The Journal of Neurophysiology Vol. 79 No. 5 May 1998, pp. 2522-2534
Copyright ©1998 by the American Physiological Society

Specificity in the Interaction of HVA Ca²⁺ Channel Types With Ca²⁺-Dependent AHPs and Firing Behavior in Neocortical Pyramidal Neurons

Juan Carlos Pineda, Robert S. Waters, and Robert C. Foehring

Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee 38163

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Pineda, Juan Carlos, Roberts S. Waters, and Robert C. Foehring. Specificity in the interaction of HVA Ca²⁺ channel types with Ca²⁺-dependent AHPs and firing behavior in neocortical pyramidal neurons. J. Neurophysiol. 79: 2522-2534, 1998. Intracellular recordings and organic and inorganic Ca²⁺ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca²⁺ channels are differentially coupled to Ca²⁺-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca²⁺ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N-type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between ~80 and 90% of the slow AHP (sAHP) were Ca²⁺ dependent. Based on the effects of specific organic Ca²⁺ channel blockers (dihydropyridines, -conotoxin GVIA, -agatoxin IVA, and -conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca²⁺ current subtypes.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

High-voltage-activated (HVA) Ca²⁺ currents have been classified according to their pharmacology or the molecular biology of underlying channel subunits, with close concordance between the two classification schemes (Birnbaumer et al. 1994). In acutely dissociated rat neocortical pyramidal neurons, L-, N-, P-type (Brown et al. 1994; Lorenzon and Foehring 1995b; Sayer et al. 1990; Ye and Akaike 1993), Q-type (McDonough et al. 1996; unpublished observations), and R-type HVA currents (unpublished observations) are expressed, leading one to question why so many subtypes of HVA Ca²⁺ channels are present in these cells. Possible reasons for coexpression include, but are not limited to, differential modulation of specific channel subtypes by transmitters (Hille 1994) and different functional roles for channel subtypes. For example, in acutely dissociated neocortical pyramidal neurons, N- and P-type currents are modulated by transmitters such as serotonin (Foehring 1996) and norepinephrine (Foehring and Lorenzon 1993).

Relatively little is known concerning the functional roles of the various subtypes of Ca²⁺ channel currents in specific cell types. Different HVA current subtypes were shown to be involved in synaptic release at different CNS synapses (e.g., Lovinger et al. 1994; Luebke et al. 1993; Wheeler et al. 1994). Less is known about the postsynaptic roles of specific Ca²⁺ channel subtypes. Postsynaptic L-type currents have been implicated in synaptic plasticity in hippocampal CA1 pyramidal neurons (Kullmann et al. 1992; Magee and Johnston 1997) and also may be coupled to gene regulation (Bading et al. 1993; Ghosh and Greenberg 1995) in neurons.

One of the physiological consequences of Ca²⁺ entry through Ca²⁺ channels is the activation of Ca²⁺-dependent K⁺ currents that underlie action potential (AP) repolarization, spike-frequency adaptation, and afterhyperpolarizations (AHPs) in various cells (Meech 1978). Studying these AHPs is thus an indirect means of examining conductances that are activated during action potentials and shape firing behavior.

Neocortical pyramidal neurons exhibit three distinct AHPs after spikes (Lorenzon and Foehring 1993; Schwindt et al. 1988b,c). The fast AHP (fAHP) follows a single spike and reflects conductances active in repolarizing the action potential (Lorenzon and Foehring 1993; Schwindt et al. 1988c). Medium and slow AHPs (mAHP and sAHP, respectively) reflect voltage- and Ca²⁺-dependent conductances activated during action potentials. A mAHP can be observed after a single spike (Lorenzon and Foehring 1993; Schwindt et al. 1988b,c), and the mAHP increases in amplitude with additional action potentials (Lorenzon and Foehring 1992, 1993, 1995a; Schwindt et al. 1988b). After several spikes, an additional slower component appears, the sAHP (Lorenzon and Foehring 1992, 1993; Schwindt et al. 1988b). These latter two AHP components differ in kinetics and pharmacology. The mAHP decays within 200 ms and is sensitive to the peptide apamin but not to transmitters (Lorenzon and Foehring 1992, 1993; Schwindt et al. 1988b). The sAHP decays with a time course of seconds and is insensitive to apamin but is modulated by several transmitters (e.g., Araneda and Andrade 1991; Foehring et al. 1989; Lorenzon and Foehring 1992, 1993; McCormick and Prince 1986; McCormick and Williamson 1989; Schwindt et al. 1988b; Spain 1994).

In various neuron types, apamin-sensitive AHPs are coupled to L- (Hernandez-Lopez et al. 1997), N- (Sah 1995), or N- and P-type (Umemiya and Berger 1994; Viana et al. 1993) currents. In cholinergic Nucleus Basalis neurons, N- and T-type currents (but not L-, P-, or Q-type) are coupled to the apamin-sensitive mAHP, and the apamin-insensitive sAHP is activated by N- and P-type currents, (but not L-, Q-, or T-type) (Williams et al. 1997). Thus no simple rules have emerged regarding association of particular Ca²⁺ channel subtypes and Ca²⁺-dependent AHPs; the relationships appear to be cell-type specific.

It is uncertain which type(s) of Ca²⁺ currents are involved in the activation of the apamin-sensitive mAHP and apamin-sensitive sAHP in neocortical pyramidal neurons. We therefore examined which aspects of APs and AHPs are Ca²⁺ dependent and which specific Ca²⁺ current subtypes are involved in the generation of the AHPs in neocortical pyramidal neurons localized to layers II/III of rat sensorimotor cortex. Preliminary aspects of this work have previously been presented in abstract form (Foehring and Waters 1995; Pineda et al. 1996).

	METHODS

Abstract Introduction Methods Results Discussion References

Intracellular recordings were made from layer II/III neocortical neurons in an in vitro brain slice preparation from immature (postnatal days 7-35) Sprague-Dawley rats of both sexes. Our protocol conformed to the guidelines of the Animal Care and Use Committee, University of Tennessee, Memphis.

