Interaction of convergent pathways that inhibit N-type calcium currents in sensory neurons

Neuroscience Vol. 65, No. 2, pp. 477-483, 1995

Pergamon

0306-4522(94)00476-5

ElsevierScienceLtd IBRO Printed in Great Britain

INTERACTION OF CONVERGENT PATHWAYS THAT INHIBIT N-TYPE CALCIUM CURRENTS IN SENSORY NEURONS M. DIVERSI~-PIERLUISSI and K. DUNLAP* Departments of Physiologyand Neuroscience, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, U.S.A. Abstraet--Norepinephrine and GABA inhibit to-conotoxin GVIA-sensitive (N-type) calcium current in embryonic sensory neurons by separate pathways. We have investigated the mechanisms that limit the modulation of N current by varying the level of activation for a single pathway or simultaneously activating multiple pathways. Calcium currents were measured with tight-seal, whole-cell recording methods. Simultaneous application of the two transmitters at saturating concentrations produced a larger inhibition of the current than either transmitter by itself, but the maximal inhibition was not linearly additive. Maximal, direct activation of GTP-binding proteins by intracellular application of guanosine 5'-(3-O-thio)-triphosphate (GTP~,S) resulted in a similar limit to the inhibition; furthermore, GTP~,S did not enhance the maximal inhibition produced by co-application of transmitters. Interventions downstream in the modulatory pathway (e.g. direct activation of protein kinase C or inhibition of protein phosphatases) were also unable to alter the maximal limit for inhibition. These results suggest that transmitter-mediated inhibition is not limited by receptor number, levels of G-protein or protein kinase C activation, or degree of phosphorylation; rather, the extent of inhibition may be limited by the structural properties of the N channels themselves.

High voltage-activated (HVA) calcium channels in neurons are established targets for inhibitory modulation by a variety of transmitters and peptides. 23'32'5zAlthough several distinct mechanisms mediate the inhibition of calcium channel function, a few common features are shared by the majority of modulatory pathways. Most reported instances of HVA calcium current inhibition in neurons indicate that the transmitters target N-type calcium channels selectively2'15'35'41 but see. 36'3s In all cases in which it has been carefully studied, modulation is also saturable, with maximal effects of transmitter usually inhibiting about 50% of the available calcium current. Such inhibition has, in addition, invariably been shown to require the activation of GTP-binding proteins (G-proteins), as evidenced by the ability of nucleotide analogs to either block (GDPflS) or potentiate (GTP~S) transmitter responses. 19'32'33 Despite these common features, however, modulation of calcium channels clearly involves a wealth of idiosyncratic detail. For example, multiple G-protein types appear to mediate the inhibition of calcium current function. Pertussis toxin (PTx)-sensitive G*To whom correspondence should be addressed. bis(o-aminophenoxy)-ethaneG-protein, GTP-binding protein; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HVA, high voltage-activated; NE, norepinephrine; OAG, oleoylacetylglycerol;OKA, okadaic acid; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PTx, pertussis toxin.

Abbreviations: BAPTA, N,N,N',N'-tetra-acetate;

NSC 65/2~G

477

proteins are considered the primary mediators, 31'33'41'53 but PTx-resistant G-proteins have also been shown to play a role in certain cases. 6'37 Further identification of the G-proteins has been attempted in only a few preparations. Protein reconstitt/tion and antisense oligonucleotide approaches in GH 3 Cells, for example, indicate that G-proteins of the PTxsensitive G-protein class link muscarinic and somatostatin receptor binding to the inhibition of calcium channel function but that different PTx-sensitive G-protein subtypes mediate the actions of the two receptors. 34 Downstream from the G-protein step, channel modulation, even within a single cell, appears to involve a variety of molecular mechanisms.6'7,9'17 In rat sympathetic neurons, a relatively slow mechanism activated by muscarinic agonists employs a diffusible second messenger whose identity is unknown (but whose mode of action does not involve PTx-sensitive G-proteins, cyclic AMP, cyclic GMP, intracellular Ca 2÷ or protein kinase C, PKC). s In these same cells, faster inhibitory mechanisms activated by muscarinic and ct-adrenergic agonists are also present. PTx-sensitive and -resistant forms of the fast pathway have been demonstrated; both are voltage-dependent and may involve direct G-proteinN channel coupling.32 A different, yet equally complex story is emerging from studies on chick dorsal root ganglion neurons in which norepinephrine (NE) and GABA utilize nonequivalent modulatory pathways to inhibit N-type

