0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO

Neuroscience Vol. 44, No. 3, pp. 663~672, 1991

Printed in Great Britain

PROPERTIES OF Ca 2+ C U R R E N T S IN A C U T E L Y D I S S O C I A T E D N E U R O N S OF THE CHICK CILIARY G A N G L I O N : I N H I B I T I O N BY SOMATOSTATIN-14 A N D SOMATOSTATIN-28 S. E. DRYER,* M. M. DOURADO a n d M. E. WISGIRDA Department of Biological Science B-157, Florida State University, Tallahassee, FL 32306-3050, U.S.A. Abstract--Whole-cell voltage-clamp recordings were made from acutely dissociated neurons obtained from the embryonic chick ciliary ganglion. Recording pipettes were filled with salines containing 120 mM CsCI or 120 mM tetraethylammonium-C1. Application of depolarizing voltage commands evoked L-type Ca 2+ currents and, at voltages positive to 0 mV, an unidentified cationic conductance. The unidentified cationic conductances made the Ca 2+ currents appear to undergo voltage-dependent inactivation and made a large contribution to tail currents present during repolarizing voltage steps. Ca 2* currents could only be isolated by digital subtraction of currents remaining in Ca 2+-free saline. These Ca 2÷ currents showed little or no sign of inactivation and did not reverse at voltages up to +60 mV. Application of somatostatin-14 or somatostatin-28 produced a reversible inhibition of Ca 2+ currents in virtually all cells, regardless of size. Somatostatin-28 (1-14) was inactive. The effects of somatostatin-14 and somatostatin-28 were attenuated by pretreatment with pertussis toxin, suggesting a role for G-proteins in mediating the response. Somatostatin-14 and somatostatin-28 had no effect on voltage-dependent K ÷ currents. The results suggest that somatostatin peptides modulate the motor output of the chick ciliary ganglion.

S o m a t o s t a t i n is a cyclic tetradecapeptide f o u n d t h r o u g h o u t the nervous system o f vertebrates. 6'7'~6'23It can be released by K +-induced depolarization ~2 a n d is capable of p r o d u c i n g a variety o f actions on n e u r o n s in areas where it is present. These include inhibition of adenylate cyclase 26 a n d m o d u l a t i o n of several ionic c o n d u c t a n c e s . 2'14"15A8'21'22'28'29 F o r example, s o m a t o s t a t i n - 1 4 causes h y p e r p o l a r i z a t i o n of several p o p u l a t i o n s o f vertebrate central neurons ~5'21'2s by activation o f a n inwardly rectifying K + conductance. ~5'2~ S o m a t o s t a t i n - 1 4 activates the Mcurrent in h i p p o c a m p a l neurons. 22 S o m a t o s t a t i n - 1 4 also causes inhibition o f Ca 2+ currents in s y m p a t h etic neurons, ~4 spinal neurons, 2 a n d neocortical neurons. 29 These effects are m e d i a t e d t h r o u g h specific m e m b r a n e receptors 7'~3'26':7 t h a t a p p e a r to be coupled to G-proteins. This n o t i o n is based o n the fact t h a t m a n y of the actions of s o m a t o s t a t i n - 1 4 are inhibited by pertussis toxin ( P T X ) 7'14'18'21'22'24'28'29 and, because the binding of labeled s o m a t o s t a t i n - 1 4 derivatives to solubilized m e m b r a n e receptors is strongly influenced by G T P , by antibodies to G-proteins, a n d by PTX. 13,24,26 S o m a t o s t a t i n - 1 4 is derived from a 28-amino acid precursor called p r o s o m a t o s t a t i n . However, b o t h so*To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; EGTA, ethyleneglycol-

tetraacetic acid; GTP, guanosine triphosphate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Mg-ATP, adenosine triphosphate Mg 2+ salt; PTX, pertussis toxin; TEA-CI, tetraethylammonium-Cl; TTX, tetrodotoxin.