Rats were anesthetized with methoxyflurane and, on evidence of areflexia, were decapitated. The brains were removed quickly and submerged in a low sodium solution at 4°C. The low-sodium solution contained (in mM) 250 sucrose, 2.5 KCl, 1 NaH₂PO₄, 11 glucose, 4 MgSO₄, 0.1 CaCl₂, and 15 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.3, osmolality 300 mOsm/l). The brain was sectioned at 400 µM with a WPI oscillating tissue slicer in the low-sodium solution. The slices were incubated for a minimum of 1 h in 32°C artificial cerebrospinal fluid (ACSF), which contained (in mM) 125 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 1.2 NaH₂PO₄, 26 NaHCO₃, and 20 dextrose (pH 7.4; osmolality 310-320 mOsm/l). The holding chamber ACSF also contained the antioxidant glutathione (200 µM: Calbiochem). Individual slices then were transferred to the recording chamber. Two different recording chambers were used: interface chamber in which the slices were bathed in ACSF (saturated with carbogen) that flowed under the slice and ran at 1 ml/min (34°C) while carbogen saturated water vapor flowed over the top of the slice. The second chamber was a submerged chamber: the slice was submerged completely in the ACSF solution (32°C) saturated with carbogen flowing at 2-2.5 ml/min. The slices were held down with a mesh net attached to a U-shaped platinum bar.

Electrophysiological recording

Sharp microelectrodes were pulled from borosilicate glass with either a 1.5 mm (WPI) or 1.2 mm (Frederick Haer) outside diameter using a Sutter (P-87) or Kopf (750) puller. They were filled with either KMeSO₄ (2 M) + HEPES (10 mM) + 1% biocytin (pH 7.2 with 1 N KOH) or KCl (3 M). The electrodes had resistances of 40-90 M. Records in continuous bridge mode were obtained using an Axoclamp-IIb electrometer. Biocytin was injected into cells, and the cells were subsequently stained as described previously (Foehring et al. 1991; Horikawa and Armstrong 1988). The signal was filtered with a four pole Bessel 5-kHz filter (Dagan), digitized (Neurodata), and stored on videotape (VCR). RC Electronics Computer-Scope was used to analyze the data off-line. Membrane potentials were determined from continuous chart recordings of DC potential.

As previously reported (Lorenzon and Foehring 1995a), the mAHP amplitude was defined operationally as the maximum hyperpolarization after one spike or a train of spikes. Single action potentials were produced by a 5-ms step of depolarizing current with intensity adjusted to threshold. The mAHP duration after a single spike was defined as the time between the maximum downstroke of the action potential and the return of the membrane potential to the baseline potential. Trains of 10 spikes were produced by a series of 10 brief (5 ms) suprathreshold positive current steps (~3 nA) at a frequency of 100 Hz. At this frequency and number of spikes, the contribution of the sAHP current to the mAHP was minimal (Lorenzon and Foehring 1992, 1993; Schwindt et al. 1988b). We measured the sAHP amplitude as the amplitude of the AHP at 500 ms after the end of a train of spikes (the mAHP fully declines within 200 ms after the spike train) (Lorenzon and Foehring 1995a). For comparison of AHPs between control solutions and solutions containing blockers, the membrane potential was manually clamped to the same potential (usually 3-4 mV below spike threshold) with DC current injection.

Drugs used

We used inorganic (Co²⁺, Cd²⁺, Ni²⁺, and Mn²⁺) and organic Ca²⁺ channel blockers to examine the roles of Ca²⁺ current subtypes in generating neuronal firing behavior. All salt solutions and inorganic blockers were obtained from Sigma. The organic blockers included nifedipine (Nif; obtained from RBI), nimodipine (RBI), -conotoxin GVIA (CgTx GVIA; RBI), -AgTx IVA (AgTx; a gift from Pfizer Research), -conotoxin MVIIC (CgTx MVIIC; Calbiochem) and the dihydropyridine agonist Bay-K 8644 (RBI). The inorganic blockers were added directly to ACSF and substituted with equimolar amounts for Ca²⁺. The conotoxins and AgTx were prepared as concentrated stock solutions in deionized H₂O and frozen. They then were thawed and added to ACSF just before recording. Concentrated stock solutions of nifedipine and Bay-K 8644 were dissolved in 95% ethanol and protected from light during the experiment. The final concentration of ethanol never exceeded 0.05%; this concentration produced no effects on AHPs when tested alone (n = 5 cells). Cytochrome C (0.01%; Calbiochem) was added to all solutions containing AgTx to avoid adherence of AgTx molecules to connecting tubing and glass walls (Bargas et al. 1994; Lorenzon and Foehring 1995b; Mintz et al. 1992). We have reported previously that this concentration of cytochrome C exerts no effects of its own on Ca²⁺ channel currents (Lorenzon and Foehring 1995b). In the present study, we observed no effects on action potentials or AHPs with application of cytochrome C alone (n = 5 cells).

For this study, we operationally defined several HVA calcium current subtypes, based on the proposed nomenclature of Birnbaumer et al. (1994), and our own work on acutely-dissociated rat neocortical pyramidal neurons (Lorenzon and Foehring 1994, 1995b; unpublished observations) and other neuronal types (Foehring and Scroggs 1994; Foehring and Armstrong 1996). L-type current was defined as that blocked by 5-10 µM Nif. N-type current was that blocked by 1 µM CgTx GVIA. AgTx blocks both P-type and Q-type currents, depending on dose (Randall and Tsien 1995). The dose for half-maximal blockade of P-type channels was ~1-3 nM in cerebellar Purkinje cells (Mintz et al. 1992; Randall and Tsien 1995) and ~100 nM for Q-type current in cerebellar granule cells (Randall and Tsien 1995). We chose 25 nM as a dose which blocked virtually all P-type current, but very little Q-type current. CgTx MVIIC has been shown to block N-, P-, and Q-type currents, but not L-type or R-type in several cell types (McDonough et al. 1996; Randall and Tsien 1995; Zhang et al. 1993). We defined Q-type current as that blocked by 1 µM CgTx MVIIC after previously blocking N- and P-type currents with 1 µM CgTx GVIA and 25 nM AgTx, respectively. In dissociated neurons, a similar proportion of current is blocked by CgTx MVIIC (after CgTx GVIA and AgTx) and 1 µM AgTx (after 25 nM AgTx), and the biophysical properties of the high AgTx- and CgTx MVIIC-sensitive currents are nearly identical (unpublished observations). R-type currents were defined as the HVA current that remains unblocked after combined application of the organic blockers (Randall and Tsien 1995).