M. Divers4-Pierluissi and K. Dunlap

478

calcium channels. A l t h o u g h b o t h p a t h w a y s require the activation of PTx-sensitive G-proteins, 33 it is a p p a r e n t t h a t inhibition p r o d u c e d by N E requires P K C activation, whereas t h a t p r o d u c e d by G A B A d o e s not. 17'42'43 As with rat sympathetic neurons, cyclic A M P , cyclic G M P a n d intracellular C a 2+ a p p e a r not to be involved for either transmitter. A variety of distinct p a t h w a y s have thus evolved to inhibit N-type calcium channels. It is interesting to note, however, t h a t in a single cell with co-existing m o d u l a t o r y mechanisms, the m a x i m a l activation of any given p a t h w a y does not eliminate N current. In chick dorsal root ganglion neurons, for example, m a x i m a l inhibition is limited to a p p r o x i m a t e l y 30%. W h a t is the underlying basis for this limit? T o begin to address this question, we have studied the simult a n e o u s activation of the N E - a n d G A B A - i n d u c e d p a t h w a y s t h a t inhibit N-type calcium c h a n n e l s in these neurons. O u r results suggested t h a t the pathways converge at a s a t u r a t i o n point t h a t lies at, or just proximal to, the N channel itself.

EXPERIMENTAL PROCEDURES

Cell culture Dorsal root ganglia were dissected from 11 12-day-old chick embryos (Spafas, Storrs, CT). Tissue was incubated at 37°C for 50 min in Ca 2+ and Mg2+-free saline and dissociated by trituration with a fire-polished Pasteur pipette. Cells were plated in plastic, collagen-coated 35 mm tissue culture dishes and grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum, 5% chick embyro extract, 50 units/ml penicillin, 50/~g/ml streptomycin and nerve growth factor.

Electrophysiologieal methods Cells were used for recording two to five days after plating. The pipette internal solution contained 150mM CsC1, 10mM HEPES, 5 m M MgATP and 5 m M bis(oaminophenoxy)-ethane-N,N,N',N'-tetra-acetate (BAPTA), pH7.2. The external saline solution contained 133mM NaCI, 1 mM CaC12, 0.8 mM MgC12, 10 mM tetraethylammonium chloride, 25 mM HEPES, 12.5 mM NaOH, 5 mM glucose and 0 . 3 # M tetrodotoxin (pH7.4). Experiments were performed at room temperature. Pipettes were prepared from Fisher microhematocrit capillary tubes. Pipette resistances prior to forming high-resistance seals ranged from 1.5 to 2.5 Mfl. A List EPC-7 amplifier (Medical Systems) was used to record calcium currents in the tightseal, whole-cell configuration. Data were acquired at I0 kHz using an Atari Mega 4 STE computer (software from Instutech Corporation, Great Neck, NY). A standard P/4 protocol was used for leakage subtraction of the currents prior to analysis. Subtracted currents were transferred to a Macintosh Quadra (Apple Computers) for further analysis. The total charge flowing through HVA calcium channels was calculated in Igor (Wave Metrics, Lake Oswego, OR) by integrating calcium current as a function of time. Charge entry from population studies is reported as the mean + S.E.M. Responses to transmitters were quite consistent from cell to cell within a given plating (with less than 10% variation among cells), but varied considerably among different platings. For this reason, data from experimental groups were always compared to controls within the same plating, measured on the same day. Interplating comparisons were limited to those platings which yielded similar control responses.

Materials Okadaic acid (OKA; ammonium salt, LC Laboratories) was prepared as a stock solution in dimethylsulfoxide and stored in aliquots at -70°C. GTPTS (Fluka), GABA and NE (Sigma) were prepared as stock solutions in distilled water. GTP7S and GABA were stored at - 2 0 and 4°C, respectively. Fresh solutions of GTPTS and OKA were prepared prior to experiment by dilution into intracellular saline. Transmitters were diluted from stock solutions into extracellular saline. OKA and GTPTS were applied intracellularly through patch pipettes; the transmitters were applied extracellularly by pressure ejection from puffer pipettes of 2-3/~m tip diameter. All salts were obtained from Fluka and tetrodotoxin from Sigma.

