Brain Research, 575 (1992) 103-112 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

103

BRES 17535

An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion George H. Fletcher and Vincent A. Chiappinelli Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, MO 63104 (USA) (Accepted 29 October 1991) Key words: Inward rectification; Presynaptic nerve terminal; Avian ciliary ganglion; Intracellular recording; Sodium-potassium channel; H-current

Inwardly rectifying voltage-sensitive channels have been detected in the cell bodies and axons of a number of excitable cells. The question of whether similar channels exist at axon terminals has been a matter of speculation for some time. We now report the first direct evidence for the existence of inward rectifiers in vertebrate presynaptic nerve terminals. Following impalement with intracellular electrodes, the large calyciform nerve terminals innervating chick ciliary ganglion neurons exhibit pronounced inward rectification upon hyperpolarization that increases with increasing current strength. The response is blocked by 2 mM Cs +, but is insensitive to Ba2+, tetraethylammonium and tetrodotoxin. The inward rectifier exhibits dependence on both Na + and K +, but is unaffected by altering extracellular Ca2+. Ciliary neurons innervated by these nerve terminals display inward rectification with similar properties. We conclude that the inward rectifier present in these presynaptic nerve terminals resembles the H-current previously described in sensory ganglion neurons and the Q-current found in hippocampal pyramidal neurons. The presence of channels that are activated by hyperpolarization may serve to enhance the excitability of the calyciform nerve terminals, which are capable of relatively high frequencies (> 100 Hz) of discharge. INTRODUCTION V e r t e b r a t e neurons possess a variety of voltage-dep e n d e n t conductances which are responsible for the typical non-linear b e h a v i o r of the neuronal m e m b r a n e 1. In particular, Katz 33 first described, in skeletal muscle, a specific increase in m e m b r a n e conductance to K + ions at m e m b r a n e potentials negative to the K + equilibrium potential (EK). The t e r m anomalous rectification was used to describe this m e m b r a n e p r o p e r t y , since it contradicted the constant field theory, as originally p r o p o s e d in the G o l d m a n - H o d g k i n - K a t z equation 23'3°. Since then, this a n o m a l o u s or inward rectification, which shows a K+-selective m e m b r a n e conductance, has b e e n found in a n u m b e r of biological m e m b r a n e s , including marine egg cells (starfish 27 and sea-squirt 41) and v e r t e b r a t e central neurons (olfactory cortex 11 and locus coeruleus32). In contrast, a n o t h e r type of inward rectification, which shows a cation-selective conductance for N a + and K + ions, has b e e n found in h i p p o c a m p a l neurons and t e r m e d Iq28, while a similar current found in spinal sensory ganglion neurons has b e e n termed /h 39. The reports published thus far have identified an inwardly rectifying response in the m e m b r a n e s of neuronal cell bodies and in the axonal m e m b r a n e s of nerve fibers 5'

19, whereas there is no available evidence to suggest whether such a conductance increase u p o n hyperpolarization is to be found at the nerve terminal. Nevertheless, the possibility that inward rectification is present at a nerve terminal region has aroused considerable interest 19'39. The failure to obtain such intracellular evidence is due in part to the technical limitations afforded by the size of most presynaptic terminal m e m b r a n e s . H o w e v e r , the chick ciliary ganglion is a p r e p a r a t i o n that provides an o p p o r t u n i t y to address this issue, since it possesses a unique anatomical feature which enables the electrophysiology of a presynaptic neuron to be explored in closer detail 37. In the ciliary ganglion of the chick, the cell bodies of ciliary neurons are innervated singly by large presynaptic nerve terminals in the form of a cup or calyx-like ending, unlike the m o r e usual b o u t o n type or diffusely branching terminal networks o b s e r v e d in other vertebrate synapses 13'29. A s the calyx terminal extends over a considerable area of the ciliary neuron, intracellular recordings can be o b t a i n e d from pre- and postsynaptic elements in intact ganglia 16A7'37, making it feasible to detect the existence of an inwardly rectifying response in the m e m b r a n e region on both sides of the synapse. Since the presynaptic calyx is a parallel sheet-like structure

Correspondence: V.A. Chiappinelli, Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, USA. Fax: (1) 314-577-8554.