In initial experiments, organic toxins (Nif, CgTx GVIA) were applied as a microdrop to the surface of the slice using a micropipette (interface chamber; hereafter referred to as drop application). Most of the experiments used bath application of blockers in the submerged chamber (hereafter referred to as bath application). One set of experiments with CgTx GVIA involved incubation of the slices with the toxin for 4-10 h before recording.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Ca2+ dependence of single action potentials and subsequent afterhyperpolarizations (AHPs). A: superimposed traces for action potentials elicited in control solution and in 400 µM Cd2+. Note: no changes in spike polarization or repolarization in this cell. Resting membrane potential: 63 mV. B: in another cell, replacement of extracellular Ca2+ with 2 mM Co2+ results in a slowing of the final phase of spike repolarization, resulting in a broader spike at threshold and at the base. Resting membrane potential: 68 mV. Note voltage-threshold (Vth) for spike initiation. Threshold duration = the width of the spike at Vth. C: control trace in a different cell at a slower time base to show the fast AHP (fAHP) and medium AHP (mAHP) after the spike. D: mAHP was blocked in the same cell as in C by Co2+-containing solution. Base duration = time between Vth and reattainment of resting potential. Box plot shows summary data for the percent mAHP block from 6 cells. In the box plot, the median value is illustrated as the vertical line within the box. Inner quartiles are shown as the edges of the box and the outer quartiles as lines extending from the box (Tukey 1977).

Statistics

Because wide variability in the amplitude and duration of the AHPs among individual cells was observed, neuronal firing characteristics were compared before and after drug administration in the same cell. For AHPs, a minimum of three AHPs were measured for each solution, and the values averaged. Data are presented as a summary of the number of cells exhibiting increases, decreases, or no change (<10% change) in the measured parameter, as median or mean ± SE for the percent changes, and box plots are used to summarize the population data in graphic form.

Box plots are an efficient way to graphically present summary data from small data sets (Tukey 1977). The median is represented by a line dividing the box. The upper and lower inner quartiles are included between the median and each edge of the box. The outer quartiles are represented by the lines extending from the edges of the box. Data points that are farther than two times the difference between the box edges are considered outliers and represented by an asterisk (Tukey 1977).

In some cases, we also present mean ± SE for absolute amplitudes. Statistical differences between control and experimental populations were determined with the nonparametric Wilcoxon signed rank test. (Differences were considered significant if   0.05).

	RESULTS

Abstract Introduction Methods Results Discussion References

Recordings were made with sharp microelectrodes to ensure stable AHPs over long recording times. Data were obtained from 88 cells (sensorimotor cortex, layer II/III) in which the records were stable for 30 min, input resistances were >20 M, and action potential (AP) amplitudes were >60 mV. All of these cells displayed physiological properties that suggested that they were regular spiking pyramidal cells (Lorenzon and Foehring 1993; McCormick et al. 1985). Twenty cells were filled with biocytin and later confirmed to be layer II/III pyramidal cells (data not shown).

Effects of inorganic Ca²⁺ blockers on the AP and AHP

To examine possible roles of Ca²⁺-dependent currents on the generation of the AP and AHPs, we replaced extracellular Ca²⁺ with inorganic Ca²⁺ channel blockers (Co²⁺, Mn²⁺, Cd²⁺, and Ni²⁺) in the bath solution. An example of the effect of 400 µM Cd²⁺ (+1.6 mM Ca²⁺) on a single AP is illustrated in Fig. 1A. We found no significant differences in AP amplitude, AP half-width (width of the spike measured at half-maximal amplitude), or AP duration measured at spike threshold (see Fig. 1B) between cells in Cd²⁺ versus control solutions (Table 1). (Throughout this paper, a significant difference refers to   0.05 in the nonparametric Wilcoxon signed rank test). Input resistance also was unchanged by Cd²⁺ (Table 1). Similar results were seen in six other cells in Co²⁺ (2 mM/0 Ca²⁺; Table 1), two other cells in the presence of Mn²⁺ (2 mM/0 Ca²⁺), and one cell in the presence of Ni²⁺ (2 mM/0 Ca²⁺) (data not shown). The only difference between these inorganic blockers was that there was a significant increase in the AP duration at spike threshold in Co²⁺ (Table 1, Fig. 1B). Because this response was unique to Co²⁺ and developed slowly (well after the effects on AHPs; see further text), we do not consider this effect to be evidence for direct Ca²⁺ dependence (see also Lorenzon and Foehring 1992; Schwindt et al. 1988c). Spike broadening at threshold was also not evident in neocortical pyramidal cells with intracellular application of the Ca²⁺ chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (Lorenzon and Foehring 1995a; Schwindt et al. 1988c, 1992b).

The spike base duration (measured as the time between spike threshold and recrossing of the original resting potential) was significantly increased by Cd²⁺ (by 100-272% in 6 of 6 cells tested; median = 141%; Table 1). Similar results were seen in six cells in the presence of Co²⁺ (median 167%; Fig. 1, C and D), two cells in Mn²⁺ (121 and 206% of control), and one cell in Ni²⁺ (217% of control). These data suggest that Ca²⁺ or Ca²⁺-dependent currents do not participate in the polarization or most of the repolarization of the AP but have a role in the final stages of return to baseline.

A single spike is shown at a longer time base in Fig. 1C to illustrate the fAHP and mAHP. Figure 1D shows that bath application of 0 Ca²⁺/2 mM Co²⁺ blocked the mAHP in the same cell shown in Fig. 1C. The box plot shown in the Fig. 1D, inset, summarizes these data. The median block of the mAHP amplitude by Co²⁺ was 76% (n = 6; Table 1). The mAHP amplitude was blocked to a similar degree by all of the inorganic antagonists tested [Mn²⁺ (n = 2), Ni²⁺ (n = 1); Cd²⁺ (n = 6); Table 1], indicating that part of the mAHP generated by a single spike is Ca²⁺ dependent (Connors et al. 1982; Lorenzon and Foehring 1992, 1993, 1995a; Schwindt et al. 1988b). On average, zero Ca²⁺/Co²⁺ significantly reduced the mAHP duration from 113 ± 5 to 37 ± 5 ms (n = 6).

