Brain Research, 579 (1992) 165-168
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
165
BRES 25155
Spontaneous miniature hyperpolarizations of 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 21 January 1992) Key words: Presynaptic terminal; Ciliary ganglion; Spontaneous hyperpolarization; Intracellular Ca2+; Ca2+-activated K÷ channel
Intracellular recordings from presynaptic nerve terminals in the chick ciliary ganglion revealed the presence of spontaneous miniature hyperpolarizations in virtually all (-86%) nerve terminals examined. These spontaneous events appeared as small, brief hyperpolarizations at resting potential and were observed to increase or decrease as the membrane potential was depolarized or hyperpolarized from rest, respectively. The hyperpolafizing potentials were sensitive to blockade by tetraethylammonium and Ba2+, while caffeine increased then abolished these events. The voltage fluctuations were unaffected by tetrodotoxin, low Ca2+ external solution or the synaptic blockers, picrotoxin and strychnine. These spontaneous, transient, miniature hyperpolarizations may be due to the brief and co-ordinated activation of between 15-60 Ca2+-dependent K + channels following the release of Ca2÷ from internal stores. Spontaneous miniature hyperpolarizations are conspicuous fluctuations in membrane potential that have been observed during intracellular recording from several types of vertebrate neurons, including bullfrog sympathetic ganglion cells 13 and mouse dorsal root ganglion neurons 11'12. These random and brief fluctuations in membrane potential are thought to occur by the release of Ca 2+ from intracellular stores, which then activates a spontaneous miniature outward current carded by K ÷ ions 1'2's'9'11-13. During a previous in vitro study 7, in which intracellular recordings were obtained from presynaptic nerve terminals of the chick ciliary ganglion, we noted the presence of periodic noise in the records of membrane potential. The nature of these random fluctuations was investigated and we now report for the first time the presence of spontaneous miniature hyperpolarizations in the membranes of presynaptic calyces of this parasympathetic ganglion, The experimental protocol has previously been outlined in detail 7. Briefly, newly hatched chicks (SPAFAS, SPF) were decapitated and one ciliary ganglion was excised and cleared of overlying connective tissue. The ciliary ganglion was pinned in a shallow (volume - 1 ml) tissue bath cured with Sylgard resin and gravity superfused (3-5 ml/min) with a pre-heated (36-37°C) tyrode solution of the following composition (mM): NaCl 150; KC1 3; CaCl 2 5; MgCl 2 2; H E P E S 10; glucose 17; buffered to pH 7.4 and saturated with 100% O2. Under these
conditions the ganglion remained viable for periods of 8-12 h. Presynaptic (oculomotor) and postsynaptic (ciliary) nerves were each taken up into a suction electrode, and orthodromic and antidromic stimuli, respectively, were delivered to identify presynaptic calyx nerve terminals following their impalement by glass microelectrodes (see below). Intracellular recordings were obtained with glass micropipettes containing 3 M KC1 (resistance 40-80 MQ), connected to a high-impedance amplifier (Axoclamp-2A, Axon Instruments) through a balanced bridge circuit. Signals were observed on an oscilloscope screen and a two-channel chart recorder (Gould RS 3400). Data was also recorded and stored using a VCR recording system (Vetter 420) for subsequent analysis. Test solutions or drug applications were delivered over ganglionic preparations by means of three-way tap valves, which interrupted the normal flow of tyrode solution. The drugs used were all obtained from Sigma (St. Louis, MO) and included tetrodotoxin (TTX), tetraethylammonium (TEA), BaC12, CdC12, caffeine, strychnine and picrotoxin. Presynaptic calyces in the ciliary ganglion were identiffed by their known electrophysiological properties 3-5' 7,10. These included generation of an action potential without an accompanying nicotinic excitatory postsynaptic potential following orthodromic or antidromic excitation of the oculomotor or ciliary nerves, respectively. The action potential was blocked by a hyperpolarizing
Correspondence: V.A. Chiappinelli, Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine,