RESULTS H V A calcium currents were evoked by 100 ms step depolarizations to 0 m V from a holding potential of -80mV. N-type calcium current is ~ 1 0 0 % of the total current m e a s u r e d in cells within 24 h of plating; a small a m o u n t ( < 15 % ) of dihydropyridinesensitive, L-type current can be observed in older cells > t h r e e days in culture. 15 Previous results have d e m o n s t r a t e d t h a t N E a n d G A B A produce c o n c e n t r a t i o n - d e p e n d e n t inhibition o f the calcium current; m a x i m a l effects are achieved at 10/~M N E 13 a n d 100~tM G A B A ? ~ We have employed these saturating c o n c e n t r a t i o n s of N E a n d G A B A in the experiments reported here.

Additivity o f responses to norepinephrine and GABA We first investigated w h e t h e r the maximal N current inhibition we observed for a single transmitter was limited by the extent of receptor activation. We tested the additivity o f responses to sequential application o f saturating c o n c e n t r a t i o n s of N E a n d G A B A , the second t r a n s m i t t e r in the c o n t i n u o u s presence of the first. Inhibition of calcium current was invariably greater when N E a n d G A B A were co-applied t h a n when either was applied alone (Fig. 1A, B, Table 1). However, responses did n o t a p p e a r to be linearly additive. In the platings used in this series of experiments, m a x i m a l inhibition was limited to 2 5 - 3 0 % when N E or G A B A was applied alone a n d to 3 5 4 0 % when the two transmitters were applied together. This m a x i m u m was i n d e p e n d e n t o f the order in which the transmitters were applied. C o m p a r i s o n of the m e a n maximal inhibition to t h a t predicted assuming linear additivity (using a S t u d e n t ' s t-test) indicated a difference t h a t was only moderately significant ( P ~ 0.1). T h a t a c o n s t r a i n t exists o n the m a x i m a l inhibition was, however, reinforced by the negative correlation in the relationship between cellular responses to one vs two transmitters (Fig. 1C). The larger the inhibition p r o d u c e d by the first transmitter, the smaller t h a t p r o d u c e d by the second. These results argue t h a t limitations in receptor activation do not underlie the s a t u r a t i o n of t r a n s m i t t e r - m e d i a t e d N current inhibition a n d t h a t N E - a n d G A B A - a c t i v a t e d

N channel modulation

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Fig. 1. Inhibitory responses to GABA and NE are additive but limited by a maximum. (A) Superimposed recordings of calcium current evoked by 50 ms step depolarizations to 0 mV in two different cells exposed first to 10/tM NE and then to a combination of 10/aM NE and 100/tM GABA (as marked). The cell whose responses are shown at the top responded robustly to NE. Subsequent application of GABA produced only a small additional inhibition. The lower traces were taken from another cell that responded less robustly to NE; subsequent application of GABA evoked additional inhibition. The combined inhibition produced by the two transmitters in this cell matched that of the cell in A. (Note: the small standing inward current remaining in the control trace following repolarization is due to some residual Ca 2+ -activated C1- current that was not eliminated by BAPTA. As the C1- equilibrium potential is 0 mV, Ca 2+ currents measured with test pulses to 0 mV are uncontaminated by this residual C1- current.) (B) Ca 2+ current inhibition as a function of time for the cell shown in the lower panel of A. The times of application of 10 # M NE and 100/~M GABA are denoted by the horizontal bars. (C) The total decrease in charge entry (in nC) produced by co-application of the transmitters (second response) is plotted as a function of the decrease in charge entry produced by application of a single transmitter (first response). The data were fitted to a straight line function; the correlation coefficient of linear regression analysis was -0.44.

m o d u l a t o r y p a t h w a y s share some c o m m o n , saturable element. Such conclusions are further s t r e n g t h e n e d by experiments with GTP~, S.