104 that envelops the postsynaptic cell body of the ciliary n e u r o n 29'38, space-clamp errors will occur when voltage-

clearly demonstrate the presence of inward rectification at the nerve terminal region. To facilitate a comparison

made by substituting TRIS-base (titrated with HCI) for NaCI. LowCaZ+/high-Mg2+ solutions were made by reducing CaCI2 to 0.25 mM and by adding 3.75 mM MgCI2 to the tyrode solution. Cesium chloride (CsC1; 0.1-2 mM), barium chloride (BaCI2; 0.5-5 mM), tetrodotoxin (TTX; 1 /~M), tetraethylammonium (TEA; 10 mM), 4-aminopyridine (4-AP; 1 mM) and cadmium chloride (CdCl2; 100 /~M) containing solutions were made by adding appropriate amounts of salt or drug to the tyrode solution without osmotic compensation.

with the postsynaptic cells upon which the presynaptic calyces impinge, this stimulus paradigm was also used in

RESULTS

ciliary neurons. O u r results provide evidence for inwardly rectifying

Physiological identification of intracellular units

clamp procedures are applied to the calyciform nerve terminal 45. Current-clamp was therefore used to offset this problem, since the main aim of this work was to

cation-selective channels both at presynaptic calyx nerve terminals and postsynaptic ciliary neurons that are similar to the I h current in mouse sensory ganglion neurons 39 and the Iq current in hippocampal pyramidal cells 2s.

MATERIALS AND METHODS

Preparation of ciliary ganglia One-day-old hatched White Leghorn chicks were decapitated and one ciliary ganglion was dissected out. After clearing adhering connective tissue and removing the external capsule, ganglionic preparations were pinned out in a Sylgard-filledplexiglass chamber (volume 1 ml) and were continuously superfused (3-4 ml.min-1) with a tyrode solution that was preheated to 36-37°C by a Peltier device prior to entry into the chamber. The oculomotor (presynaptic) and ciliary (postsynaptic) nerves were each taken into a suction electrodes and orthodromic and antidromic stimuli were delivered to the ciliary ganglion by means of a Grass S-88 stimulator and SIU-5 isolation unit. The supeffusion of ganglionic preparations with tyrode solution could be switched to a test solution by manually controlled valves. The test solutions entered the recording chamber within 30 s of turning the tap, the delay being necessary for passage of the new solution through the heat exchanger.

Many characteristics distinguish calyx terminals from ciliary neurons and some of these, such as responses to orthodromic and antidromic nerve stimulation, have previously been described 16'17'37 (see Fig. 1). Fig. 1 shows additional properties that distinguish pre- and postsynaptic cells electrophysiologically. Depolarizing constantcurrent pulses (0.6-2.0 nA) applied to presynaptic calyces generally elicit a single action potential (amplitude = 84.9 +_ 1.2 mV, duration = 1.00 + 0.03 ms, n = 79), while ciliary neurons fire single and multiple spikes (amplitude = 89.0 + 3.1 mV, duration = 1.32 + 0.06 ms, n = 52) in response to current steps of lower intensity (0.2-0.8 nA). We have also found that whereas spontaneous miniature excitatory postsynaptic potentials are regularly observed in ciliary neurons TM (Fig. 1B), they are never seen in calyces, which instead almost always ( - 8 6 % ) display spontaneous miniature hyperpolarizing potentials (Fig. 1A) that appear to be due to a Ca 2÷d e p e n d e n t K+-conductance (unpublished observations).

General cell properties Electrophysiological recording Intracellular recordings were made using glass microelectrodes filled with 3 M KCI and with DC tip resistances ranging from 40 to 80 MQ. The step current command of a high impedance DC amplifier (Axoclamp-2A, Axon Instruments Inc.) in bridge circuit mode was used to deliver hyperpolarizing and depolarizing constant-current pulses of up to 200 ms duration through the recording microelectrode. The current steps and resulting membrane responses were monitored from the balanced bridge outputs of the amplifier by continuous display on an oscilloscope (Tektronix 5111), from which experimental records were photographed directly from the screen. These voltage and current signals were simultaneously recorded on a two channel Gould RS 3400 chart recorder and a Vetter 420 video cassette recorder for future analysis. Data stored on tape were viewed and analyzed on a Nicolet 3091 digital oscilloscope connected to an X - Y plotter (Hewlett Packard 7015). When exposure to the test solution resulted in a hyperpolarization or depolarization of cells, holding current (positive or negative DC current) was applied through the microelectrode to bring the membrane potential back to control values prior to studying currentvoltage relationships of the neurons in the bathing medium.