1402 South Grand Boulevard, St. Louis, MO 63104, USA. Fax: (1) (314) 577-8554.
166 A
- 3 5 " " l l l ~ l " ~ ' d ~ I t ~ ~ J l l ~~ ~
-
4
5
forward and backward directions 1°. A further criterion for classification was the presence of a single action potential (80-90 mV in height) in response to prolonged (65 ms) depolarizing current pulses of 0.6-1.0 nA. Intracellular recordings from 112 out of 131 (86%) of presynaptic calyces revealed the presence of small, discrete hyperpolarizing potentials of 2-6 mV at resting membrane potential (-62 to -68 mV), which appeared to resemble inhibitory synaptic potentials. These hyperpolarizing events were sensitive to membrane potential (Fig. 1). When the presynaptic calyx was depolarized from the resting potential both the frequency and the amplitude of the spontaneous hyperpolarizations increased, whereas they decreased when the cell was hyperpolarized from rest. No spontaneous hyperpolarizations were detected below -85 to -90 mV. Perfusion with 10 mM TEA completely eliminated the voltage fluctuations in presynaptic calyces (n = 7; Fig. 2). This effect was reversed upon washing with normal tyrode. Similar results have been demonstrated for postsynaptic cell bodies in other neuronal systems exhibiting spontaneous miniature hyperpolarizations 11-13. Since TEA is known to block K + conductances in a variety of excitable cells, and in particular, those ion channels requiring C a 2+ for their activation TM, the results raise the possibility that random voltage events in caly-
~
-ss ~"~ ~ r ~r V '" -"' l v~ """ r~- " 'r ' ~ r ""- r~' - - r~r "" " ~" " *~ " "~' ''¢" ~T~ ' -6e -Ts
~_-
-
8
s
~
lOmV B
500 ms
RMP-62
~
.... -'v'~ "
"10
mV
~
] ~
I 20 ms
Fig. I. The effect of membrane potential on spontaneous hyperpolarizations. The calyx was depolarized or hyperpolarized to the membrane potential indicated to the extreme left of the traces (A). Note the increase in frequency and amplitude of the voltage fluctuations at depolarized potentials, which disappear when the nerve terminal is driven to membrane potentials more negative than rest. In the cell shown in B, the spontaneous hyperpolarizing potentials are observed with an expanded time-base as discrete events with excursions up to 5 mV (resting membrane potential = -62 mV).
c i f o r m n e r v e terminals are also d u e t o a Ca2÷-dependent K + flux.
Caffeine is known to potentiate the frequency of Ca2+-dep endent K÷ fluxes responsible for spontaneous miniature hyperpolarizations in many excitable cells 2'8'9' 12, presumably by either crossing the cell membrane t o release Ca 2÷ from internal stores 8 or by activation of a caffeine-sensitive receptor in the cell membrane to cause Ca 2+ store release internally2'8. In our hands, the external perfusion of presynaptic calyces with 10 mM caffeine initially increased the frequency and amplitude of these random fluctuations in membrane voltage (n = 3; Fig.
pulse of 10-20 mV, leaving a small depolarization known as the coupling potential. The coupling potential is expected only in the presynaptic terminal, as the calyx-type synapse of the chick is electrically coupled in both the
RMP
- 6 6 m V
NT ~ i f - ~ -
10
TEA
N
- -
T
._- . . . . .
-~ - . . . .
~ ~
J
10 mV
5 0 0 ms Fig. 2. Effect of 10 mM TEA on spontaneous hyperpolarizations in presynaptic calyx nerve terminals. 10 min perfusion with TEA (10 TEA) blocked these spontaneous fluctuations in membrane voltage. Records were taken in normal tyrode (NT) before application of TEA (Top) and after washing out the drug (Bottom). Membrane potential was -66 mV in all three records.
167 A B C ~
|
10 mV .,J
100 ms Fig. 3. Effect of external perfusion of caffeine on presynaptic calyx nerve terminals. Spontaneous miniature hyperpolarizations were observed in normal tyrode solution (A). Bath application of caffeine (10 mM) initially caused (1-2 min) an increase in frequency and amplitude of these transient voltage events (B), which were subsequently abolished (C) during a prolonged (5 min) exposure to caffeine. Membrane potential was -66 mV.