m a t o s t a t i n - 1 4 a n d somatostatin-28 are biologically active. ~4'2z28'29 In some cases, the actions of s o m a t o statin-28 a n d s o m a t o s t a t i n - 1 4 are different in a given p o p u l a t i o n o f neurons. F o r example, s o m a t o s t a t i n - 14 a n d somatostatin-28 produce opposite effects on delayed rectifier K + currents in m a m m a l i a n neocortical neurons. 28 Moreover, somatostatin-28 a n d somatostatin-14 have a differential distribution within the brain. 23 It is therefore possible that s o m a t o s t a t i n - 1 4 a n d s o m a t o s t a t i n - 2 8 represent distinct t r a n s m i t t e r systems t h a t m a y utilize distinct subtypes of receptors. 7 One p o p u l a t i o n of n e u r o n s t h a t is k n o w n to contain s o m a t o s t a t i n - 1 4 (or a related peptide) is the small c h o r o i d n e u r o n s f o u n d in the chick ciliary ganglion. 8't2 These cells are the sole source of the p a r a s y m p a t h e t i c m o t o r o u t p u t to the s m o o t h muscle c h o r o i d layer in the back o f the eye. 9'2° The larger ciliary n e u r o n s project to striated muscle in the iris a n d ciliary body 19 a n d do not express s o m a t o s t a t i n i m m u n o r e a c t i v i t y Y 2 The n a t u r e of the s o m a t o s t a t i n t h a t gives rise to the i m m u n o r e a c t i v i t y in choroid cells is not known. S o m a t o s t a t i n i m m u n o r e a c t i v i t y can be released from superfused p r e p a r a t i o n s of chick c h o r o i d ~2 a n d s o m a t o s t a t i n - 1 4 has been reported to inhibit the K + - i n d u c e d release of acetylcholine ( A C h ) from the same preparation. ILl2 These results suggest t h a t some form of s o m a t o s t a t i n m a y function as a n e u r o t r a n s m i t t e r or n e u r o m o d u l a t o r in c h o r o i d neurons. The purpose o f the present study was to examine the effects o f s o m a t o s t a t i n - 1 4 a n d somatostatin-28 on

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Ca :÷ currents in acutely dissociated chick ciliary ganglion neurons. This p o i n t was of interest because inhibition o f Ca 2÷ currents would be expected to result in inhibition of n e u r o t r a n s m i t t e r release and because n e u r o n s t h a t express a particular receptor o n a nerve terminal often express the same receptor on the cell body. Moreover, at least one previous study has suggested t h a t chick ciliary ganglion n e u r o n s can release n e u r o t r a n s m i t t e r s from the cell b o d y ) 7 As a prelude, it was necessary to perform biophysical a n d p h a r m a c o l o g i c a l experiments to characterize the Ca 2÷ currents present in these cells. EXPERIMENTAL

for 30 min to 4 h at 37cC under an atmosphere containing 5% CO 2. Under these conditions, ciliary ganglion neurons were essentially free of neurites, although some process outgrowth could be observed with longer times in cell culture. Whole-cell recordings

PROCEDURES

Cell isolation

Chick ciliary ganglia were obtained from embryos on embryonic days 10-14. At this stage of development, choroid neurons have been reported to express somatostatin immunoreactivity. 8 Ganglia were dissected and placed in a saline nominally free of divalent cations and containing 1 mg/ml collagenase (Sigma type II). Ganglia were incubated for 10-30 min at 37°C. The collagenase was removed by aspiration, and the ganglia were rinsed once in a cell culture medium consisting of Eagle's Minimal Essential Medium supplemented with 10% heat-inactivated horse serum, 2 mM glutamine, 50 U/ml penicillin, 50/~g/ml streptomycin, and 3% chick embryo eye extract. The ganglia were resuspended in cell culture medium and triturated with eight to 12 passes through a fire-polished Pasteur pipette. Dissociated neurons were plated onto poly-D-lysine-coated glass coverslips (two ganglia per dish) and allowed to settle A.

Control

Recordings were made with a commercially available patch-clamp amplifier (Axopatch IC, Axon Instruments, Foster City, CA). Pipettes were pulled in two steps from Boralex glass micropipettes (Rochester Scientific), coated to within 100#m of the tip with Sylgard 184 resin (Dow Corning), and fire polished. Pipettes were filled with a solution consisting of (in mM): CsC1, 120; MgCI:, 10; EGTA, 10; HEPES, 10; Mg-ATP, 5; pH 7.4. In some experiments, the CsCI was replaced with tetraethylammonium-C1 (TEA-CI). Differences between these pipette solutions will be described in Results. Ciliary ganglion neurons were mounted in a 500-/~1 chamber and perfused with a saline consisting of (in mM); NaCI, 148; KCI, 5.4; MgCI v 0.8; CaC12, 5.8; HEPES, 13; glucose, 5; TEA-CI, 5; 4-aminopyridine, 2; and 0.5#M tetrodotoxin at a pH of 7.4. In some experiments, cells were exposed to salines identical to that described above except that CaCI 2 was replaced on an equimolar basis by MgCI: (Ca:+-free saline). Cells were perfused at 2-3 ml/min. All experiments were performed at room temperature. Peptides were applied by total bath perfusion from separate reservoirs controlled by valves. The whole-cell recording configuration was obtained with standard techniques. Briefly, a high-resistance seal ( > 10GO) was obtained by application of a brief suction immediately after contact between the recording micropipette and the cell. The fast component of capacitance current was compensated for, and additional suction was