Solutions and drugs The normal recording medium was a modified tyrode solution containing (in mM): NaC1 150, KC1 3, CaCI2 5, MgC12 2, HEPES 10, glucose 17, titrated to pH 7.4 and equilibrated with 100% 02. When the extracellular K+ concentration was removed equimolar amounts of NaC1 were substituted for KC1. Na÷-free solutions were

The results of this study were obtained in 131 currentclamped cells from 81 ciliary ganglia. In normal tyrode solution pre- and postsynaptic cells were considered suitable for experimentation if the resting m e m b r a n e potential was between - 5 0 and - 7 0 mV (presynaptic calyces, mean + S.E.M. was -65.2 + 0.2 mV, n = 79; ciliary neurons, mean + S.E.M. was -56.3 + 0.9 mV, n = 52) and the direct spike amplitude was greater than 80 mV. These values are directly comparable to previous reports from other studies 16'17'37. In presynaptic calyx nerve terminals (Fig. 2A) and ciliary neurons (Fig. 2B), hyperpolarizing current steps of 200 ms duration elicited time-dependent relaxations or sags in the recorded voltage deflections, which were more p r o m i n e n t at greater negative current pulses. After the electrotonic potentials had reached a peak size (5-25 ms in pre- and postsynaptic cells), there was a subsequent decline to a plateau level which lasted until the end of the pulse (Fig. 2A,B). This suggests that at hyperpolarized levels beyond the resting m e m b r a n e potential a time-dependent inward current is activated.

105

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Fig. 1. Identification of intracellular units in the chick ciliary ganglion. A: those cells that were classified as presynaptic calyx nerve terminals responded to orthodromic and antidromic stimulation (40-50 V, 0.04 ms) with an action potential but no excitatory postsynaptic potential (EPSP). The orthodromic and antidromic spikes were blocked by a hyperpolarizing pulse (~ 100 pA) of 5-10 mV, leaving a small depolarization known as the coupling potential (insets, A). The presence of such a coupling potential is expected in the nerve terminal, as the calyx-type synapse of the chick is electrically coupled in both the forward and backward directions37. The presence of the subthreshold coupling potentials identified this unit as a calyciform nerve terminal and not as a pre- or postganglionic axonal fiber. Presynaptic calyces were also distinguished from postsynaptic ciliary neurons by the presence of a single action potential in response to a prolonged (65 ms, 1 Hz) depolarizing current pulse (t>0.6 nA) and by the appearance of spontaneous miniature hyperpolarizations in almost all recorded cells (-86%). B: neuronal elements that were classified as ciliary neurons elicited an action potential followed by a prominent nicotinic EPSP in response to pre- and postsynaptic nerve stimulation. The two records in B, obtained from the same ciliary cell, indicate that the EPSP following antidromic spikes was due to transmitter release from the presynaptic terminal, which is consistent with the view that the calyx-type synapse is coupled in the forward and backward directions. Further criteria for identification of ciliary neurons included a burst of action potentials in response to depolarizing current pulses of 0.2-0.8 nA and the conspicuous presence of spontaneous miniature EPSPs in all cells tested.

This p h e n o m e n o n , which is termed inward rectification, has been described in a n u m b e r of excitable cells 11'12'19. At the end of the hyperpolarizing current pulse, the repolarizing phase was accompanied by a r e b o u n d depolarization which overshot the resting potential (Fig. 2). C u r r e n t - v o l t a g e relationships derived from these records are displayed in Fig. 2. The values for m e m b r a n e potential were obtained either at the peak of the hyperpolarizing responses or at the steady-state, near the end of the pulse (times indicated 1 and 2 respectively in Fig. 2). These plots show a decrease in slope in the hyperpolarizing direction for the steady-state curves, indicating an apparent increase in m e m b r a n e conductance with

increasing current strength. In both presynaptic calyces and ciliary neurons, inward rectification was already present near the resting potential, and was still present but more p r o n o u n c e d during hyperpolarizing excursions of more than 50 mV, to levels of m e m b r a n e potential near -120 mV.

Cesium Since Cs + is k n o w n to block the K + conductance of the anomalous rectifier in skeletal muscle 22 and olfactory cortex neurons 11, as well as the mixed Na+-K + conductance of Iq in hippocampal pyramidal cells28 and lh in dorsal root ganglion neurons 39, we investigated the ef-

106

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Fig. 2. Inward rectification in presynaptic nerve terminals and ciliary neurons. Superimposed voltage traces in response to a series of hyperpolarizing current steps recorded from a presynaptic calyx (A) and a ciliary neuron (B) in standard tyrode solution. Note the sagging of the hyperpolarizing electrotonic potentials. The peak (O) and the steady-state (0) voltage responses (as indicated in points 1 and 2, respectively) for these cells were plotted against the amplitude of the current step. The decreasing slope of the steady-state curves with hyperpolarization indicates the presence of inward rectification in these synaptic elements of the ciliary ganglion.