3). This increase was subsequently followed by their abolition, until a recovery was obtained some minutes after removal of caffeine from the bathing medium. Under these conditions, the methylxanthine is considered to produce a massive release of Ca 2÷ from caffeine-sensitive internal stores, which could then act on Ca2+-acti vated K + channels z'8'9. The results further support the hypothesis that spontaneous miniature hyperpolarizations in chick calyces are due to a Ca2+-dependent K ÷ current. The spontaneous fluctuations were completely blocked by 5 mM Ba 2÷ (n = 5; results not shown), again indicating an important role for K ÷ ions. When presynaptic calyces were bathed in a low Ca 2+ (0.25 mM Ca 2÷, 3.75 mM Mg 2÷) environment (n = 3) or in 100/tM Cd 2÷ (n = 4), there was no effect on the frequency or amplitude of the voltage fluctuations, suggesting that influx of extracellular Ca z+ is not required for these spontaneous hyperpolarizations (results not shown). This agrees with findings in frog sympathetic neurons 13, but not with results in mouse dorsal root ganglion cells ~2. 1 ktM TTX was ineffective in abolishing spontaneous hyperpolarizations in presynaptic calyces (n = 7; results not shown), indicating that these voltage fluctuations are not due to changes in Na ÷ permeability along the nerve terminal. Blockers of fast inhibitory synaptic transmission, such as strychnine and picrotoxin, were similarly without effect at concentrations up to 50 #M (n = 3; resuits not shown).
miniature hyperpolarizations. The hyperpolarizing potentials are blocked by T E A and Ba 2+, potentiated by caffeine, and unaffected by q'TX, low-Ca2÷-containing saline and blockers of inhibitory synaptic events. Thus, nerve terminals in the chick ciliary ganglion have spontaneous hyperpolarizations that resemble those previously observed in neuronal somas in other systems 1'2's' 9,11-13, which are considered to be due to the activation of a CaE+-dependent K + current occurring as a result of the release of Ca 2+ from intracellular stores. In cultured bullfrog neurons, spontaneous hyperpolarizations are due to the co-ordinated, brief activation of 10-5,000 channels 13. The magnitude of spontaneous hyperpolarizations we have observed in presynaptic calyces indicates that they are also due to the nearly simultaneous openings of multiple channels. An estimate of the number of channels opened during a 5 mV event can be obtained using Ohm's Law and an average value for the input resistance of a calyx (60 Mr)). The current necessary to account for the event is then 83 pA. Assuming a driving force of 30 mV at rest, the conductance associated with the event would be 2.8 nS. In chick neurons, unitary chord conductances of Ca2+-activated K + channels have been reported to range from 4 5 - 1 9 0 pS 6, and thus such an event is likely to involve the opening of between 15-60 channels in the membrane of the presynaptic terminal. Interestingly, whereas spontaneous hyperpolarizations have been detected in several types of autonomic ganglia neurons 12'13, we have not observed such events in postsynaptic ciliary neurons, which instead displayed only nicotinic miniature excitatory postsynaptic potentials 5. Dryer et al, 6 have recently demonstrated that acutely dissociated ciliary ganglion neurons exhibit several different types of Ca2÷-activated K + channels, some of which are activated by depolarization of the membrane. However, these do not exhibit spontaneous co-ordinated, brief activations such as would be required to produce hyperpolarization events similar to those seen in the presynaptic calyces. Thus, the internal, discretely releaseable Ca 2+ stores that appear to be present in the presynaptic terminals in the avian ciliary ganglion may not be present in the postsynaptic ciliary neurons.
The results of this study demonstrate that presynaptic calyces of the chick ciliary ganglion display spontaneous
We thank Dr. Rodrigo Andrade and DI Stuart E. Dryer for many helpful comments and discussions an Melody Mance for secretarial assistance. This work was support d by National Institutes of Health Grant EY06564 to V.A.C.