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Membrane potential (mV) Fig. 1. Ca 2+ currents and nonselective currents in acutely dissociated neurons of the chick ciliary ganglion. (A) Families of averaged ionic currents evoked by depolarizing voltage commands to - 6 0 , - 3 0 , and - 1 0 mV (top) and 0, + 20, and + 40 mV (bottom) from a holding potential of - 8 0 inV. Records were made from a single cell in normal saline (control) and Ca 2+ -free saline (0 Ca). Currents that were strictly dependent on the presence of Ca 2+ were obtained by digital subtraction (control - 0 Ca). All recordings were automatically leak-subtracted according to a P/4 protocol (see text). Each record shown is the average of five individual sweeps. Note presence of nonselective outward current in Ca 2 + -free saline. (B) Current-voltage diagram of data obtained from the same cell. Data are plotted for control saline (triangles), Ca2+-free saline (diamonds), and the difference between the two (circles). Note that the Ca 2 +-dependent currents do not reverse.

Somatostatin inhibition of neuronal calcium currents applied to produce intracellular contact. Additional capacitance and series resistance were then compensated for iteratively. The usual series resistance was 4-7 Mr2. In most cases, it was possible to compensate for 90% of this series resistance before oscillation of the current output of the clamp amplifier. Voltage commands and data acquisition were controlled by an AST 286 personal computer equipped with a Labmaster 12-bit, 125-kHz A/D converter using commercially available software (P-Clamp version 5.5, Axon Instruments, Inc.). Data were filtered at 2 5 kHz with a four-pole Bessel filter prior to digitization at 20-50kHz. This software allowed for current averaging, current subtraction, and subtraction of capacitance and linear leakage currents. In general, subtraction of linear leakage and capacitance currents was performed according to a P/4 or P/6 protocol. ~ With this protocol, four or six subpulses were applied prior to application of a main experimental pulse that was four or six times as large. Currents evoked by the subpulses were added together and subtracted from currents evoked by the main pulse to yield leak-subtracted traces. Care was taken to insure that subpulses did not activate voltage-sensitive ionic currents. Average currents could be fitted with exponential functions according to a Simplex nonlinear least-squares routine. Materials PTX, tetrodotoxin (TTX), TEA-C1, nifedipine, and 4aminopyridine were obtained from Sigma (St Louis, MO). All cell culture media were obtained from Whittaker Bioproducts (Walkersville, MD). Somatostatin-14 was obtained from Calbiochem (San Diego, CA) and from Sigma. Somatostatin-28 and somatostatin-28 (1-14) were obtained from Sigma.

RESULTS Results in this study are due to recordings made from 137 acutely dissociated ciliary ganglion neurons. Properties o f Ca 2 + currents

Application of depolarizing voltage commands from a holding potential of - 8 0 m V resulted in inward currents, as shown in Fig. 1. When the recording micropipettes were filled with CsC1, these inward currents were maximal at around 0 mV. At more positive potentials, the currents became smaller and appeared to reverse between + 4 0 and + 6 0 mV (Fig. 1A, bottom left). At potentials positive to around + 10 mV, the inward currents showed distinct outward relaxations. This result initially suggested the presence of both L- and N-type Ca 2 + currents. However, it was never possible to find a c o m m a n d potential at which the current remained at zero throughout the duration of the pulse. This result suggested instead that, with CsCI recording pipettes, Ca 2 ~ currents were imperfectly isolated. The result is not consistent with the idea that Cs + was flowing outward through Ca 2+ channels. 9 This point was confirmed when the same series of voltage commands was applied after perfusion with Ca2+-free salines (Fig. IA, middle). U n d e r these conditions, depolarizing voltage commands failed to evoke inward currents but did produce slowly activated outward currents at potentials positive to around 0 mV (Fig. 1A, middle, bottom). The activation kinetics of these currents were substantially slower than those of the