Potentiation of transmitter responses of intracellular GTPTS T o test w h e t h e r limitations in G - p r o t e i n activ a t i o n play a pivotal role in s a t u r a t i o n of N c h a n n e l inhibition. G - p r o t e i n s were directly activated by GTPTS. The nucleotide was a d d e d to the internal solution a n d cells were dialysed d u r i n g whole cell recording prior to testing with transmitter. O n its own, G T P ? S could p r o d u c e inhibition o f calcium currents d u r i n g this application period. The higher the nucleotide c o n c e n t r a t i o n , the m o r e rapid the onset o f i n h i b i t i o n (Fig. 2). T h e m a x i m a l inhibition p r o d u c e d by 100 # M G T P T S was, o n average, larger t h a n t h a t p r o d u c e d by N E or G A B A , b u t the effect still fell s h o r t of complete inhibition (Fig. 2, T a b l e 2). If G T P 7 S-dialysed cells were tested with t r a n s m i t t e r after the nucleotide h a d p r o d u c e d its full i n h i b i t o r y effect, no further reduction in

calcium current was observed (Fig. 3A). Lower concentrations of GTP?S produced partial occlusion (Fig. 3B). As reported previously, maximal responses produced by N E or G A B A could be e n h a n c e d by intracellular GTPTS. ]9'z3 If cells were tested with t r a n s m i t t e r s prior to the onset o f G T P ? S - i n d u c e d inhibition (i.e. within 1 m i n o f whole cell access), the presence of the nucleotide p o t e n t i a t e d t r a n s m i t t e r effects. A t low c o n c e n t r a t i o n s o f transmitters, this e n h a n c i n g effect of GTP~,S was most p r o m i n e n t , but p o t e n t i a t i o n was significant w h e n the cells were tested with s a t u r a t i n g c o n c e n t r a t i o n s o f t r a n s m i t t e r

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Table 1. Inhibition of calcium current by coapplication of norepinephrine (10pM) and GABA (100#M) % Inhibition

Treatment

No. of cells

(of charge entry)

NE GABA NE~JABA GABA-NE

7 8 10 13

20 + 6 26 + 7 36 _+ 6 37 + 7

time (s)

Fig. 2. GTP 7 S inhibits calcium current in a concentrationdependent manner. Total charge entry through N channels (normalized to the maximum in each cell) is plotted as a function of time after breakthrough for three cells, each dialysed with a different concentration of GTPTS (as marked) and typical for the sample.

480

M. Divers~-Pierluissi and K. Dunlap

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control Fig. 3. Intracellular GTP7 S occludes transmitter-induced inhibition of calcium current. Calcium currents were isolated according to methods described and were evoked by step depolarizations from a holding potential of - 8 0 mV to 0 mV for 100 ms. Only the first 50 ms of the records are displayed. Cells were dialysed with 100/tM (A) or 10 # M (B) GTPTS (as marked). After 10 min of dialysis, when the calcium current amplitudes had reached a steady state, the cells were tested with I0 # M NE. Traces taken under control, GTP 7S-dialysed or NE-treated conditions are superimposed and labeled accordingly.

(Table 2). W h e n responses o f the n e u r o n s to nucleotide or to co-application o f the transmitters were c o m p a r e d in cells from the same plating, the maximal inhibition p r o d u c e d by GTP~,S alone was n o t different f r o m t h a t p r o d u c e d by co-application of the transmitters. G T P 7 S (100 p M ) inhibited the current by 44 + 4 % , whereas N E a n d G A B A , applied together, p r o d u c e d a 40 4 - 5 % inhibition. Evalulation o f these data with a S t u d e n t ' s t-test indicated no difference between the two groups ( P > 0.1). T a k e n together, these results suggest t h a t s a t u r a t i o n o f the m o d u l a t o r y p a t h w a y likely occurs d o w n s t r e a m from G-proteins.

Enhancement of protein kinase C-mediated inhibition We k n o w from o u r previous work j7'42 t h a t N E a n d G A B A use different p a t h w a y s to produce N c h a n n e l inhibition. N E - i n d u c e d inhibition requires the activation of P K C , while t h a t induced by G A B A does not. T o test w h e t h e r s a t u r a t i o n o f N c h a n n e l inhibition is, in part, associated with limitations in P K C activation, we have used two m e t h o d s of e n h a n c i n g P K C - m e d i a t e d m o d u l a t i o n : direct activation of P K C by oleoylacetylglycerol ( O A G ) a n d inhibition of