feet of this ion on the inward rectifier of the presynaptic calyx terminal and the ciliary neuron. While lower concentrations (0.1-0.5 mM) of this cation proved to be ineffective (n = 12, data not shown), the application of 2 mM Cs ÷ to the bathing solution produced a rapid, virtual elimination of the prominent relaxations in the hyperpolarizing electrotonic potentials for presynaptic calyces (Fig. 3A, n = 8) and ciliary neurons (Fig. 3B, n = 7). The effect of Cs + exhibited some degree of voltagedependence, with more pronounced blockade occurring during larger hyperpolarizing pulses. This voltage-dependence has previously been reported for Cs ÷ blockade of other inward rectifiers n'22. As shown in the I - V relationships (Fig. 3A,B), Cs ÷ increased the slopes of both the peak and the steady-state curves indicating that in addition to largely abolishing inward rectification, Cs ÷ produced an overall increase in membrane resistance. Inward rectification was quickly restored when Cs ÷ was removed from the external solution by peffusion with

normal tyrode (results not shown). Barium Unlike Cs +, Ba z+ blocks the K+-selective current of the anomalous rectifier at micromolar concentrations TM 25, while having no effect on the Na+-K + current of lh 47 and lq z8 at concentrations in the millimolar range. We therefore tested the effects of Ba 2+ over varying concentrations in order to facilitate a comparison with Cs +. In both presynaptic calyces (Fig. 4A, n = 4) and ciliary neurons (Fig. 4B, n -- 7) the relaxations of the electrotonic potentials in response to a series of hyperpolarizing current pulses were not blocked when Ba 2+ at a concentration of 5 mM was present in the bathing medium. In Fig. 4A,B, the I - V relationship shows that in 5 mM Ba 2+ there was clear evidence of an increase in input resistance, as well as a greater amplitude of rebound depolarization, but no decrease in inward rectification (Fig. 4). Thus, the effects of Cs + and Ba 2+ on inward rectifi-

107

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Control

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Fig. 3. Effects of cesium on inward rectification. Electrotonic potentials produced by hyperpolarizing current pulses were recorded from a presynaptic calyx (A) and ciliary neuron (B) in control solution and in the presence of 2 mM Cs ÷ (30 min exposure). Cs ÷ abolished the sag in the electrotonic potentials and produced a marked increase in input resistance. The current-voltage relationships for these same cells in control solution (O, peak; O, steady-state) and 2 mM-Cs+ (El, peak; II, steady-state) reveal that Cs + largely abolished inward rectification over the range of hyperpolarizing current tested.

cation in these neuronal elements are similar to those reported f o r lh 39 and Iq2S. In order to generate hyperpolarizing voltage traces (Fig. 4A,B), it was first necessary to inject negative current (150-250 pA) so that the membrane potential was stabilized at control values, as the highest concentration of Ba 2+ (5 mM) invariably caused a 15-20 mV depolarization in all neurons that were tested. When hyperpolarizing current steps were applied to pre- and postsynaptic cells under the condition of no holding current, it was c o m m o n to observe the presence of anode-break excitation at the closure of the hyperpolarizing pulse (not illustrated). All of these effects were readily reversed by washing out barium with a normal tyrode solution.

Sodium and potassium Since the current that is responsible for I h and Iq is due to an increase in both Na ÷ and K + conductance 28' 34,39 we studied the effects of removing external Na + or K + on the inward rectification of pre- and postsynaptic elements of the ciliary ganglia. The contribution of Na +

to inward rectification in a presynaptic calyx (n = 3) is shown in Fig. 5A. In the presence of 150 mM [Na]out hyperpolarizing current pulses elicited inward relaxations of the electrotonic potentials, which became essentially flat in Na+-free medium. The current-voltage relationships in Fig. 5A (presynaptic calyx) and 5C1 (ciliary cell) indicate that the inward conductance activated at hyperpolarizing levels of membrane potential could no longer be sustained when [Na+]out was replaced by Tris-base. A Na+-free solution also caused a reduction in cell input resistance for the presynaptic calyx, with little or no change in this membrane parameter for the postsynaptic ciliary neuron, while the rebound depolarizations normally observed were absent (calyx, Fig. 5A; ciliary cell, not shown). Similar results were obtained when [Na+]out was replaced by choline instead of Tris-base (n = 2, data not shown). W h e n external K ÷ was eliminated by equimolar substitution with Na +, there were two noticeable effects on the electrotonic potentials of presynaptic calyces (Fig. 5B; n = 12). These included an increase in cell input resistance and a decline in the sag in the electrotonic po-

108

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Fig. 4. Effects of barium on inward rectification. Superimposed electrotonic potentials from a presynaptic calyx (A) and a ciliary neuron (B) produced by negative current pulses (200 ms, 0.5 Hz) in control solution and in the presence of Ba 2+ (5 mM; 30 min). In these pre- and postsynaptic cells the sagging of the hyperpolarizing electrotonic potentials remained in the presence of Ba z÷. The current-voltage relationships for these neurons before (O, peak; O, steady-state) and during (Tq, peak; II, steady-state) bath perfusion with 5 mM Baz+ indicate no abolition of inward rectification in a presynaptic calyx and a ciliary neuron.