665

inward Ca 2+ currents observed in normal saline. Digital subtraction of currents evoked in Ca 2 +-free salines from those present in control conditions yielded those currents that were strictly dependent on extracellular Ca 2+ (Fig. 1A, right). The Ca 2+dependent inward currents could be evoked at voltages positive to around - 4 0 mV. They showed very little inactivation during pulses of up to 50 ms duration and never showed a true reversal at voltages up to + 6 0 m V . It was not possible to make good recordings beyond this potential. The results of these experiments are summarized in the current-voltage ( I - V ) diagrams shown in Fig. lB. These show steady-state current plotted against command potential in normal saline (triangles) and Ca 2+ free saline (diamonds) and the difference between the two (circles). The results are consistent with a single homogeneous population of Ca 2+ channels corresponding to the L-type channels described previously. 1° They also suggest that the use of CsC1 pipettes is insufficient to provide complete isolation of Ca 2+ currents, even when extracellular salines contained 5 m M TEA-CI and 2 m M 4-aminopyridine. A similar result could be obtained by examination of the tail currents apparent at the break of the depolarizing voltage step (Fig. 2). These recordings were obtained from the same cell shown in Fig. 1. The tail currents represent the deactivation of the steadystate currents and were recorded at - 80 mV. Arrows in Fig. 2A show the portions of the tail currents that were subjected to kinetic analysis. Note that inward tail currents were apparent in both normal and Ca 2+-free salines. Tail currents evoked by depolarizing commands to + 2 0 and 0 mV are shown in Fig. 2B. Fitted exponential curves are superimposed on the digitized data. In normal salines (Fig. 2B), the decay of the tail currents could only be fitted with double-exponential curves with time constants of around 0.15 and 0.9 ms. The two components were of approximately equal weight, and the time constants did not depend systematically on the voltages used to evoke the currents. In Ca 2+-free salines, the decay of the tail currents could be fitted with single-exponential curves with time constants of around 1.0 ms (Fig. 2B, middle). Digital subtraction of currents evoked in Ca2+-free salines from those obtained in normal salines yielded the portion of the tail currents that were dependent on extracellular Ca 2+ (Fig. 2B, right). These could be fitted with single-exponential curves with time constants of around 0.15 ms. We have fitted the Ca 2+-dependent tail currents in seven other cells. The mean time constant was 0.17 + 0.03 ms (mean _+ S.E.M.). These data are consistent with the notion that dissociated ciliary ganglion neurons express a single, kinetically homogeneous population of Ca 2+ channels but that isolation is often imperfect with CsCl-filled recording pipettes. In general, better isolation of Ca 2 + currents was achieved when recording pipettes filled with TEA-C1

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Fig. 2. Properties o f tail currents in acutely dissociated chick ciliary ganglion neurons. Data were obtained from the same cell as in Fig. 1. (A) Currents evoked by depolarizing steps to + 10mV from a holding potential o f - 8 0 mV. Data were obtained in normal saline (control) and in Ca2+-free saline (0 Ca). Currents strictly dependent on external Ca 2+ were obtained by digital subtraction ( c o n t r o l - 0 Ca). Arrows mark the portions o f the tail currents subjected to kinetic analysis. (B) Tail currents evoked by depolarizing commands to + 20 (top) and 0 mV (bottom). Membrane potential at time o f tail currents was - 8 0 mV. Recordings were automatically leak-subtracted according to a P/4 protocol. Each record shown is the average o f five individual sweeps. Averaged data (points) are shown along with superimposed fitted exponential curves (solid lines). Data were obtained in normal saline (left) and Ca 2+-free saline (middle). The Ca 2 +-dependent tail currents were obtained by digital subtraction and are shown to the right. Tail currents in normal saline could only be fitted as the sum o f two exponential curves. Tail currents in Ca 2+ -free saline, and the Ca 2+ -dependent tail currents, could be fitted with single-exponential curves. Time constants of fitted curves are shown in each panel. A.