Table 2. GTPTS potentiates GABA- and norepinephrineinduced inhibition of charge entry through N-type calcium channels Treatment O.5 # M GABA control 100pM GTPvS I00 # M GABA control 100 p M GTP'fS I # M NE control 100/~M GTPyS 10/tM NE control 100 # M GTPvS

No. of cells

% Inhibition (of charge entry)

5 5

10 _ 2 40_+ 5

6 6

35 + 5 47 __+6

4 8

7_ 1 25 ___3

10 10

25 + 7 35 4- 6

p h o s p h a t a s e activity by O K A . W h e n applied alone, O A G p r o d u c e d 23 ___6 % inhibition of N current and occluded further inhibition by N E (data not shown; see Rane a n d Dunlap42). W h e n co-applied with G A B A , a 38.5 + 7 % inhibition was observed; the m a x i m u m inhibition was, again, constrained by a n u p p e r limit indistinguishable from t h a t observed with G T P T S or with co-application of N E a n d G A B A (Table 3). O K A , w h e n applied intracellularly to dorsal root ganglion n e u r o n s at 0.1 p M , p o t e n t i a t e d the calcium current inhibition p r o d u c e d by N E (Table 3) or O A G . n The extent of inhibition in the presence o f O K A was similar to that observed during coapplication o f G A B A with N E or O A G . To determine w h e t h e r further e n h a n c e m e n t of N channel m o d u l a t i o n could be achieved, we tested the effects of intracellular O K A o n responses to co-applied agonists. N o further inhibition was p r o d u c e d u n d e r these conditions (Table 3). The similarity in m a x i m a l response u n d e r all o f these conditions suggests t h a t some element b e y o n d the level of P K C is responsible

Table 3. Okadaic acid potentiates the inhibition of charge entry through N-type calcium channels produced by norepinephrine Treatment 100 ~M GABA control 100 nM OKA 100pM NE control 100 nM OKA NE + GABA control 100 nM OKA GABA + NE control 100 nM OKA 60 p M OAG + GABA control 100 nM OKA

No. of cells

% Inhibition (of charge entry)

10 10

32 + 7 29 4- 8

10 7

12 __+6 40 +_ 5

10 10

36 4- 6 37 _+ 5

7 7

37 + 7 38 + 4

7 7

37 +_ 6 35 + 5

N channel modulation for saturation of calcium current inhibition. Perhaps it is the calcium channel itself that limits the extent of inhibition. DISCUSSION

We have studied the interaction of modulatory pathways activated by NE and GABA that inhibit N-type calcium currents in dorsal root ganglion neurons. Our results demonstrate that two distinct pathways converge on N channels to produce additive inhibition, but N currents are not eliminated, even with maximal stimulation. Increases in receptor activation produced by simultaneous activation of two receptor types produces greater inhibition than that produced by a single receptor type, but the responses are not linearly additive, suggesting that N channel inhibition is limited by something beyond the level of the receptors. Likewise, enhancement of G-protein activation produced by intracellular GTPTS inhibits N channel function on its own and potentiates individual transmitter responses, but produces no more inhibition than that induced by co-application of the transmitters. This suggests that saturation of the modulatory response occurs beyond the level of the G-proteins as well. Finally, the saturation point observed in the presence of both transmitters was also unaffected when G-protein activation was bypassed through direct activation of PKC (with OAG) or when PKC-dependent phosphorylation was enhanced with the phosphatase inhibitor OKA. This argues that the saturable element shared by the modulatory pathways lies beyond the kinase. As the modulation produced by GABA saturates similarly despite its being PKCindependent, limitations on the inhibition may be controlled at the level of the N channels themselves.

Direct biochemical alteration of calcium channels The hypothesis that the modulation is limited by the structural properties of the channel is not surprising in view of the phosphorylation-dependent modulation of L-type calcium channel function. Within the calcium channel subunits in skeletal muscle Ttubule membranes, the pore-forming ~-subunit is the preferred substrate for phosphorylation by, among others, protein kinases A (PKA) and C. 14'39"44'55 Reconstitution of such phosphorylated calcium channels into lipid vesicles demonstrated enhanced dihydropyridine-sensitive calcium influx, t4 Studies on intact skeletal muscle argue, additionally, that phosphorylation is likely to play a significant role in regulating muscle contractility during stimulus trains. 45 In cardiac muscle, it has long been known that L-type calcium current is also enhanced by agents that raise intracellular cyclic AMP, such as noradrenaline or forskolin, 29'5j leading to increased force of contraction. This action of cyclic AMP is produced via PKA-dependent phosphorylation