1 Benham, C.D, and Bolton, T.B., Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of rabbit, J. Physiol., 381 (1986) 385-406. 2 Bolton, T.B. and Lim, S.P., Properties of calcium stores and transient outward currents in single smooth muscle cells of rab-
bit intestine, J. Physiol., 409 (1989)385-401. 3 Dryer, S.E. and Chiappinelli, V.A., Substance P depolarizes nerve terminals in an autonomic ganglion, Brain Res., 336 (1985) 190-194. 4 Dryer, S.E. and ChiappineUi, V,A., Properties of choroid and
168
5
6
7
8
9
ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter, J. Neurosci., 5 (1985) 26542661. 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. Dryer, S.E., Dourado, M.M. and Wisgirda, M.E., Characteristics of multiple Ca2+-activated K ÷ channels in acutely dissociated chick ciliary ganglion neurons, J. Physiol., 443 (1991) 601-627. Fletcher, G.H. and Chiappinelli, V.A., An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion, Brain Res., 575 (1992) 103-112. Komori, S. and Bolton, T.B., Actions of guanine nucleotides and cyclic nucleotides on calcium stores in single patch-clamped smooth muscle cells from rabbit portal vein, Br. J. Pharmacol., 97 (1989) 973-982. Komori, S. and Bolton, T.B., Role of G-proteins in muscarinic
10
11
12
13
14
receptor inward and outward currents in rabbit jejunal smooth muscle, J. Physiol., 427 (1990) 395-419. Martin, A.R. and Pilar, G., Dual mode of synaptic transmission in the avian ciliary ganglion, J. Physiol., 168 (1963) 443463. Mathews, D.A. and Barker, J.L., Spontaneous hyperpolarizations at the membrane of cultured mouse dorsal root ganglion cells, Brain Res., 211 (1981) 451-455. Mathews, D.A. and Barker, J.L., Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells, Brain Res., 293 (1984) 35-47. Satin, L.S. and Adams, P.R., Spontaneous miniature outward currents in cultured bullfrog neurons, Brain Res., 401 (1987) 331-339. Smart, T.G., Single calcium-activated potassium channels recorded from cultured rat sympathetic neurons, J. Physiol., 389 (1989) 337-360.
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
165
BRES 25155
Spontaneous miniature hyperpolarizations of 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 21 January 1992) Key words: Presynaptic terminal; Ciliary ganglion; Spontaneous hyperpolarization; Intracellular Ca2+; Ca2+-activated K÷ channel
Intracellular recordings from presynaptic nerve terminals in the chick ciliary ganglion revealed the presence of spontaneous miniature hyperpolarizations in virtually all (-86%) nerve terminals examined. These spontaneous events appeared as small, brief hyperpolarizations at resting potential and were observed to increase or decrease as the membrane potential was depolarized or hyperpolarized from rest, respectively. The hyperpolafizing potentials were sensitive to blockade by tetraethylammonium and Ba2+, while caffeine increased then abolished these events. The voltage fluctuations were unaffected by tetrodotoxin, low Ca2+ external solution or the synaptic blockers, picrotoxin and strychnine. These spontaneous, transient, miniature hyperpolarizations may be due to the brief and co-ordinated activation of between 15-60 Ca2+-dependent K + channels following the release of Ca2÷ from internal stores. Spontaneous miniature hyperpolarizations are conspicuous fluctuations in membrane potential that have been observed during intracellular recording from several types of vertebrate neurons, including bullfrog sympathetic ganglion cells 13 and mouse dorsal root ganglion neurons 11'12. These random and brief fluctuations in membrane potential are thought to occur by the release of Ca 2+ from intracellular stores, which then activates a spontaneous miniature outward current carded by K ÷ ions 1'2's'9'11-13. During a previous in vitro study 7, in which intracellular recordings were obtained from presynaptic nerve terminals of the chick ciliary ganglion, we noted the presence of periodic noise in the records of membrane potential. The nature of these random fluctuations was investigated and we now report for the first time the presence of spontaneous miniature hyperpolarizations in the membranes of presynaptic calyces of this parasympathetic ganglion, The experimental protocol has previously been outlined in detail 7. Briefly, newly hatched chicks (SPAFAS, SPF) were decapitated and one ciliary ganglion was excised and cleared of overlying connective tissue. The ciliary ganglion was pinned in a shallow (volume - 1 ml) tissue bath cured with Sylgard resin and gravity superfused (3-5 ml/min) with a pre-heated (36-37°C) tyrode solution of the following composition (mM): NaCl 150; KC1 3; CaCl 2 5; MgCl 2 2; H E P E S 10; glucose 17; buffered to pH 7.4 and saturated with 100% O2. Under these
