suggest that artifacts resulting from the dissociation procedures likely do not account for the observed differences (Table 3) in Ca2+ channel distributions and densities in different types of visual cortical neurons. It is tempting to speculate about the physiological role of voltage-gated Ca2+ channels in visual cortical neurons in general and about the observation that different Ca2+ channel types are differentially distributed in functionally distinct types of visual cortical neurons. By analogy to other cells (Llinas and Yarom, 1981; Lovinger and White, 1989; White et al., 1989), the presence of LVA or T-type Ca2+ channels might be expected to result in the firing of “bursts” of action potentials, rather than a single action potential, in response to brief current injection. Interestingly, “intrinsically bursting” cortical neurons have been observed in in vitro recordings from slices prepared from several cortical areas (Connors et al., 1982; McCormick et al., 1985; Agmon and Connors, 1989; Connors and Gutnick, 1990), including the rat primary visual cortex (Chagnac-Amitai et al., 1990; Mason and Larkman, 1990). These intrinsically bursting cells are large, excitatory pyramidal neurons whose cell bodies reside in cortical layers 4 and 5 (Connors et al., 1982; McCormick et al., 1985; Agmon and Connors, 1989; Chagnac-Amitai et al., 1990; Mason and Larkman, 1990). Because all colliculus-projecting and many callosal-projecting cells are found in layer 5 of the rat primary visual cortex (Schofield et al., 1987; Hallman et al., 1988), it seems possible that the subsets of these cells with LVA Ca2+channels could correspond to the bursting cells identified in slices (Chagnac-Amitai et al., 1990; Mason and Larkman, 1990). The threshold for activation of the LVA currents measured in visual cortical neurons, however, is more depolarized than that measured in other cells (McCleskey et al., 1986; Bean, 1989; Lovinger and White, 1989; White et al., 1989), and the currents inactivate rapidly. In addition, LVA current densities in these cells are very low. Taken together, these results do not provide a convincing argument to suggest that LVA channels play an important role in controlling burst firing in subsets of cortical projection neurons. It is possible, however, that LVA channels are present at higher densities in the dendrites (Harris et al., 1989) of these cells and that dendritic LVA channels underlie burst firing. Because the experiments here were performed on freshly isolated cells that were largely devoid of processes, this possibilitywas not examined. Additional experiments will be necessary to determine whether colliculusprojecting cells correspond to the bursting cells of layer 5 of the rat primary visual cortex (ChagnacAmitai et al., 1990; Mason and Larkman, 1990) and whether only those cells with LVA Ca2+ channels are capable of burst firing. In contrast to the variable distribution of LVA chan-

nels, currents through HVA Ca2’ channels were measured in all three types of visual cortical neurons examined. There were, however, differences in peak HVA current densities among cell types, and it is possible that these differences will be reflected in firing properties. Variations in HVA Cal+ channel densities could, for example, underlie differences in action potential durations, as well as in the durations and amplitudes of action potential afterhyperpolarizations observed in different types of cortical neurons (McCormick et al., 1985; Huettner and Baughman, 1988; Connors and Gutnick, 1990). The finding that peak HVA current densities are significantly lower in VCl.l+ than in projection neurons is particularly interesting since intrinsic, CABAergic inhibitory cortical neurons display”fast spiking”properties (McCormick et al., 1985; Huettner and Baughman, 1988; Connors and Cutnick, 1990). Experiments aimed atdetermining whether differences in HVA channel densities contribute to determining whether cells respond to prolonged depolarizing inputs by displaying “fast spiking” or “regular spiking” firing patterns (McCormick et al., 1985; Connors and Gutnick, 1990) are underway. Althoughthe precedingdiscussion hasemphasized differences in the types and densities of voltage-gated Ca2+ channels in different visual cortical cell types, our results also reveal that there are differences among cells within each type. For example, LVA Ca2+channels were measured only in subsets of projection neurons. Similarly, HVAchannel densities and the relative contribution of the inactivating and the noninactivating HVA components to the peak IB, also varied within each population. Since callosal-projecting cells are known to be heterogeneous in terms of laminar distributions and morphologies (Hallman et al., 1988; Peters et al., 1990), finding differences in the membrane properties of these cells may not be that surprising. For colliculus-projecting cells, however, these results are somewhat more puzzling, sinc:e by all other criteria, these cells appear to be quite homogeneous (Schofield et al., 1987; Hallman et al., 1988). Although the functional significance of these differences is not clear, these results do suggest that neurons which participate in the same types of cortical circuits have distinct electrophysiological properties. Whether the observed differences in Ca2+ channel currents are translated into differences in the firing or the response properties of these cells or the roles that individual cells play in the functioning of specific cortical circuits remains to be determined. The results presented here also do not exclude the possibility that HVA or LVA Ca2+channels are present at higher densities, or are distributed differently, in the dendrites of visual cortical neurons. Because the voltage-clamp experiments here were performed on freshly isolated cells (
Neuron 330