481

as demonstrated by its elimination with inhibitors selective for PKA. 29 Although limited biochemical evidence has emerged to directly demonstrate phosphorylation of non-muscle L-type channels,3° numerous electrophysiological studies clearly indicate cyclic AMP-dependent up-regulation of L channel function in neurons and neuroendocrine cells as well.3,4, 28

Direct demonstration of N channel phosphorylation is also limited. In vitro experiments have shown that proteins from rabbit brain that are labeled with [~25I]-og-conotoxin GVIA and immunoprecipitated with antibodies to the skeletal muscle Ca 2+ channel serve as substrates for PKA and PKC. ~ Although the functional consequences of PKA-dependent phosphorylation of N channels is unknown, the effects of PKC-dependent phosphorylation can be inferred from electrophysiological studies. In some vertebrate neurons, activation of PKC inhibits N channel f u n c t i o n . 11'17'18'42'43 If such inhibition occurs on N channels in nerve terminals, it may explain the action of certain neurotransmitters thought to control synaptic transmission through presynaptic inhibition,l° Phosphorylation is likely not the only mechanism responsible for alterations in N channel function. In fact, the majority of studies on N channel modulation have ruled out an involvement of most known second messengers and kinases. Our experiments with GABA indicate that cyclic AMP, cyclic GMP and phospholipase A 2 are not involved (unpublished observations). Such negative evidence, coupled with the widespread involvement of G-proteins in receptor-mediated inhibition of N channels and the absence of modulation of channels in the patch by application of transmitters to the extrapatch membrane, has lead to the notion that direct G-protein binding inhibits N channels. 12,27,32

Underlying basis for partial transmitter-mediated inhibition Whatever the precise structural mechanisms underlying N channel inhibition, our results demonstrate that maximal activation of the multiple modulatory pathways leaves a significant fraction of N current unmodified. At this point we do not know whether such partial inhibition is due (i) to heterogeneity among N channels (some that can and others that cannot be inhibited) or (ii) to incomplete inhibition of a homogeneous population of N channels (all of which can be only partially inhibited). Limited information exists regarding the former of these two possibilities. Molecular cloning and expression studies have established that class B calcium channel, when expressed in mammalian cells, code for a ~o-conotoxin GVIA-sensitive calcium current. 2°,54 Although splice variants of class B calcium channel subunits have not been reported, alternative splicing is common in the calcium current gene family.49'~ It is possible, therefore, that incomplete inhibition

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M. Divers6-Pierluissi and K. Dunlap

o f N currents observed in our study results from the presence o f N channel subtypes that c a n n o t be modulated. Precedent in the m o d u l a t i o n literature favors the possibility o f partial inhibition o f the channel. Studies o f b o t h L- and N-type single calcium channel currents have d e m o n s t r a t e d that m o d u l a t i o n is m o s t often associated with alterations in channel gating, producing either increases 5° or decreases 16'36'47 in the probability o f opening, depending u p o n the channel type and m o d u l a t o r y p a t h w a y activated. These alterations are rarely all-or-none; rather, more subtle shifts in open or closed times are generally observed. Future investigations o f single N channels in chick dorsal root ganglion neurons during activation o f the separate m o d u l a t o r y pathways are required to determine whether activation o f distinct biochemical pathways by N E and G A B A produces similar or different functional effects on N channels.

CONCLUSIONS Multiple m o d u l a t o r y pathways converge to inhibit N-type Ca 2+ channels in embryonic chick sensory neurons. In this paper, we have tested several means o f simultaneously activating the pathways, or o f increasing the activation o f a single pathway, to investigate the mechanisms that limit the extent o f Ca 2+ channel inhibition. We conclude that the maximal inhibition is not a function o f receptor occupancy, G - p r o t e i n activation or kinase-dependent phosphorylation. Rather, it appears likely that limitations on maximal inhibition are imposed at the level o f the N channels themselves. Acknowledgements--The authors thank Michael Goy for

his critical comments on an earlier and more complex version of this manuscript. This work was supported by NS16483 and a supplement from the Program for Underrepresented Minorities from the National Institutes of Health.

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