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Fig. 4. Properties of Ca 2+ currents in acutely dissociated ciliary ganglion neurons. (A) Effects of bath application of 5 # M nifedipine on Ca 2+ currents in a ciliary ganglion cell. Currents were evoked by a depolarizing step to - 1 0 m V from a holding potential of - 8 0 mV. Records were not automatically leak-subtracted but are the average of five sweeps. Records on left are before (C) and after (N) application of nifedipine. Records on the right are the nifedipine-sensitive currents obtained by digital subtraction. (B) Effects of hyperpolarizing prepulses on Ca 2+ currents in a different cell. Ca 2+ currents were evoked by depolarizing steps to - 1 0 m V . These depolarizing steps were preceded by 50-ms prepulses to the potentials indicated to the left of each record. The holding potential was - 6 0 mV. Voltage-clamp protocol is indicated above the current records. These records were automatically leak-subtracted.

were used (Fig. 3). N o t e t h a t these recordings were not leak-subtracted. The difference was a p p a r e n t from a n e x a m i n a t i o n o f currents in control saline a n d Ca 2+ -free saline a n d calculation o f the difference between the two. In every case, it was still possible to d e m o n s t r a t e " r e v e r s a l " o f the currents with TEA-C1 pipettes, b u t the o u t w a r d currents observed in Ca 2 +free saline were m u c h smaller, a n d there was substantially less o u t w a r d relaxation in the Ca z+ currents. Similar results were o b t a i n e d in seven o t h e r cells. U n f o r t u n a t e l y , the TEA-CI pipettes seemed to produce a m u c h m o r e rapid deterioration of ciliary ganglion cells, so they were n o t utilized routinely. F u r t h e r evidence for identification o f these currents comes f r o m experiments using d i h y d r o p y r i d i n e C a 2 + -channel blockers. A p p l i c a t i o n o f 5 p M nifedipine p r o d u c e d a reversible reduction in Ca 2 ÷ current (Fig. 4A). A t this c o n c e n t r a t i o n , b l o c k a d e was never complete (38 _+ 9 % , mean_+ S.E.M., nine cells tested). In addition, virtually identical Ca 2+ currents were evoked by pulses to - 10 m V in the presence o f a series of 50-ms c o n d i t i o n i n g prepulses to voltages between - 4 0 a n d - 8 0 m V (Fig. 4B). Again, these results suggest t h a t ciliary ganglion n e u r o n s express only L-type Ca 2 + channels.

Effects of somatostatin and related peptides The effects of s o m a t o s t a t i n - 1 4 o n Ca 2+ currents evoked in ciliary ganglion n e u r o n s are s h o w n in Fig. 5. S o m a t o s t a t i n - 1 4 reversibly inhibited C a 2 + currents

evoked by pulses to 0 m V from a holding potential of - 6 0 m V (Fig. 5A). This was true w h e t h e r pipettes were filled with CsCI or TEA-CI. The effects of s o m a t o s t a t i n - 1 4 o n Ca 2+ currents evoked by a series of depolarizing c o m m a n d s are s h o w n in Fig. 5B. S o m a t o s t a t i n - 1 4 reversibly reduced inward currents w i t h o u t p r o d u c i n g any effect on o u t w a r d currents p r o d u c e d by pulses to m o r e positive potentials. This result suggests t h a t the effects of s o m a t o s t a t i n - 1 4 were due to inhibition o f Ca 2÷ currents a n d not to p o t e n t i a t i o n o f nonselective o u t w a r d currents. T o be certain o f this last point, we also e x a m i n e d the effect of s o m a t o s t a t i n - 1 4 o n families of Ca 2+ currents isolated by digital s u b t r a c t i o n o f Ca 2 + - i n d e p e n d e n t currents (Fig. 5C). W i t h these procedures it was possible to exclude possible actions of s o m a t o s t a t i n 14 o n c o n t a m i n a t i n g o u t w a r d currents t h a t might have been present prior to digital subtraction. The Ca 2+-dependent currents evoked by a series of depolarizing c o m m a n d s are s h o w n in the presence o f 100 n M s o m a t o s t a t i n - 1 4 (left) a n d after 1 min of washing in n o r m a l saline (middle). The family of records o n the fight represent the somatostatin-14sensitive currents o b t a i n e d by digital subtraction. The same data are s h o w n as I-V diagrams below the actual current traces. The d a t a indicate t h a t somatostatin-14 evoked a reversible inhibition of Ca 2+ currents at all of the voltages where Ca 2 + currents were detectable. Moreover, the somatostatin-14-sensitive currents showed the same I-V b e h a v i o r as n o r m a l