tential in response to hyperpolarizing pulses. The current-voltage relationships in Fig. 5B (presynaptic calyx) and Fig. 5C2 (ciliary neuron) confirm the effect of a K+-free solution on inward rectification by showing that removal of K + reduces inward rectification over the range of hyperpolarizating current levels tested. Thus, inward rectification in presynaptic calyx terminals and ciliary neurons is d e p e n d e n t on the level of both extracellular Na ÷ and K ÷. Pharmacological agents blocking Na + and K + channels Various Na + and K ÷ channel blocking agents, such as T I ' X , T E A and 4 - A P are known to block inward rectifiers in different neuronal systems 6'31, In the presence of 10 m M T E A (Fig. 6 A , B , n = 9) the inward relaxations induced by hyperpolarizing current steps were unchanged in presynaptic calyces. H o w e v e r , the m e m b r a n e potential of all cells tested depolarized by 5-10 mV under T E A , which was c o m p e n s a t e d for by negative current injection prior to generation of the voltage records. Similar results (not shown) were obtained for ciliary neurons

in 10 m M T E A (n = 4). Also without effect on inward rectification were 1 /~M T T X (calyces, n = 7; ciliary cells, n = 3) and 1 m M 4 - A P (calyces, n = 3; ciliary cells, n = 6). Calcium C a 2+ is involved in the inward-going rectification of hippocampal neurons 31. To investigate the possible contribution of Ca 2+ to inward rectification of calyces and ciliary cells, ganglionic preparations were perfused with a low-Ca2+/high-Mg 2+ solution. The perfusion of presynaptic calyces (n = 4) with a low-CaZ+/high-Mg 2+ solution did not modify the inward rectifier in these cells, as shown by the I - V plot in Fig. 6C. Similar results (not shown) were obtained with ciliary neurons (n = 5) in low-CaZ+/high-Mg 2+ solution. The CaZ+-channel blocker Cd 2+ (0.1 mM, n = 8) also had no effect on the observed inward rectifiers. On this evidence it seems unlikely that C a 2+ contributes to inward rectification in either pre- or postganglionic cells of the ciliary ganglia.

109

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Fig. 5. Effect of removing external sodium or potassium on inward rectification. A: superimposed records show voltage traces elicited by steps of hyperpolarizing current and the resulting current-voltage relationships for a presynaptic calyx in control (O, peak; 0, steady-state) and in Na+-free solution ([~, peak; I , steady-state). When Na+-free or K+-free solutions altered resting membrane potential, positive or negative DC current was applied through the microelectrode to bring the potential back to the control value prior to obtaining the 1 - V data. B: responses and current-voltage relationships for a presynaptic calyx in control solution (O, peak; O, steady-state) and in K+-free solution (13, peak; I , steady-state). C: current-voltage relationships for a ciliary neuron before (O, peak; O, steady-state) and during (n, peak; II, steady-state) bath perfusion with Na÷-free (C1) and K+-free (C2) buffer.

DISCUSSION

Current-clamp studies on m a m m a l i a n and o t h e r vertebrate neurons have identified two forms of inward rectification that exhibit t i m e - d e p e n d e n t sags of electrotonic potentials in response to hyperpolarizing current steps. Sympathetic ganglion neurons show inward rectification which disappears with increasing hyperpolarizing cur-

rent 1°. This response is considered to be the result of deactivation of a voltage-sensitive K + conductance sensitive to muscarinic agonists (M-current) which carries an outward current at resting potential 2. However, in sensory ganglion neurons, inward rectification increases with increasing levels of hyperpolarizing current 39. It appears that activation of this inward rectifier is due to the opening of a voltage-sensitive channel carrying inward cur-

110

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Low-

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Current Injected (nA)

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Fig. 6. Effects of TEA and low-Ca2+/high-Mg2+ on inward rectification in a presynaptic calyx. A: electrotonic potentials produced by hyperpolarizing current pulses were recorded from a presynaptic calyx in control solution, and in the presence of TEA (10 mM; 30 min). B: current-voltage relationship in control (O, peak; O, steady-state) and 10 raM-TEA (D, peak; B, steady-state) for the calyx depicted in A. TEA did not decrease inward rectification. C: current-voltage relationship for a presynaptic calyx in control (©, peak; O, steady-state) and low-Ca2+/high-Mg2+ (3 min; n, peak; B, steady-state) solution, showing no increase or decrease of the inward rectifier.