thwarted by the clear lack of spatial control of the membrane voltage. Howevqr, recent electrophysiological studies on substantia nigra zona compacta cells have demonstrated that low-threshold spiking is eliminated following removal of the pars reticulata, suggesting that LVA Ca*+channels are localized in the apical dendrites of nigral neurons (Harris et al., 1989). It may also be that LVA Ca*+ channels are present at higher densities in the dendrites of cortical projection neurons. If so, this could explain why LVA current amplitudes were small in isolated colliculusand callosal-projecting cells and did not increase with increasing cell size. It is also possible that the densities of HVA Ca2+ channels are different in the dendrites of cortical neurons, or that the distributions of N- and L-type channels in the processes of these cells are different from those measured in the cell bodies. Alternative experimental approaches will be necessary to evaluate these possibilities directly in neurons of the mammalian visual cortex. Experimental

Procedures

identification of Cortical Cell Types Callosal-projecting and colliculus-projecting cortical neurons were identified in vitro following in vivo retrograde labeling with fluorescent latex microspheres (Lumafluor) or beads (Katz et al., 1984; Huettner and Baughman, 1986; Katz and larovici, 1990). For labeling callosal-projecting neurons, P5 animals were anesthetized with isoflurane, and beads were pressure injected into the left hemisphere of the primary visual cortex (area 17) from a micropipette; in general, one or two (-50 nl) injections were made. To label colliculus-projecting neurons, similar injections were made into the right superior colliculus. In double-labeling experiments (see Figure I), rhodamine beads (Katz et al., 1984) were injected into the superior colliculus and fluorescein beads (Katzand larovici, 1990) were injected into thecortex. In all cases, animals were allowed to recover from the anesthesia before being returned to the mother. Survival times ranged from 3 to 9 days. To examine the distribution of bead-labeled callosal-projetting and colliculus-projecting cortical neurons, animals were perfused transcardiallywith 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were removed and placed in the same fixative with 30% sucrose until they sank (~18 hr). Coronal sections (50 wrn) were then cut on a freezing microtome. These sections were mounted on gelatinized slides and coverslipped for examination. Alternate sections were counterstained with cresyl violet or reacted for cytochrome oxidase activity (Wang-Riley, 1979). This revealed the distinctive cytological (Paxinos and Watson, 1986) and metabolic (Zilles et al., 1984) architecture of different cortical areas and allowed us to determine whether the injection sites were confined to area 17. Similar procedures were employed to evaluate the locations of the tissue removal sites for the preparation of dissociated cultures (see below) following bead injections into the contralateral area I7 or the ipsilateral superior coliiculus. A subset of GABAergic interneurons in the rat primary visual cortex was identified using the monoclonal antibody VC1.l (Arimatsu et al., 1987; Naegele et al., 1988). To evaluate whether VCl.l+ neurons have the typical nonpyramidal morphology of CABAergic neurons, VCl.l-immunoreactive cells were examined in tissue sections. For this purpose, PI0 animals were perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After the brains were equilibrated in phosphate-buffered 30% sucrose, frozen sections were cut in the coronal plane. These sections were then incubated with VC1.l (ascites fluid, diluted 1:4000) for 24 hr, and positive cells were visualized using a biotinylated goat anti-mouse IgM secondary