conditions the ganglion remained viable for periods of 8-12 h. Presynaptic (oculomotor) and postsynaptic (ciliary) nerves were each taken up into a suction electrode, and orthodromic and antidromic stimuli, respectively, were delivered to identify presynaptic calyx nerve terminals following their impalement by glass microelectrodes (see below). Intracellular recordings were obtained with glass micropipettes containing 3 M KC1 (resistance 40-80 MQ), connected to a high-impedance amplifier (Axoclamp-2A, Axon Instruments) through a balanced bridge circuit. Signals were observed on an oscilloscope screen and a two-channel chart recorder (Gould RS 3400). Data was also recorded and stored using a VCR recording system (Vetter 420) for subsequent analysis. Test solutions or drug applications were delivered over ganglionic preparations by means of three-way tap valves, which interrupted the normal flow of tyrode solution. The drugs used were all obtained from Sigma (St. Louis, MO) and included tetrodotoxin (TTX), tetraethylammonium (TEA), BaC12, CdC12, caffeine, strychnine and picrotoxin. Presynaptic calyces in the ciliary ganglion were identiffed by their known electrophysiological properties 3-5' 7,10. These included generation of an action potential without an accompanying nicotinic excitatory postsynaptic potential following orthodromic or antidromic excitation of the oculomotor or ciliary nerves, respectively. The action potential was blocked by a hyperpolarizing
Correspondence: V.A. Chiappinelli, Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine,
1402 South Grand Boulevard, St. Louis, MO 63104, USA. Fax: (1) (314) 577-8554.
166 A
- 3 5 " " l l l ~ l " ~ ' d ~ I t ~ ~ J l l ~~ ~
-
4
5
forward and backward directions 1°. A further criterion for classification was the presence of a single action potential (80-90 mV in height) in response to prolonged (65 ms) depolarizing current pulses of 0.6-1.0 nA. Intracellular recordings from 112 out of 131 (86%) of presynaptic calyces revealed the presence of small, discrete hyperpolarizing potentials of 2-6 mV at resting membrane potential (-62 to -68 mV), which appeared to resemble inhibitory synaptic potentials. These hyperpolarizing events were sensitive to membrane potential (Fig. 1). When the presynaptic calyx was depolarized from the resting potential both the frequency and the amplitude of the spontaneous hyperpolarizations increased, whereas they decreased when the cell was hyperpolarized from rest. No spontaneous hyperpolarizations were detected below -85 to -90 mV. Perfusion with 10 mM TEA completely eliminated the voltage fluctuations in presynaptic calyces (n = 7; Fig. 2). This effect was reversed upon washing with normal tyrode. Similar results have been demonstrated for postsynaptic cell bodies in other neuronal systems exhibiting spontaneous miniature hyperpolarizations 11-13. Since TEA is known to block K + conductances in a variety of excitable cells, and in particular, those ion channels requiring C a 2+ for their activation TM, the results raise the possibility that random voltage events in caly-
~
-ss ~"~ ~ r ~r V '" -"' l v~ """ r~- " 'r ' ~ r ""- r~' - - r~r "" " ~" " *~ " "~' ''¢" ~T~ ' -6e -Ts
~_-
-
8
s
~
lOmV B
500 ms
RMP-62
~
.... -'v'~ "
"10
mV
~
] ~
I 20 ms
Fig. I. The effect of membrane potential on spontaneous hyperpolarizations. The calyx was depolarized or hyperpolarized to the membrane potential indicated to the extreme left of the traces (A). Note the increase in frequency and amplitude of the voltage fluctuations at depolarized potentials, which disappear when the nerve terminal is driven to membrane potentials more negative than rest. In the cell shown in B, the spontaneous hyperpolarizing potentials are observed with an expanded time-base as discrete events with excursions up to 5 mV (resting membrane potential = -62 mV).
c i f o r m n e r v e terminals are also d u e t o a Ca2÷-dependent K + flux.