rent. The results obtained from presynaptic calyces and ciliary neurons are in close agreement with the second form of inward rectifier, since inward rectification was more pronounced with increasing levels of hyperpolarizing current. However, many biological membranes that show inward rectification upon hyperpolarization do not share a common conductance mechanism. For instance, the inward rectifier of marine egg cells 25, skeletal muscle 44 and olfactory cortex neurons 11 is associated with a K+-selec tive conductance, which is blocked by Ba 2+ ( < 1 mM) and Cs + (1 mM). This conductance depends on the difference between the membrane and K + equilibrium potentials (V-EK), rather than on membrane potential alone 1]'26'35. By contrast, the conductance responsible for inward rectification in sensory ganglia 39, hippocampal pyramidal neurons 28 and cardiac sino-atrial node TM is carried by both Na + and K + ions. Furthermore the inward rectifiers of these membranes are sensitive to Cs +, but not to Ba 2+. The inward rectifiers of presynaptic calyces and ciliary neurons are blocked by Cs + (2 mM) but are resistant to Ba 2+ up to 5 mM. In addition to the pharmacological sensitivity, ion-replacement experiments are also

suggestive of a mixed Na + and K + conductance in the inward rectifiers of calyces and ciliary cells. Thus, the inward-going rectifier in the membranes on both sides of the ciliary neuron synapse resembles those of sensory ganglia 39 and hippocampal pyramidal cells 2s. The reports concerning the functional role of inward rectifiers are numerous and dependent on which cell system is tested. The inward rectifier is responsible for the depolarization associated with the pacemaking mechanism in cardiac sino-atrial node 9, the buffering of [K+]out by retinal glial cells 3'4's, the setting of resting potential close to E K in macrophages 2t and the counterbalancing of the hyperpolarizing action of light in retinal photoreceptors 2°. So what is the functional significance of inward rectification at the calyx-type synapse? Three possibilities exist. First, an inwardly rectifying response present in the nerve terminal region may counterbalance a prolonged hyperpolarization of the membrane brought about by Ca 2+ entry during an action potential which activates an outward CaZ+-dependent K + current 39. Calcium influx through voltage-gated channels of the N- and L-types appears to be responsible for the release of transmitters 36'42. In ciliary calyces of the chick two subtypes of

111 Ca 2+ channels have b e e n distinguished as corresponding to the N- and L-types and are considered to b e involved in excitation-secretion coupling 45'48. It therefore seems possible that activation of the inward rectifier of the chick calyx terminates the afterhyperpolarization following an action potential and t h e r e b y brings the m e m b r a n e potential back to resting values. A similar situation m a y also exist at the postsynaptic ciliary neuron, where Ca 2+d e p e n d e n t K + conductances have b e e n detected 15. Second, the inward rectifier of the calyx and the ciliary neuron m a y be involved in the d e t e r m i n a t i o n of resting m e m b r a n e potential. This m a y occur by the counter-balancing of two v o l t a g e - d e p e n d e n t conductances. In frog sympathetic ganglion neurons, speculation exists that the I h current exerts the depolarizing influence, while the other is a non-inactivating outward current exerting the hyperpolarizing influence, possibly Im46. In the ciliary ganglion of the chick, M-currents a p p e a r to be present in some calyces 16 suggesting that along with the inward rectifiers they may play a permissive role in the setting of m e m b r a n e potential. Finally, axonal excitability of rat optic nerve is increased by inward rectification elicited by hyperpolarization, since the depolarization associated with the conductance increase brings these axons closer to threshold 19. This potential role for the inward rectifier is especially i m p o r t a n t in calyces and ciliary neurons since they have among the highest discharge frequencies yet d e t e c t e d in the autonomic nervous system (67 Hz and 40 Hz, respectively, at 20-21°C according to Martin and PilaraS; often above 100 Hz at 36-37°C in the present study). Activation of the inward rectifier during the hyperpolarizing after-potential could counteract impulse b l o c k a d e brought about by hyperpolarization and might

thereby protect impulse transmission 19'39.