The mAHP and sAHP after a train of 10 spikes also exhibited Ca²⁺ dependence. Two millimolar Co²⁺/zero Ca²⁺ significantly reduced mAHP amplitude by 10-100% (median =61%; Table 1, Fig. 2A) and significantly reduced sAHP amplitude (Table 1, median = 100%, Fig. 2A) in 6 of 6 cells tested. Figure 2B illustrates that 400 µM Cd²⁺/1.6 mM Ca²⁺ had a similar effect (4 of 4 cells tested). The median block of the mAHP was 41% and of the sAHP was 72% (Table 1). The combined data from all inorganic blockers (Co²⁺ = 6; Cd²⁺ = 4, Mn²⁺ = 2) indicate that 93 ± 4% of the sAHP (median = 100%) and 55 ± 8% of the mAHP (median = 55%), are Ca²⁺ dependent (both significant; Table 2).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Ca2+ dependence of the mAHP and sAHP after multiple spikes. A: superimposed traces taken in control solution and in 2 mM Co2+ illustrating mAHP and sAHP elicited by 10 suprathreshold stimuli at 100 Hz (see protocol). mAHP was defined as the peak response immediately after the train of spikes. Amplitude of the slow AHP (sAHP) was defined as the amplitude at 500 ms after the spike train. Note elimination of the sAHP and substantial reduction (51% in this cell) of the mAHP in Co2+. Bottom: box plot illustrating population data for the percent of the mAHP and sAHP blocked by 2 mM Co2+ (n = 6 cells). B: superimposed traces taken in control solution and in 400 µM Cd2+ to illustrate the effects of Cd2+ on the mAHP and sAHP elicited by 10 suprathreshold stimuli at 100 Hz. Note substantial reduction of the sAHP (82% in this cell, 1 of the smallest blocks seen) and of the mAHP (39% in this cell). Bottom: box plot illustrating population data for the percent of the mAHP and sAHP blocked by 400 µM Cd2+ (n = 4 cells).

Ca²⁺ current subtypes

To determine which subtypes of Ca²⁺ currents are involved in the generation of AP repolarization and AHPs, we elicited APs and AHPs in control solution and again in the presence of selective organic Ca²⁺ current blockers using bath or drop applications (see METHODS). During the time course of these experiments (30-60 min), the effects of Nif were reversible and those of CgTx GVIA, CgTx MVIIC and AgTx were essentially irreversible [as would be expected based on their effects on isolated cells (Lorenzon and Foehring 1995b; McDonough et al. 1996; Mintz et al. 1992; Zhang et al. 1993].

SINGLE ACTION POTENTIAL AND SUBSEQUENT AHP. None of the organic antagonists used had any significant effect on input resistance or action potential amplitude, half-width, or width at threshold; the effect of Co²⁺ on AP threshold width was not mimicked by any of the organic blockers tested (data not shown).

Figure 3 shows the effect of organic Ca²⁺ current blockers on the mAHP after a single action potential. On average, CgTx GVIA (1 µM) had no effect on the mAHP after one spike (3.8 ± 1 vs. 3.7 ± 1 mV; Fig. 3, A and B; n = 9: 7 tested with bath application, 2 tested with drop application). Nif also had no effect on the mAHP (6 of 6 cells tested; data not shown), suggesting that L- and N-type currents do not participate in generating the mAHP.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Effects of -conotoxin (CgTx GVIA) and -AgTx IVA (AgTx) on the mAHP after a single action potential. A: single spike and subsequent AHP in control solution. Spike is truncated in this and subsequent panels to allow visualization of the AHP at high gain. B: single spike and subsequent AHP in same cell as in A except in the presence of 1 µM CgTx GVIA (in bath). There was no change in the amplitude of the mAHP. AP duration was unchanged at half-amplitude and at threshold but was longer at the base. Similar data were observed in 12 cells. C: data from a different cell in control solution. D: same cell as in C in the presence of 25 nM AgTx (in bath). Note reduction of mAHP amplitude (37% in this cell). In this cell, the spike base width was increased by AgTx, however, this was not a consistent finding. Box plot shows summary data from 8 cells.

CgTx GVIA significantly increased spike base duration (from 12 ± 2 to 24 ± 6 ms; Fig. 3, A and B), in six of six cells tested suggesting a role for N-type Ca²⁺-dependent outward currents in the final phase of repolarization. Nif(n = 6 with bath application and n = 3 with drop application; data not shown) and AgTx (n = 13 with bath application) had no consistent effect on AP base duration. Cytochrome C alone caused no changes in APs or AHPs (n = 5; data not shown).

Bath application of AgTx (25 nM) reduced the mAHP after a single AP by 10-100% (median = 27%) in 8 of 13 cells tested (Fig. 3, C and D). AgTx increased the amplitude of the mAHP (by 53 ± 5%) in three cases, and there was no change in the mAHP in two other cells. The average effect for all 13 cells was a reduction in the mAHP by15 ± 15% (median = 18%). If the three cells showing an increase in the mAHP in AgTx are removed, the reduction of the mAHP becomes significant (38 ± 12%; median = 22%, n = 10). Taken together, single spike data suggest that P-type, but not N- and L-type, currents participate in the generation of the mAHP in some, but not all cells. CgTx MVIIC, in the presence of AgTx and CgTx GVIA, caused no further changes in the mAHP after a single spike or in action potential parameters (n = 3; data not shown).

AHPs after trains of spikes

We next tested which calcium currents underlie the generation of the mAHP and sAHP observed after multiple action potentials. Because both AHPs vary depending on the number and frequency of spikes used to elicit them and we wished to make quantitative comparisons in control and antagonist solutions, we elicited the AHPs with a standardized protocol of 10 suprathreshold stimuli of 5-ms duration, at a frequency of 100 Hz (see METHODS). This protocol was repeated once every minute. Several control trials were obtained, and then the protocol was repeated throughout the application of blockers to observe the rate of change and to ensure that a stable endpoint was reached.