antibody followed by an avidin-biotinylated horseradish peroxidase complex (Vectastain ABC Kit, Vector) and reacted with diaminobenzidine. After mounting on slides and coverslipping, these sections were examined under the microscope. For identification of VCl.l+ cells in dissociated cultures prior to electrophysiological recordings, cultures were exposed to VC1.l (diluted 1:5000) in low glutamine (0.5 mM) Eagle’s minimum essential medium (MEM; GIBCO) containing 10% heat-inactivated horse serum (Hyclone) for 30 min at 37X After washingwith MEM, these cultures were subsequently incubated (for 30 min at 370C) with biotinylated goat anti-mouse IgM (Sigma), followed by a 15 min (at 37°C) exposure to rhodamine-conjugated streptavidin (40 pg/ ml; Jackson Laboratories). After washing with MEM, recording solution (see below) was added to the culture dish, and VCl.l+ cells were identified under epifluorescence illumination. Approximately 15% of the cells in cultures prepared at PB-PI2 were identified as VCl.l+. Because we were uncertain whether the staining procedure altered the electrophysiological properties of VCl.l+ cells (see text), an alternative procedure was used in some experiments. In this case, whole-cell Ca*+ channel currents were recorded in randomly selected cortical neurons in dissociated cultures plated on glass-etched gridded coverslips (Bellco), and the locations of the cells (on the grids) from which the recordings were obtained were noted. Following electrophysiological recordings, cultures were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in PBS for 1 hr at room temperature. After washing with PBS, cultures were exposed sequentially to VC1.l, biotinylated goat anti-mouse IgM, and rhodamine-conjugated streptavidin as described above, except that these incubations were for 1 hr each at room temperature. VCl.l+ cells were identified under epifluorescence illumination. Approximately 15% of the cells in these cultures were also identified as VCl.l+, and as would be expected, approximately 15% of the cells selected at random for recordings were VCl.l+. Dissociated Ceil Cultures Visual cortical neurons weredissociated from PB-P14Long Evans rat pups using a procedure based on that described by Huettner and Baughman (1986). Briefly, a 2 x 3 m m piece of area 17 was removed, chopped, and incubated at 34YI-37”C in 4 ml of Earle’s balanced salt solution (EBSS; CIBCO) containing 20 U/ml papain (Worthington), activated by the addition of 2.8 m M cysteineHCI. After 30 min, approximately 3 ml of fresh enzyme solution was added and the incubation was continued. This procedure was repeated again 30 min later. Following the incubation (total time 90 min), the enzyme solution was removed, and the tissue pieces were rinsed with a wash solution of EBSS containing 1 mg/ml trypsin inhibitorand 1 mg/ml (essentiallyfattyacid-free) bovine serum albumin (BSA) and triturated. The resulting cell suspension was layered over 5 ml of EBSS containing 10 mg/ml BSA and 10 mglml trypsin inhibitor and spun at 50 x g for 15 min. Pelleted cells were resuspended in 0.5 ml of wash solution and diluted to a final concentration of 1.5 x IO5 cells per ml in low glutamine (0.5 mM) MEM supplemented with 100 U/ml penicillin/streptomycin and plated on a monolayer of cortical astrocytes (see below) at a density of 3 x IO3 to 5 x 10’ cells per cm*. Approximately 1-3 hr after plating, cultures were fed with low glutamine MEM supplemented with 10% heat-inactivated horse serum. Cortical neurons prepared in this manner can be maintained in culture for several weeks U. P. Doyle and J. M. Nerbonne, unpublished data). Cortical glial cultures were prepared from P6 Long Evans rat pups using described procedures (Raff et al., 1979). Briefly, cortices were removed, chopped into 1 mm2 pieces, and incubated for 15 min in 10 ml of MEM containing 0.5 mglml type I trypsin (Sigma) and 6 mg/ml BSA at 37°C. AFter removing the enzyme solution, the tissue pieces were rinsed with MEM containing 6 mglml BSA, 50 pg/ml trypsin inhibitor, and 80 mg/ml DNAase (Sigma) and dispersed by gentle trituration. Following filtration through 25 pm Nitex, the resulting cell suspension was layered over Dulbecco’s modified Eagle’s medium (GIBCO) containing 4% BSA and spun at 180 x g for 10 min. Pelleted cells were