Caffeine is known to potentiate the frequency of Ca2+-dep endent K÷ fluxes responsible for spontaneous miniature hyperpolarizations in many excitable cells 2'8'9' 12, presumably by either crossing the cell membrane t o release Ca 2÷ from internal stores 8 or by activation of a caffeine-sensitive receptor in the cell membrane to cause Ca 2+ store release internally2'8. In our hands, the external perfusion of presynaptic calyces with 10 mM caffeine initially increased the frequency and amplitude of these random fluctuations in membrane voltage (n = 3; Fig.
pulse of 10-20 mV, leaving a small depolarization known as the coupling potential. The coupling potential is expected only in the presynaptic terminal, as the calyx-type synapse of the chick is electrically coupled in both the
RMP
- 6 6 m V
NT ~ i f - ~ -
10
TEA
N
- -
T
._- . . . . .
-~ - . . . .
~ ~
J
10 mV
5 0 0 ms Fig. 2. Effect of 10 mM TEA on spontaneous hyperpolarizations in presynaptic calyx nerve terminals. 10 min perfusion with TEA (10 TEA) blocked these spontaneous fluctuations in membrane voltage. Records were taken in normal tyrode (NT) before application of TEA (Top) and after washing out the drug (Bottom). Membrane potential was -66 mV in all three records.
167 A B C ~
|
10 mV .,J
100 ms Fig. 3. Effect of external perfusion of caffeine on presynaptic calyx nerve terminals. Spontaneous miniature hyperpolarizations were observed in normal tyrode solution (A). Bath application of caffeine (10 mM) initially caused (1-2 min) an increase in frequency and amplitude of these transient voltage events (B), which were subsequently abolished (C) during a prolonged (5 min) exposure to caffeine. Membrane potential was -66 mV.
3). This increase was subsequently followed by their abolition, until a recovery was obtained some minutes after removal of caffeine from the bathing medium. Under these conditions, the methylxanthine is considered to produce a massive release of Ca 2÷ from caffeine-sensitive internal stores, which could then act on Ca2+-acti vated K + channels z'8'9. The results further support the hypothesis that spontaneous miniature hyperpolarizations in chick calyces are due to a Ca2+-dependent K ÷ current. The spontaneous fluctuations were completely blocked by 5 mM Ba 2÷ (n = 5; results not shown), again indicating an important role for K ÷ ions. When presynaptic calyces were bathed in a low Ca 2+ (0.25 mM Ca 2÷, 3.75 mM Mg 2÷) environment (n = 3) or in 100/tM Cd 2÷ (n = 4), there was no effect on the frequency or amplitude of the voltage fluctuations, suggesting that influx of extracellular Ca z+ is not required for these spontaneous hyperpolarizations (results not shown). This agrees with findings in frog sympathetic neurons 13, but not with results in mouse dorsal root ganglion cells ~2. 1 ktM TTX was ineffective in abolishing spontaneous hyperpolarizations in presynaptic calyces (n = 7; results not shown), indicating that these voltage fluctuations are not due to changes in Na ÷ permeability along the nerve terminal. Blockers of fast inhibitory synaptic transmission, such as strychnine and picrotoxin, were similarly without effect at concentrations up to 50 #M (n = 3; resuits not shown).