L-TYPE CURRENTS. Bath application of the dihydropyridine L-current blocker Nif (5 µM) did not change the mAHP or the sAHP following a train of 10 spikes (Fig. 4, A and B; n = 9 cells: 7 bath application and 2 drop application; Table 2). These results suggest that L-type channels do not participate in the generation of the mAHP or sAHP. Consistent with this observation, another dihydropyridine antagonist nimodipine (10 µM) had no effect on the mAHP or sAHP(n = 3, data not shown), and the L-type current agonist BAYK 8644 did not affect the mAHP or sAHP (n = 2, data not shown). Nif did alter the firing properties of these cells, as illustrated in Fig. 4C, 1-3. L-channel blockade led to a decrease in firing frequency in response to long (1 s) suprathreshold current injections in five of six cells (1 cell showed no change). The change in firing was reversible in response to short applications of Nif. The median slope of the plot of average steady-state firing frequency (last 500 ms of response to 1-s current injection) versus injectedcurrent (f-I slope) was 29 Hz/nA in control solution, 20 Hz/nA in Nif, and 36 Hz/nA after wash (n = 5). Rheobase and the voltage threshold for spikes were unchanged by Nif (n = 6).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Effects of nifedipine (Nif). A: response of neuron in control solution to 10, 5-ms suprathreshold current injections at 100 Hz. B: trace from the same cell and protocol as in A except in the presence of 5 µM Nif. Nif had no effect on the mAHP or sAHP. Box plots summarize population data for the mAHP and sAHP in control vs. Nif (n = 9 cells). C: effects of Nif on repetitive firing. Resting potential = 75 mV. C1: repetitive firing in control solution in response to 0.5-nA, 1-s current injection. C2: repetitive firing in the same cell as in B (same current protocol) in the presence of 10 µM Nif. Note slowing of firing frequency. C3: Nif effect reversed on wash in control media.

N-TYPE CURRENTS. We next studied the effects of microdrop application of CgTx GVIA (1 µM) onto the surface of the slice in the interface chamber. Figure 5A shows that the peak amplitude of the mAHP was not reduced by CgTx GVIA (<10% reduction). Similar results were obtainedfor all cells tested (n = 8). The mAHP generated after a10-spike train was also not reduced by bath application of CgTx GVIA (n = 2). Because the results were similar for both bath and drop data, the data were combined to generate the box plot in Fig. 5C (also Table 2).

View larger version (34K): [in this window] [in a new window]   FIG. 5. Effects of CgTx GVIA on the AHPs after multiple spikes. A: superimposed control (CTL) and 1 µM CgTx GVIA traces elicited by 10 suprathreshold 5-ms current injections at 100 Hz. Note minimal reduction in the mAHP (9%) and substantial reduction of the sAHP (61% in this cell). Box plot illustrates summary data for percent sAHP reduction. B: plot of sAHP amplitude measurements as a function of time. Same cell as in A. AHPs were elicited at 1-min intervals. Note: stable values in control solution (no "run-down" of response) and abrupt change attaining a new, lower amplitude in CgTx GVIA. C: box plots illustrate summary data for mAHP amplitude in CTL and CgTx GVIA solutions (n = 9 cells). D: box plots illustrate summary data for sAHP amplitude in CTL and CgTx GVIA solutions (n = 9 cells). E: repetitive firing in a different cell in response to a 1-nA, 500-ms current injection (CTL solution). F: repetitive firing in the same cell as in E (in response to 1-nA, 500-ms current injection), in the presence of 1 µM CgTx GVIA. Note increased firing frequency and reduction in spike frequency adaptation.

In contrast, the sAHP was reduced in seven of these same eight cells (by 29-100%, median = 52%) by drop application of CgTx GVIA (Fig. 5A). One cell did not respond to CgTx GVIA. With bath application of CgTx GVIA, the sAHP was reduced in the two cells tested (by 42 and 64%). The data obtained with drop and bath application were pooled, and for all 10 cells, the average effect was a significant reduction of the sAHP (median = 52%; Fig. 5D; Table 2). In Fig. 5B, the sAHP amplitude is plotted as a function of time for the same cell as in Fig. 5A. Note the nearly constant amplitude during the control period and reduction by CgTx, reaching a new stable level after ~3 min. This stability and time course was consistent over all the cells tested. Figure 5, E and F, shows that the CgTx GVIA application resulted in altered firing properties in response to long (500 ms) current injections: spike-frequency adaptation was reduced and firing frequency was increased (greater number of spikes in response to same current amplitude). Similar effects were seen in all four cells tested.

To examine whether the lack of effect on the mAHP was a result of a failure of CgTx GVIA to reach equilibrium during the acute application of toxin, we preincubated cells in 1 µM CgTx GVIA for 2 h. Consistent with the data obtained from acute application of CgTx GVIA, the sAHP, but not the mAHP, was smaller (by 47 ± 15%) in preincubated neurons compared with nonpreincubated neurons, recorded from on the same day (3 control cells and 10 preincubated neurons: data not shown). Thus with both acute application and preincubation, the sAHP was reduced and the mAHP was unaffected. Collectively, these data suggest that N-type currents are responsible, in part, for generation of the sAHP in these pyramidal neurons.

P-TYPE CURRENTS. As shown in Fig. 6, A and B, 25 nM AgTx influenced both the mAHP and the sAHP after 10 spikes. In 8 of 13 cells tested (bath application), the mAHP was reduced by 10-86% (median 54%). Cytochrome C alone caused no changes (n = 5; data not shown). The mAHP was not changed by AgTx in two cells, and AgTx increased the mAHP amplitude by an average of 68 ± 5% in three cells (the same 3 cells in which the mAHP after 1 spike was increased by AgTx: see earlier text and DISCUSSION). For all 13 cells, the median effect was a 17% reduction of the mAHP (Fig. 6C; Table 2). If the three cells showing an increased mAHP are removed from the data set, the average reduction becomes significant (median = 46%). These results suggest that P-type currents participate in the generation of the mAHP in most cells but with considerable cell-to-cell variability.