Caz+ Channels 331

in Identified

Cortical

Neurons

resuspended in MEM containing25 m M KCI, 100 U/ml penicillin/ streptomycin, and 10% heat-inactivated fetal calf serum (K.C. Supplies) and plated at 101 cells per cm2 on polylysine-coated coverslips in modified tissueculture dishes (Bray, 1970). Cultures fed every other day reached confluence within 4-5 days after plating. At this time, the cultures were treated with MEM containing 1O..5M cytosine arabinoside (Sigma) for 24 hr to eliminate fibroblasts and other rapidly dividing cells. The resulting cultures contain 295% astrocytes and are useful as substrates for cortical neurons for at least 4 weeks (j. P. Doyle and J. M. Nerbonne, unpublished data). Electrophysiological Recordings Macroscopic current recordings were obtained from isolated cortical neurons at room temperature (22OC-23OC) within the first 16 hr after isolation using the whole-cell variation of the patch-clamp recording technique (Hamill et al., 1981). Small cells (GO urn in diameter), which were largely devoid of processes, were selected for recordings to ensure adequate voltage control. The voltage-clamp circuit was provided by a Dagan (Model 8900; 1 G&2 feedback resistor) whole-cell/patch-clamp amplifier. Recording pipettes were fabricated from soda lime glass, and the shanks, up to the tips, were coated with Sylgard (Dow Corning) to reduce pipette capacitance. Pipettes, after fire-polishing, had tip diameters of 0.5-I urn and resistances of 2-8 MD when filled with recording solution (see below). Experiments were controlled and data were collected on an IBM-PC or an IBM-AT equipped with an analog/digital interface (Tecmar Labmaster, Scientific Solutions) to the electrophysiological equipment. Experimental variables were manipulated and data were acquired using pClamp (Axon Instruments). After formation of a high resistance (giga) seal between the recording electrode and the cell membrane, electrode capacitance was compensated electronically prior to rupturing the membrane patch; seal resistances were 2-10 CD. In the wholecell configuration, series resistances were approximately 1.5-2 times the pipette resistance (Marty and Neher, 1983). Because series resistances were compensated electronically by 295% and peak current amplitudes were 62nA, voltage errors arising from the uncompensated series resistancewere minimal ((2 mV) and were not corrected. Cell input resistances were in the range of l-5 CD, and capacitative transients (after series resistance compensation) decayed to baseline in <0.5 ms. Capacitative currents were measured during small (5 mV) depolarizing or hyperpolarizing voltage steps from a holding potential of -60 mV. IBawaveforms were routinely measured during voltage steps to potentials between -60 and +50 mV from holding potentials of -80 and -40 mV; voltage steps were presented in 10 mV increments at 5 s intervals. Sampling frequencies ranged from 2 to 20 kHz, depending on the particular experimental paradigm employed. Linear leakagecurrents,although small,weresubtracted on line, and current signals were low-pass-filtered at 3-5 kHz prior to digitization and storage. For experiments, bath solutions contained 5 m M Bach, 4 m M KCI, 2 m M MgCI,, 140 m M TEA-Cl, 5 m M glucose, IO m M HEPES, (pH 7.4, ~320 mOsm), and 1 uM tetrodotoxin. Recording pipettes contained 140 m M CsCI, 5 m M glucose, 10 m M ECTA, 10 m M HEPES, 3 m M ATP, and 0.1 m M CTP (pH 7.4, ~320 m0s.m). After establishing the whole-cell configuration, dialysis of the pipette solutionwiththecellinteriorwasallowedtoproceedforapproximately 1 min before recordings were initiated. This period appeared to be sufficient for establishing ionic equilibrium between the cell interior and the recording electrode. Although generally negligible in these cells, if run down of the currents was observed, experiments were terminated. Data Analyses Datawere compiled and analyzed using Lotus l-2-3 (Lotus Corporation) and pClamp. For quantitative analyses of It,= waveforms measured in isolated, identified cortical neurons, only those recordings in which seal resistances were 82 CD and cell input resistances were >,I G62 were analyzed. Also, as noted above, small cells (<30 urn), largely devoid of processes, were selected

for recordings to ensure adequate spatial control of the membrane voltage. If inadequate space clamp, indicated by the appearance of regenerative events during small depolarizations, was noted, data obtained from that cell were not subsequently analyzed. In addition, only those cells in which the capacitative transients decayed over a single exponential time course are included in the analyses presented here. Peak I,, amplitudes were measured as the differences between the maximal inward current amplitude evoked during depolarizing voltage steps and the zero current level. Plateau currents were measured as the difference between the amplitude of thecurrent remaining 125 ms after the onset of thedepolarizationsand thezerocurrent level.Thecontribution oftheinactivating current component to the peak HVA IHawas determined in individual cells by subtracting the plateau current amplitude evoked at +I0 mV from a holding potential of -80 mV from the peak current evoked during the same voltage step and dividing this difference by the peak current (Figure 5A). Whole-cell membrane capacitances were determined from integration of the capacitative transients evoked during small (* 5 mV) voltage deflections. Peak current densities were calculated by dividing the peak current amplitude evoked at +I0 mV from a holding potential of -80 mV by the whole-cell membrane capacitance. All averaged peak current amplitude and peak current density data are presented as mean + SD. Normalized current-voltage data are presented as mean * SEM. The statistrcal significance of observed differences between different populations of cells was examined using Student’s t test; p values (are provided in parentheses in the text. Acknowledgments We wish to thank Joe Doyle and John Doyle for their expert technical assistance. We also thank Joe Henry Steinbach for his careful and critical reading of this manuscript. In addition, we thank Dr. Janice Naegele for providing the VC1.l antibody and Dr. David Cottlieb for providing the antibody against glutamic acid decarboxylase. This work was supported by the Epilepsy Foundation of America, the National Institute of Mental Health, the National Science Foundation (BNS-8809823). and the National Institutes of Health (T32-HL07275). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemem’in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

13, 1990; revised

December

31, 1990

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