REFERENCES

diate K ÷ buffering, Nature, 324 (1986) 466-468. 9 Brown, H. and DiFrancesco, D., Voltage-clamp investigation of membrane currents underlying pacemaker activity in rabbit sino-atrial node, J. Physiol., 308 (1980) 331-351. 10 Constanti, A. and Brown, D.A., M-currents in voltage clamped mammalian sympathetic neurones, Neurosci. Lett., 24 (1981) 288-294. II Constanti, A. and Galvan, M., Fast inward-rectifying current accounts for anomalous rectification in olfactory cortex neurones, J. Physiol., 335 (1983) 153-178. 12 Crepel, R. and Penit-Soria, J., Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells, J. Physiol., 372 (1986) 1-23. 13 DeLorenzo, A.J., The fine structure of synapses in the ciliary ganglion of the chick, J. Biophys. Biochem. Cytol., 7 (1960) 31-36. 14 DiFrancesco, D. and Ojeda, C., Properties of the current if in the sino-atrial node of the rabbit compared with those of the current ira in purkinje fibres, J. Physiol., 308 (1980) 353-364. 15 Dryer, S.E., Na÷-activated K ÷ channels and voltage-evoked ionic currents in brain stem and parasympathetic neurones of the chick, J. Physiol., 435 (1991) 513-532. 16 Dryer, S.E. and Chiappinelli, V.A., Substance P depolarizes nerve terminals in an autonomic ganglion, Brain Res., 336

1 Adams, ER., Voltage-dependent conductances of vertebrate neurons, Trends Neurosci., 5 (1982) 116-119. 2 Adams, ER., Brown, D.A. and Constanti, A., M-currents and other potassium currents in bullfrog sympathetic neurones, J. Physiol., 330 (1982) 537-572. 3 Atwell, D. and Wilson, M., Behaviour of rod network in tiger salamander retina mediated by membrane properties of individual rods, J. Physiol., 309 (1980) 287-315. 4 Bader, C.R. and Bertrand, D., Effects of changes in intra- and extraceUular sodium on the inward (anomalous) rectification in salamander photoreceptors, J. Physiol., 347 (1984) 611-631. 5 Baker, M., Bostock, H., Grafe, P. and Martius, P., Function and distribution of three types of rectifying channel in rat spinal root myelinated axons, J. Physiol., 383 (1987) 45-67. 6 Bauer, C.K., Meyerhof, W. and Schwarz, J.R., An inwardlyrectifying K + current in clonal rat pituitary cells and its modulation by thyrotrophin-releasing hormone, J. Physiol., 429 (1990) 169-189. 7 Bobker, D.H. and Williams, J.T., Serotonin augments the cationic current I h in central neurons, Neuron, 2 (1989) 535-540. 8 Brew, H., Gray, P.T.A., Mobbs, P. and Atwell, D., Endfeet of retinal glial cells have higher densities of ion channels that me-

The inward rectification that we have observed in the chick ciliary ganglion m a y be under some form of modulatory control. I n d e e d , in thalamocortical neurons, both n o r e p i n e p h r i n e (NE) and 5-hydroxytryptamine (5-HT) enhance a hyperpolarization-activated current (Ih) carried by both N a + and K + ions, an effect which may be m e d i a t e d by an increase in intracellular c A M P 4°. Similar augmentation of I h by N E and 5-HT via activation of adenylate cyclase has been observed in the heart 24 and in brain stem neurons 7. Since N E and 5-HT are not known to be active in this parasympathetic ganglion, likely o r suitable candidates for m o d u l a t i o n of inward rectification include the n e u r o t r a n s m i t t e r A C h itself, and any of the known n e u r o p e p t i d e s (substance P, the enkephalins and vasoactive intestinal peptide) present in presynaptic nerve terminals 43. A n examination of the possible m o d u l a t o r y effects of these substances on inward rectifiers in the ciliary ganglion is currently underway in this laboratory. In conclusion, the present study has d e m o n s t r a t e d for the first time the presence of an inwardly rectifying N a + - K + current in chick presynaptic calyx nerve terminals and the existence of a similar current in the membrane of the ciliary neuron located across the synapse.

Acknowledgements. We thank Dr. Rodrigo Andrade for many helpful discussions and comments on the manuscript; Dr. Stuart E. Dryer for providing a copy of his paper prior to publication; and Melody Mance for secretarial assistance. This research was supported by a grant from the National Institutes of Health (Grant EY06564 to V.A.C.).