View larger version (29K): [in this window] [in a new window]   FIG. 6. Effects of AgTx on the AHPs after multiple spikes. A: superimposed control (CTL) and 25 nM AgTx traces elicited by 10 suprathreshold 5-ms current injections at 100 Hz. Note reduction in the mAHP (60% in this cell) and sAHP (70% in this cell). Box plot illustrates summary data for percent sAHP reduction. B: plot of sAHP amplitude measurements as a function of time. Same cell as in A. AHPs were elicited at 1-min intervals. Note: stable values in control solution (no run-down of response) and at new, lower level in AgTx. C: box plots illustrate summary data for mAHP amplitude in CTL and AgTx solutions (n = 13 cells). D: box plots illustrate summary data for sAHP amplitude in CTL and AgTx solutions (n = 13 cells). E: repetitive firing in a different cell in response to a 0.5-nA, 500-ms current injection (CTL solution). Note strong spike-frequency adaptation. F: repetitive firing in the same cell as in C (in response to 0.5-nA, 500-ms current injection), in the presence of 25 nM AgTx. Note increased firing frequency and reduction in spike frequency adaptation.

The sAHP was reduced in 12/13 cells (15-100%, Fig. 6A) by bath application of AgTx; one cell showed no change. For these 13 cells, the median decrease was 47% (inset in Fig. 6A; Fig. 6D), which was statistically significant (Table 2). Figure 6B is a plot of sAHP amplitude as a function of time to illustrate the stability of control sAHP amplitude and the time course of its reduction by AgTx. Figure 6, E and F, shows that application of AgTx resulted in increased firing frequency in response to a 500-ms current injection. Similar data were obtained from three cells. Thus P-type channels coupled to the sAHP and, in some cells, to the mAHP.

The median block of the sAHP by AgTx was 47% and that by CgTx was 52% (see preceding text). We therefore hypothesized that the combination of AgTx plus CgTx GVIA should block the entire sAHP. In 10 cells where we first applied AgTx and then the combination of AgTx plus CgTx GVIA, the median block of the sAHP by AgTx alone was 47% (Fig. 7, A and B; Table 2). In all 10 cells, subsequent application of AgTx plus CgTx GVIA blocked additional current, with a median block of 62% (Fig. 7, A and B; Table 2). In these same cells, the median mAHP reduction was 12%. Thus the combined toxins did not add linearly.

View larger version (24K): [in this window] [in a new window]   FIG. 7. Effects of combination of CgTx GVIA, AgTx, and CgTx MVIIC. A: superimposed AHPs in CTL, 25 nM AgTx, and AgTx plus 1 µM CgTx GVIA in response to 10 spikes at 100 Hz. Note reduction of mAHP and sAHP (47%) in AgTx and further reduction in AgTx + CgTx GVIA (to 58%, no further change in the mAHP). B: box plots (n = 9 cells) show summary data for percent block of sAHP amplitude by AgTx and AgTx + CgTx GVIA (*, outliers: see METHODS). C: AHPs in response to 10 spikes at 100 Hz. Superimposed traces taken from the same cell in CTL solution and in the presence of 25 nM AgTx plus 1 µM CgTx GVIA. Note reduction in the mAHP and sAHP. Combination of the 2 toxins blocked 41% of the mAHP and 61% of the sAHP in this neuron. Box plot shows summary data for sAHP block in AgTx + CgTx GVIA (n = 6 cells). D: superimposed traces from the same cell in CTL solution, in the presence of 25 nM AgTx plus 1 µM CgTx GVIA and after addition of 1 µM CgTx MVIIC (plus AgTx plus CgTx GVIA). Note nearly complete elimination of sAHP and no further block of the mAHP. Box plot shows summary data for sAHP block in AgTx + CgTx GVIA + CgTx MVIIC (n = 6 cells).

Q-TYPE CURRENTS. Because the combination of P- andN-type blockade did not block the entire sAHP, we tested whether another type of Ca²⁺ current could elicit the sAHP. Possible candidates include Q- or R-type currents, or the low-threshold T-type currents. Applications of 25-50 µM Ni²⁺ (n = 3), which is reported to block T-type (Tsien et al. 1988) and R-type currents (Randall and Tsien 1995), had no effect on either the mAHP or sAHP (data not shown). We therefore tested for the involvement of Q-type currents.

CgTx MVIIC blocks N-, P-, and Q-type currents in different neuronal types, including pyramidal neocortical neurons (McDonough et al. 1996; unpublished observations). Therefore in six cells we first applied the combination of CgTx GVIA (1 µM) + AgTx (25 nM) to block N- and P-type currents, respectively. This combination partially blocked the mAHP (median block = 14%) in all six cells (Fig. 7C; Table 2). Subsequent application of 1 µM CgTx MVIIC + 1 µM CgTx GVIA + 25 nM AgTx had no consistent effect on the mAHP: Two cells exhibited considerable additional block compared with AgTx plus CgTx GVIA (31%, 85%: e.g., Fig. 7D), two cells showed no additional block by CgTx MVIIC, and the remaining two cells showed only 3% further reduction in the mAHP. The median reduction from the control mAHP was 30% (Table 2).

The combination of CgTx GVIA + AgTx also reduced the sAHP amplitude by 24-77% in these same six cells (median = 67%; Fig. 7C; Table 2). Subsequent application of 1 µM CgTx MVIIC + 1 µM CgTx GVIA + 25 nM AgTx to these six cells increased the block of the sAHP to 71-100% (median 100%; Fig. 7D; Table 2). This is similar to the maximal block with inorganic blockers, suggesting that blockade of N-, P-, and Q-type Ca²⁺ currents is sufficient to eliminate all of the Ca²⁺-dependent portion of the sAHP. Like AgTx and CgTx GVIA, CgTx MVIIC similarly produced an increase in the frequency of firing in response to depolarizing current injections (500 ms, data not shown, n = 4). In two of the six cells tested with all three toxins, the sAHP was completely eliminated and an afterdepolarization (ADP) followed the mAHP (data not shown). This ADP is of unknown mechanism and was not studied further.

Consistent with the known slow kinetics of block ofQ-type currents by CgTx MVIIC (McDonough et al. 1996; Randall and Tsien 1995; unpublished observations), the onset of the block of the sAHP by CgTx MVIIC was much slower than the block by CgTx GVIA or AgTx. The effects of the latter two blockers were usually maximal within3-4 min after changing solutions, but the CgTx MVIIC effect always took >8 min to stabilize (often >20 min).

Taken together, these results suggest that Q-, P-, andN-type currents are involved in the generation of the sAHP, and P-type currents are involved in eliciting the mAHP in layer II/III neocortical pyramidal neurons.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study was designed to determine which aspects of APs and AHPs are Ca²⁺ dependent in layer II/III neocortical pyramidal neurons and which subtypes of calcium currents are involved in generating these events.