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Fig. 5. Effects of somatostatin-14 (St,) on Ca 2+ currents in acutely dissociated ciliary ganglion neurons. (A) Effects of somatostatin-14 on three different ciliary ganglion cells. Currents were evoked by a depolarizing step to 0 mV from a holding potential of - 6 0 mV. Recordings are shown before (C), during (St4), and after (R) exposure to 100 nM somatostatin-14. Records were automatically leak-subtracted according to a P/4 protocol. Each trace shown is the average of five sweeps. Somatostatin-14 caused a reversible reduction in the evoked Ca 2+ currents. (B) Effects of somatostatin-14 on families of Ca 2+ currents evoked by a series of depolarizing commands. Records were not automatically leak-subtracted. Records were obtained before (C), during (Sl4), and after (R) exposure to 100 nM somatostatin-14. Somatostatin-14 reduced peak inward currents but had no effect on maximal outward currents. (C) Effects of somatostatin-14 on Ca2+-dopendent currents obtained by digital subtraction. Currents evoked by a series of depolarizing commands were obtained in normal saline, normal saline containing 100 nM somatostatin-14, and Ca 2+ -free saline. Ca 2+ -dependent currents in the presence of somatostatin-14 (S~*) and after restoration of normal saline (R) were obtained by digital subtraction. Portion of total currents suppressed by somatostatin- 14 was also obtained by digital subtraction (R - St4). Note that somatostatin14 caused suppression of inward Ca z+ currents. (D) Current-voltage diagrams of data shown in C. Data plotted were obtained in the preseace of somatostatin-14 (circles) and after restoration of normal saline (triangles). Plot on the right (diamonds) is the somatostatin-14-sensitive current obtained by digital subtraction. The form of this curve is identical to that shown in Fig. 1 and indicates that somatostatin-14 causes a reversible reduction of L-type Ca 2+ currents.

Ca 2+ currents. The inhibitory effects of somatostatin14 were observed in virtually every celt tested (n = 60) regardless of the size of the recorded cell. This result suggests that somatostatin-14 is capable o f inhibiting Ca 2+ currents in both choroid and ciliary neurons. The effect o f 200 n M somatostatin-28 on Ca 2+ currents evoked by pulses to 0 mV from a holding potential of - 6 0 mV is shown in Fig. 6. Results are shown for four different cells. As with somatostatin14, somatostatin-28 caused a reversible inhibition of the Ca 2+ currents. The effects of somatostatin-28 seemed to persist slightly longer than somatostatin-14 after return to normal salines but were otherwise identical. Somatostatin-28 effects were similar in each of 14 cells tested (mean inhibition 23.9 + 8.2%).

As mentioned above, somatostatin-14 is a cleavage product of somatostatin-28. The other peptides resulting from this cleavage have not been shown to have biological activity and are not believed to bind to somatostatin receptors. 26 We have examined the effect of the peptide somatostatin-28 (1-14) on Ca 2+ currents in dissociated chick ciliary ganglion neurons (not shown). Somatostatin-28 (1-14) did not produce inhibition in any of eight cells tested. In two cells, somatostatin-28 (1-14) actually seemed to produce a slight increase in the Ca 2+ currents, but this effect was not reversible in either cell. Clearly, somatostatin-28 (1-14) does not activate somatostatin receptors in chick ciliary ganglion neurons. In other preparations, the effects of somatostatin

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peptides are t h o u g h t to be mediated t h r o u g h Gproteins that are sensitive to modification by PTX. 7 We have examined the effect o f P T X on inhibition o f Ca 2* currents p r o d u c e d by somatostatin-14 and somatostatin-28. Cells were exposed to 100 ng/ml P T X for 4 h at 37°C immediately following dissociation. P T X was dissolved in normal cell culture medium. C o n t r o l cells were from sister cultures examined on

the same day as the P T X - t r e a t e d cultures. Typical recordings are s h o w n in Fig. 7, and the results from all o f the experiments are summarized in Table 1. P r e t r e a t m e n t with P T X inhibited the responses to both 100 n M somatostatin-14 and 200 n M somatostatin-28. The cells that did r e s p o n d to somatostatin14 following P T X p r e t r e a t m e n t showed a much smaller response t h a n normal. M o r e typically, the

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Fig. 7. Effects of PTX pretreatment on responses to somatostatin-14 (St4) and somatostatin-28 (S2s). Cells were exposed to 100 ng/ml PTX for 4 h prior to recording. Each trace shows the leak-subtracted average of five sweeps. Currents were evoked by depolarizing pulses from a holding potential of - 60 mV. (A) Examples of responses to somatostatin-14 in four different cells after exposure to PTX. Currents are shown before (C) or after (R) exposure to somatostatin-14 and in the presence of somatostatin-14 (S~4). (B) Responses to somatostatin-28 in four cells after exposure to PTX. Recordings were made before (C) or after (R) exposure to somatostatin-28 and in the presence of somatostatin-28 ($28). PTX attenuated and in many cases eliminated the effect of somatostatin-14 and somatostatin-28 on Ca 2+ currents.