miniature hyperpolarizations. The hyperpolarizing potentials are blocked by T E A and Ba 2+, potentiated by caffeine, and unaffected by q'TX, low-Ca2÷-containing saline and blockers of inhibitory synaptic events. Thus, nerve terminals in the chick ciliary ganglion have spontaneous hyperpolarizations that resemble those previously observed in neuronal somas in other systems 1'2's' 9,11-13, which are considered to be due to the activation of a CaE+-dependent K + current occurring as a result of the release of Ca 2+ from intracellular stores. In cultured bullfrog neurons, spontaneous hyperpolarizations are due to the co-ordinated, brief activation of 10-5,000 channels 13. The magnitude of spontaneous hyperpolarizations we have observed in presynaptic calyces indicates that they are also due to the nearly simultaneous openings of multiple channels. An estimate of the number of channels opened during a 5 mV event can be obtained using Ohm's Law and an average value for the input resistance of a calyx (60 Mr)). The current necessary to account for the event is then 83 pA. Assuming a driving force of 30 mV at rest, the conductance associated with the event would be 2.8 nS. In chick neurons, unitary chord conductances of Ca2+-activated K + channels have been reported to range from 4 5 - 1 9 0 pS 6, and thus such an event is likely to involve the opening of between 15-60 channels in the membrane of the presynaptic terminal. Interestingly, whereas spontaneous hyperpolarizations have been detected in several types of autonomic ganglia neurons 12'13, we have not observed such events in postsynaptic ciliary neurons, which instead displayed only nicotinic miniature excitatory postsynaptic potentials 5. Dryer et al, 6 have recently demonstrated that acutely dissociated ciliary ganglion neurons exhibit several different types of Ca2÷-activated K + channels, some of which are activated by depolarization of the membrane. However, these do not exhibit spontaneous co-ordinated, brief activations such as would be required to produce hyperpolarization events similar to those seen in the presynaptic calyces. Thus, the internal, discretely releaseable Ca 2+ stores that appear to be present in the presynaptic terminals in the avian ciliary ganglion may not be present in the postsynaptic ciliary neurons.
The results of this study demonstrate that presynaptic calyces of the chick ciliary ganglion display spontaneous
We thank Dr. Rodrigo Andrade and DI Stuart E. Dryer for many helpful comments and discussions an Melody Mance for secretarial assistance. This work was support d by National Institutes of Health Grant EY06564 to V.A.C.
1 Benham, C.D, and Bolton, T.B., Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of rabbit, J. Physiol., 381 (1986) 385-406. 2 Bolton, T.B. and Lim, S.P., Properties of calcium stores and transient outward currents in single smooth muscle cells of rab-
bit intestine, J. Physiol., 409 (1989)385-401. 3 Dryer, S.E. and Chiappinelli, V.A., Substance P depolarizes nerve terminals in an autonomic ganglion, Brain Res., 336 (1985) 190-194. 4 Dryer, S.E. and ChiappineUi, V,A., Properties of choroid and
168
5
6
7
8
9
ciliary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter, J. Neurosci., 5 (1985) 26542661. 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. Dryer, S.E., Dourado, M.M. and Wisgirda, M.E., Characteristics of multiple Ca2+-activated K ÷ channels in acutely dissociated chick ciliary ganglion neurons, J. Physiol., 443 (1991) 601-627. Fletcher, G.H. and Chiappinelli, V.A., An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion, Brain Res., 575 (1992) 103-112. Komori, S. and Bolton, T.B., Actions of guanine nucleotides and cyclic nucleotides on calcium stores in single patch-clamped smooth muscle cells from rabbit portal vein, Br. J. Pharmacol., 97 (1989) 973-982. Komori, S. and Bolton, T.B., Role of G-proteins in muscarinic
10
11
12
13
14
receptor inward and outward currents in rabbit jejunal smooth muscle, J. Physiol., 427 (1990) 395-419. Martin, A.R. and Pilar, G., Dual mode of synaptic transmission in the avian ciliary ganglion, J. Physiol., 168 (1963) 443463. Mathews, D.A. and Barker, J.L., Spontaneous hyperpolarizations at the membrane of cultured mouse dorsal root ganglion cells, Brain Res., 211 (1981) 451-455. Mathews, D.A. and Barker, J.L., Spontaneous voltage and current fluctuations in tissue cultured mouse dorsal root ganglion cells, Brain Res., 293 (1984) 35-47. Satin, L.S. and Adams, P.R., Spontaneous miniature outward currents in cultured bullfrog neurons, Brain Res., 401 (1987) 331-339. Smart, T.G., Single calcium-activated potassium channels recorded from cultured rat sympathetic neurons, J. Physiol., 389 (1989) 337-360.