Ca²⁺ dependence

We found no Ca²⁺-dependent component to the major part of spike repolarization in rat layer II/III neocortical pyramidal neurons (see also Lorenzon and Foehring 1995a; Schwindt et al. 1988c; Zhou and Hablitz 1996). Our results confirm previous studies in rat (Connors et al. 1982; Lorenzon and Foehring 1993, 1995a) and cat (Schwindt et al. 1988b,c) cortical pyramidal neurons in that ~75% of the mAHP after a single AP and 30-60% of the mAHP and 80-90% of the sAHP observed after a train of spikes are Ca²⁺ dependent. Our data also suggest involvement ofN-type current in activating outward currents responsible for the final phase of spike repolarization (after the fAHP).

Ca²⁺ current subtypes

We found that N-, P-, and Q-type currents converge to activate the Ca²⁺-dependent K⁺ currents underlying the sAHP (apamin-insensitive) and are sufficient to account for the entire sAHP. P-type currents contributed to the apamin-sensitive mAHP (but not in all cells). We found no evidence for involvement of T- or R-type currents in the generation of these AHPs. L-type currents played no role in the activation of the mAHP or sAHP despite the fact that L-type current accounts for substantial current in dissociated pyramidal neurons (Lorenzon and Foehring 1995b). It is unlikely that these results are due to poor access by Nif because there was a similar lack of response to nimodipine and BAY K 8644, other studies have shown effects of Nif in slices at similar doses (Kullmann et al. 1992; Magee and Johnston 1997), and Nif reduced neuronal firing rate, suggesting that L-type channels contribute to the inward currents underlying steady firing.

In three cells, AgTx increased the mAHP by an unknown mechanism. Possibilities include Ca²⁺-independent postsynaptic effects of AgTx IVA on other than Ca²⁺ channels, presynaptic Ca²⁺ channel blockade resulting in reduced tonic release of a neuromodulator that normally inhibits the mAHP, and a Ca²⁺-dependent inward current coupled toP-type currents that overlaps in time with the mAHP. We have found no published evidence for any of these possibilities, although in rat trigeminal motoneurons, P-type channels were found to selectively reduce the ADP seen after a single spike (Kobayashi et al. 1997).

In neocortical pyramidal neurons, the combined application of AgTx and CgTx GVIA resulted in less than additive effects. This could indicate that both Ca²⁺ channel types activate the same K⁺ channels or that there is cooperativity among Ca²⁺ channels and a threshold for K⁺ current activation. The AHP amplitudes and effects of all of the organic blockers were highly variable between cells, presumably due to recording from a heterogeneous population of pyramidal neurons. Because of the variability observed between different cells, the use of current-clamp measurements, possible dendritic shunting of currents, and the unknown subcellular localization of the Ca²⁺ and Ca²⁺-dependent K⁺ channels in pyramidal neurons, we do not place much emphasis on the absolute values for the percent of the AHPs blocked by a given toxin and do not have enough confidence in the quantitative aspects of our data to resolve this issue. Our data provide strong evidence for the involvement of particular channel subtypes in AHPs and firing, but are not likely to be accurate quantitative estimates of the proportions of the AHPs affected by each Ca²⁺ channel subtype.

It is unlikely that the nonadditive effects were due to cross-reactivity of the blockers because data from dissociated pyramidal cells (Brown et al. 1994; Lorenzon and Foehring 1995b; McDonough et al. 1996; Mintz et al. 1992; Sayer et al. 1990) suggest that at the doses used, AgTx and CgTx GVIA are highly selective for P- and N-type channels, respectively. Slice studies on neurotransmitter release also have shown selectivity between GVIA and AgTx in this dosage range (Lovinger et al. 1994; Wheeler et al. 1994).

Comparative data

In neocortex, we found that N-, P-, and Q-type channels (but not L type) could elicit the sAHP. In cholinergic neurons from rat N. Basalis, N- and P-type channels (but not L, T, or Q type) coupled to a similar apamin-insensitive sAHP (Williams et al. 1997). In rat superior cervical ganglion neurons (Davies et al. 1996) and rat CA1 pyramidal neurons (Torres et al. 1996), the sAHP was dependent on internal stores of Ca²⁺ (but see Zhang et al. 1995). The sAHP in superior cervical ganglion neurons was not sensitive to organic Ca²⁺ channel blockers (Davies et al. 1996). In the present study, we cannot rule out a role for internal stores secondary to Ca²⁺ entry through N-, P-, and Q-type channels.

In neocortical pyramidal neurons, the apamin-sensitive mAHP was only coupled to P-type channels. In contrast, apamin-sensitive AHPs in vagal motoneurons (Sah 1995), CA1 pyramidal neurons (Higashi et al. 1990), and superior cervical neurons (Davies et al. 1996) were found to be coupled to N-type channels. In hypoglossal motoneurons (Umemiya and Berger 1994; Viana et al. 1993), the apamin-sensitive AHP was due to N- and P-type but not L-type currents (Umemiya and Berger 1994; Viana et al. 1993). In trigeminal motoneurons, the mAHP was coupled to N- but not L- or P-type channels (Kobayashi et al. 1997). P-type channels underlie an afterdepolarization in those cells (Kobayashi et al. 1997). In cholinergic N. basalis neurons,N- and T-type currents, but not P-, Q-, or L-type currents, couple to the mAHP (Williams et al. 1997). L-type channels underlie the apamin-sensitive AHP in rat substantia nigra pars compacta neurons (Nedergaard et al. 1993), N. basalis neurons (Williams et al. 1997), and striatal medium spiny neurons (Hernandez-Lopez et al. 1997). L-type currents also elicit I_C-type K⁺ currents in chick sympathetic and parasympathetic neurons (Wisgirda and Dryer 1994). In chick parasympathetic neurons, N-type currents also activate I_C-type K⁺ currents (Wisgirda and Dryer 1994). Thus there does not seem to be a simple pattern of relationships across cell types between particular Ca²⁺ channels and Ca²⁺-dependent K⁺ channels.