S. E. DRYERet

670

al.

Table I. Effects of pertussis toxin pretreatment on responses of ciliary ganglion neurons to somatostatin Control

Somatostatin-14 Somatostatin-28

PTX-treated

Mean per cent inhibition

Number of cells responding*

Mean per cent inhibition

Number of cells responding*

23.6 + 8.23 25.9 __+10.87

12/12 9/9

3.3 __+6.34? 1.4 _+4.33t

3/12 1/9

*Number of cells with detectable responses to somatostatin over total number of cells tested. ?Significantly different from control (P < 0.05). Values represent mean percentage inhibition of Ca2÷ current + S.D. response was completely abolished. PTX completely eliminated responses to somatostatin-28 in eight out of nine cells tested. This result indicates that somatostatin receptors are coupled to G-proteins in chick ciliary ganglion neurons. These data do not allow us to determine whether the G-proteins are directly coupled to the Ca 2÷ channels or whether some soluble second messenger is required. Attempts to examine somatostatin effects on single Ca 2÷ channels in cell-attached patches have proved inconclusive. Previous studies have shown that somatostatin peptides can effect K + conductances in vertebrate central neurons. ~5~':2,2sIn six cells, we have examined the effects of somatostatin-14 and somatostatin-28 on delayed rectifier K + currents (observed with normal, KCI recording pipettes). No effects were observed, and there was no effect on holding current at a holding potential of - 1 0 0 m V (not shown). This result suggests that somatostatin-14 or somatostatin28 has no effect on voltage-dependent K ÷ currents in chick ciliary ganglion neurons. DISCUSSION Two main conclusions emerge from this study. Firstly, acutely dissociated chick ciliary ganglion neurons express a single, kinetically homogeneous population of L-type Ca 2÷ channels. Secondly, these Ca 2÷ channels are inhibited reversibly by somatostatin-14 and somatostatin-28 because of activation of receptors that are coupled to PTX-sensitive Gproteins. The identification and characterization of the Ca 2÷ currents were based on their voltage dependence of activation, lack of rapid inactivation, tail-current kinetics, and sensitivity to nifedipine. The initial characterization was complicated by the fact that these currents were not completely isolated by'CsCl recording pipettes, even when the external saline contained 0.5 g M TTX, 5 mM TEA-CI, and 2 mM 4-aminopyridine. This problem was especially apparent at membrane potentials positive to around -10mV, where a contaminating outward current was observed in Ca: ÷-free salines. The most troublesome consequence is that these contaminating currents make a large contribution to the tail currents present at the break of the depolarizing voltage command. This contribution is clearly apparent upon examination of the amplitude and kinetics of tail

currents in normal and Ca 2+-free salines (Fig. 2). We have observed that the tail currents in Ca2+-free salines increase monotonically with depolarizing commands to at least + 60 mV. In other words, Ca 2÷ channels make a proportionally decreasing contribution to tail currents in normal salines as the membrane potential is made more positive, The use of tail currents to analyse the voltage dependence of drug effects on Ca 2+ currents is, therefore, potentially misleading, especially when CsC! pipettes are employed. We have found that bath application of metal cations, such as Co 2+ and especially La 3+, may produce some blockade of the contaminating outward current and probably should not be used in the isolation of Ca 2+ currents by digital subtraction. These results are similar to those reported previously in snail neurons.3'4 In those studies, outward currents observed following Ca 2÷-channel blockade were attributed to ionic flux through nonselective cationic channels that could be suppressed by both Cd 2+ and La 3÷. We believe a similar situation exists in ciliary ganglion neurons for two reasons. Firstly, there is never a voltage where the current remains at zero throughout the duration of the voltage step. This result suggests the presence of at least two currents of opposite polarity with different activation kinetics. Secondly, outward currents could be evoked in Ca 2÷ free salines with either CsC1 or TEA-C1 recording pipettes. Outward currents were always smaller with TEA-CI pipettes, suggesting that the outward currents were due to ionic flux through channels that are relatively nonselective but that are more permeable to Cs ÷ than to TEA-CI. The major conclusion of these analyses is that dissociated chick ciliary ganglion neurons express a homogeneous population of L-type Ca 2÷ channels. 5,~° These currents display extremely fast deactivation following repolarization of the cell membrane, as expected for L-type Ca ÷ channels?5a The inhibition of Ca 2÷ currents by somatostatin14 and somatostatin-28 was apparent at nanomolar concentrations and was fully reversible in less than 1 min. The inhibition could be detected at all voltages where Ca 2÷ currents could be measured. Pretreatmerit with PTX markedly attenuated responses to both somatostatin-14 and somatostatin-28, indicating involvement of G-proteins in these responses. This last result was not surprising in light of considerable biochemical and physiological data supporting a role for G-proteins in the responses to somatostatin-