112 (1985) 190-194. 17 Dryer, S.E. and Chiappinelli, V.A., Properties of choroid and ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter, J. Neurosci., 5 (1985) 26542661. 18 Dryer, S.E. and Chiappinelli, V.A., Analysis of quantal content and quantal conductance in two populations of neurons in the avian ciliary ganglion, Neuroscience, 20 (1987) 905-910. 19 Eng, D.L., Gordon, T.R., Kocsis, J.D. and Waxman, S.G., Current-clamp analysis of a time-dependent rectification in rat optic nerve, J. Physiol., 421 (1990) 185-202. 20 Fain, G.L. and Lisman, J.E., Membrane conductances of photoreceptors, Prog. Biophys. Mol. Biol., 37 (1981) 91-147. 21 Gallin, E.K., Voltage clamp studies on macrophages from mouse spleen cultures, Science, 21 (1981) 458-460. 22 Gay, L. and Stanfield, P.R., Cs ÷ causes a voltage-dependent block of inward K ÷ currents in resting skeletal muscle fibers, Nature, 267 (1977) 169-170. 23 Goldman, D.E., Potential, impedance and rectification in membranes, J. Gen. Physiol., 27 (1943) 37-60. 24 Hagiwara, N. and Irisawa, H., Modulation by intracellular Ca 2÷ of the hyperpolarization-activated inward current in rabbit single sino-atrial node cells, J. Physiol., 409 (1989) 121-141. 25 Hagiwara, S., Miyazaki, S., Moody, W. and Patlak, J., Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg, J. Physiol., 279 (1978) 167-185. 26 Hagiwara, S., Miyazaki, S. and Rosenthal, N.P., Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish, J. Gen. Physiol., 67 (1976) 621-638. 27 Hagiwara, S. and Takahashi, K., The anomalous rectification and cation selectivity of the membrane of a starfish egg cell, J. Memb. Biol., 18 (1974) 61-80. 28 Halliwell, J.V. and Adams, P.R., Voltage clamp analysis of muscarinic excitation in hippocampal neurones, Brain Res., 250 (1982) 71-92. 29 Hess, A., Developmental changes in the structure of the synapse on the myelinated cell bodies of the chicken ciliary ganglion, J. Cell Biol., 25 (1965) 1-19. 30 Hodgkin, A,L. and Katz, B., The effect of sodium ions on the electrical activity of the giant axon of the squid, J. Physiol., 108 (1949) 37-77. 31 Hotson, J.R., Prince, D.A. and Schwartzkroin, P.A., Anomalous rectification in hippocampal neurons, J. NeurophysioL, 42 (1979) 889-895. 32 Inoue, M., Nakajima, S. and Nakajima, Y., Somatostatin induces an inward rectification in rat locus coeruleus neurones through a pertussis toxin-sensitive mechanism, J. Physiol., 407 (1988) 177-198.

33 Katz, B., Les constantes de la membrane de muscle, Arch. Sci. Physiol., 3 (1949) 285-300. 34 Lacey, M.G., Mercuri, N.B. and North, R.A., Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids, J. Neurosci., 9 (1989) 1233-1241. 35 Leech, C.A. and Stanfield, P.R., Inward rectification in frog skeletal muscle fibres and its dependence on membrane potential and external potassium, J. Physiol., 319 (1981) 295-309. 36 Lemos, J.R. and Nowycky, M.C., Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals, Neuron, 3 (1989) 1419-1426. 37 Martin, A.R. and Pilar, G., Dual mode of synaptic transmission in the avian ciliary ganglion, J. Physiol., 168 (1963) 443463. 38 Martin, A.R. and Pilar, G., Transmission through the ciliary ganglion of the chick, J. Physiol., 168 (1963) 464-475. 39 Mayer, M.L. and Westbrook, G.L., A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones, J. Physiol., 340 (1983) 19-45. 40 McCormick, D.A. and Pape, H.-C., Noradrenergic and serotonergic modulation of a hyperpolarization-activated cation current in thalamic relay neurones, J. Physiol., 431 (1990) 319-342. 41 Miyazaki, S., Takahashi, K., Tsuda, K. and Yoshii, M., Analysis of non-linearity observed in the current-voltage relation of the tuuicate embryo, J. Physiol., 238 (1974) 55-77. 42 Perney, T.M., Hirning, L.D., Leeman, S.E. and Miller, R.J., Multiple calcium channels mediate neurotransmitter release from peripheral neurons, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 6656-6659. 43 Reiner, A., Erichsen, J.T., Cabot, J.B., Evinger, C., Fitzgerald, M.E.C. and Karten, H.J., Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion, Vis. Neurosci., 6 (1991) 451-472. 44 Standen, N.B. and Stanfield, P.R., A potential- and time-dependent blockade of inward rectification in frog skeletal muscle fibres by barium and strontium ions, J. Physiol., 280 (1978) 169-191. 45 Stanley, E.E and Goping, G., Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal, J. Neurosci., 11 (1991) 985-993. 46 Tokimasa, T. and Akasu, T., Cyclic AMP regulates an inwardrectifying sodium-potassium current in dissociated bull-frog sympathetic neurones, J. Physiol., 420 (1990) 409-429. 47 Yanagihara, K. and Irisawa, H., Inward current activated during hyperpolarization in the rabbit sinoatrial node cell, Pfliigers Arch., 385 (1980) 11-19. 48 Yawo, M., Voltage-activated calcium currents in presynaptic nerve terminals of the chicken ciliary ganglion, J. Physiol., 428 (1990) 199-213.