In neocortical pyramidal neurons, we found that blocking N- and P-type currents led to reduced spike-frequency adaptation and increased firing rate and L-channel blockade led to a decrease in firing frequency in response to long suprathreshold current injections. This latter effect is similar to the effect of L-type currents on turtle spinal motoneurons (Hounsgaard and Mintz 1988) and rat medium spiny striatal neurons (Hernandez-Lopez et al. 1997). In substantia nigral neurons, blockade of L-type currents leads to an increase in firing frequency (Nedergaard et al. 1993).

Potential mechanisms

The dynamics of the relationship between Ca²⁺ currents and Ca-dependent K⁺ currents depends on the subcellular localization of these channels and on intracellular Ca²⁺ accumulation during neural activity (Ghosh and Greenberg 1995; Hernandez-Cruz et al. 1990; Schwindt et al. 1992a). The low Ca²⁺-sensitivity of BK type K⁺ channels and strong intracellular Ca²⁺ buffering (McBurney and Neering 1987), suggest that Ca²⁺ channels must be in close physical proximity to these effectors (Gola and Crest 1993; Robitaille et al. 1993), and there is narrow colocalization of Ca²⁺-dependent K⁺ channels (BK) and Ca²⁺ channels in the frog neuromuscular junction (Robitaille et al. 1993) and in Helix neurons (Gola and Crest 1993). Calcium-dependent K⁺ channels of the small conductance (SK) type are more sensitive to Ca²⁺ than BK type channels (Blatz and Magleby 1987), suggesting that the spatial separation of SK type channels from their sources of Ca²⁺ could be greater.

A possible mechanism for the differential coupling ofN-, P-, and Q-type channels to the sAHP, P-type to the mAHP and L-type to neither AHP would be differences in the subcellular distribution of these Ca²⁺ channels and/or the Ca²⁺-dependent K⁺ channels. Virtually nothing is known about the subcellular distribution of Ca²⁺-dependent K⁺ channels in neocortical pyramidal neurons. For the mAHP, the underlying Ca²⁺-dependent K⁺ channels are apamin-sensitive (Lorenzon and Foehring 1993; Schwindt et al. 1988b), SK type channels (perhaps SK2 channels) (Kohler et al. 1996). Apamin-binding sites (Gehlert and Gackenheim 1993) and SK2 mRNA (Kohler et al. 1996) are present in rat sensorimotor cortex but the subcellular localization is unknown. The sAHP in cortical pyramidal neurons is Ca²⁺ dependent but apamin insensitive (Sah 1996; Schwindt et al. 1988b). The channels underlying the sAHP in pyramidal neurons might be apamin-insensitive SK channels (e.g., SK1 channel) (Kohler et al. 1996), although the slow activation of the sAHP suggests that the sAHP could be due to a novel channel type (Sah 1996; Schwindt et al. 1992a). In CA1 pyramidal neurons, Sah and Bekkers (1996) suggested that the sAHP channels may largely be found on the proximal apical dendrite within ~200 µm of the soma (see also Andreasen and Lambert 1995). The photolytic manipulations of intracellular Ca²⁺ levels in CA1 pyramidal cells by Lancaster and Zucker (1994) suggest that the source of Ca²⁺ for the sAHP channels is likely from HVA Ca²⁺ channels in proximity to the K⁺ channels.

The data on the distribution of Ca²⁺ channels in pyramidal neurons are also conflicting. Dendritic and somatic recordings (Kim and Connors 1993; Reuvani et al. 1993; Schiller et al. 1996) and Ca²⁺ imaging studies (Yuste et al. 1994) of neocortical pyramidal cells suggest that HVA Ca²⁺ channels have a nonuniform distribution in dendrites and somas. Immunocytochemical and imaging data (Elliot et al. 1995; Mills et al. 1994; Westenbroeck et al. 1990, 1992, 1995) suggest that N- and P/Q-type channels are in high density in the apical dendrites of pyramidal neurons (as well as the soma), but L-type channels are restricted to the soma and proximal apical dendrite. On-cell patch recordings from apical dendrites (Magee and Johnston 1995) and whole cell recordings from putative dissociated dendrites (dendrosomes) (Kavalali et al. 1997) suggest that L-type currents are found in abundance in dendrites. Thus although we favor the hypothesis (cf. Kobayashi et al. 1997; Williams et al. 1997) that the basis for differential coupling of Ca²⁺ channels to Ca²⁺-dependent K⁺ channels is their subcellular colocalization, we know of no evidence for a cell region with a channel distribution that would match our data.

Functional significance

The mAHP and sAHP reflect the presence of Ca²⁺-dependent K⁺ currents activated during APs that serve to regulate the firing behavior of neocortical pyramidal cells (Lorenzon and Foehring 1993, 1995a; Schwindt et al. 1988b). The mAHP currents are important in controlling interspike intervals and the sAHP current in regulating spike-frequency adaptation. We found convergent and divergent actions of HVA Ca²⁺ currents on the firing behavior of neocortical pyramidal cells. L-type channels contribute to inward currents underlying firing, and N-, P-, and Q-type currents also contribute to spike-frequency adaptation. Ca²⁺-dependent outward currents also may play a role in shaping the transfer of dendritic synaptic events to the soma, particularly with strong repetitive activation, such as that used to elicit long-term potentiation or long-term depression (e.g., Sah and Bekkers 1996). A dendritic location for Ca²⁺ and Ca²⁺-dependent K⁺ channels could contribute to shaping synaptic events in dendrites (Magee and Johnston 1995; Magee et al. 1996; Markham et al. 1995), modulate transfer of excitation to the soma, and allow temporary isolation of subcellular regions (cf. Yuste et al. 1994). The differential coupling of Ca²⁺ channel types to AHPs and firing would allow specific alterations in cell behavior by neuromodulators targeted to particular calcium channel types.

	ACKNOWLEDGEMENTS

  The authors thank Drs. W. Armstrong and R. Scroggs for critical reading of an earlier version of this manuscript, B. Harms and B. Mattix for excellent technical assistance, and Dr. C. J. Wilson for assistance with the biocytin-labeled cells.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-33579 to R. C. Foehring and National Science Foundation Grant IBN-9400318 to R. S. Waters.

	FOOTNOTES

  Address for reprint requests: R. C. Foehring, Dept. of Anatomy and Neurobiology, University of Tennessee, Memphis, 855 Monroe Ave., Memphis, TN 38163.

  Received 17 October 1997; accepted in final form 13 January 1998.

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