Somatostatin inhibition of neuronal calcium currents 14 and somatostatin-28] ,7'14'26,29That somatostatin-28 (1-14) was inactive was also consistent with previous studies suggesting that this peptide does not bind to solubilized somatostatin receptors. 26 Previous studies have suggested that somatostatin can be released from choroid nerve terminals by K + - i n d u c e d depolarization. ~2 Moreover, somatostatin caused a PTX-sensitive inhibition of A C h release from parasympathetic nerve terminals in the choroid coat of the eye, but not from striated muscle in the iris or ciliary body. n This last observation is consistent with the fact that somatostatin immunoreactivity is restricted to the small choroid neurons of the chick ciliary ganglion, sa2 In contrast, we observed inhibition of Ca 2÷ currents in all cells tested, regardless of size. One possible explanation is that our preparations of acutely dissociated ciliary ganglion neurons did not contain ciliary cells. We consider this possibility to be unlikely on the basis of the essentially bimodal distribution of cell sizes in our preparations (unpublished observations). A second possibility is that ciliary neurons express receptors for somatostatin that are located on cell bodies but not nerve terminals. It is also possible that somatostatin peptides inhibit the release of A C h from the cell bodies but not the nerve terminals of the ciliary neurons. ~7 Previous studies suggest that K+-induced A C h release from chick choroid is dependent on N-type Ca 2÷ channels on the basis of resistance to blockade by nifedipine.H In contrast, indirect evidence suggests that release of somatostatin is dependent on dihydropyridine-sensitive L-type Ca 2÷ channels. ~2 In the present study we observed a single, homogeneous population of Ca 2 ÷ channels, in cell bodies, corresponding to L-type Ca 2÷ channels. However, these channels were not particularly sensitive to blockade by nifedipine. Recent studies suggest that several populations of cells express L-type Ca 2÷ channels that are resistant to blockade by either ~o-conotoxin or dihydropyridinesY It is difficult to see how N-type Ca 2+ channels can be involved in K ÷ -induced release of neurotransmitter, as they tend to become inacti-

671

vated. 1° Therefore, although we hhve not made whole-cell recordings from nerve terminals of the choroid neurons, we would nevertheless be very cautious about assigning a role to N-type Ca 2÷ channels in chick ciliary ganglion solely on the basis of pharmacological criteria. The actions of somatostatin peptides on neurons of the chick ciliary ganglion are strikingly similar to those reported previously in sympathetic ganglia, 14 pituitary cell lines, TM and spinal motoneurons. 2 In all of these preparations, somatostatin-14 was found to produce a G-protein-dependent inhibition of L-type Ca 2÷ channels. However, neither somatostatin-14 nor somatostatin-18 had any effect on zero-current level or on evoked Ca 2÷-independent outward currents in ciliary ganglion ceils. They differ in this way from hippocampal cells, 22 neoeortical cells, 28 locus coeruleus cells, 15 and submucosal plexus cells, 2~ where somatostatin peptides produce stimulatory ~5'2~'22'28 and occasionally inhibitory 28 effects on various K ÷ currents. These results suggest that somatostatin peptides can influence the m o t o r output of the chick ciliary ganglion. Along with previous studies, 8'H'~2'17 they suggest that a somatostatin peptide functions as co-transmitter with A C h to regulate release of A C h from parasympathetic neurons of the choroid coat. It is also possible that somatostatin released from choroid neuron somata produces paracrine effects throughout the ciliary ganglion.

CONCLUSION

Acutely dissociated chick ciliary ganglion neurons express a kinetically homogeneous population of Ltype Ca 2÷ channels. Application of somatostatin-14 or somatostatin-28 produces a reversible, PTXsensitive inhibition of Ca 2 ~ currents. Somatostatin28 (1-14) is inactive. Somatostatin peptides may modulate the m o t o r output of the chick ciliary ganglion.

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