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Somatostatin inhibits nicotinic cholinoceptor mediated-excitation in rat ambigual motoneurons in vitro
236
A'eurosciencc Lci,'er;, i 2~ ( 19~)1~2~0 239 t;~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940 91,$ 03.50 d DONIS 030439409100137R
NSL 07574
Somatostatin inhibits nicotinic cholinoceptor Mediated-excitation in rat ambigual motoneurons in vitro Yu Tian Wang, Richard S. N e u m a n and Detlef Bieger Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland (Canada) (Received 14 August 1990; Revised version received 23 October 1990; Accepted 21 November 1990)
Key words: Somatostatin; Acetylcholine; Nicotinic receptor; Nucleus ambiguus; Brainstem slice; Intracellular recording The interaction between somatostatin and acetylcholine, two putative transmitters in the nucleus ambiguus, was investigated on single ambigual neurons in a brainstem slice preparation. Somatostatin reversibly inhibited the nicotinic cholinoceptor-mediated depolarization and inward current induced by acetylcholine. This inhibition persisted in the presence of tetrodotoxin (TTX) or Mn 2+. In contrast, somatostatin enhanced both the glutamate-evoked depolarization and spiking discharges generated by current injection. These results suggest that somatostatin exerts a differential action in modulating excitatory inputs to the nucleus ambiguus at the level of postsynaptic receptors.
Somatostatin (SST) is regarded as a neurotransmitter or neuromodulator in the peripheral and central nervous system (CNS) [7], although the nature of its synaptic action is still unclear. Electrophysiological evidence regarding the effects of SST on CNS neurons has been equivocal, both inhibitory and excitatory effects having been observed in hippocampal slices [6, 14-16]. Since the inhibitory effects of SST were abolished by muscarinic agonists in hippocampal neurons [13, 20], excitatory effects of SST have been attributed by some investigators to disinhibition elicited by the presence of exogenous or endogenous acetylcholine (ACh) [12, 13]. SST also reportedly decreases ACh release [8, 9]. Recent immunohistochemical studies have demonstrated that a group of motoneurons forming the compact formation of the nucleus ambiguus (AMB¢) in the rat [3] receives SST and ACh inputs from the subnucleus centralis of the solitary complex [4] and the nucleus reticularis parvicetlularis intermedialis of the medulla oblongata [17], respectively. Electrophysiological and pharmacological studies have revealed ACh-evoked excitations in AMBc neurons, which are mediated by nicotinic cholinoceptors [2, 19]. In addition, preliminary work has demonstrated a fast depolarizing response to SST in AMBc neurons [18]. These findings prompted us to investigate the possible interaction between SST and ACh in brainstem transverse slice preparations. Furthermore, the interaction with glutamate was examined in light of the observation Correspondence: D. Bieger, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6.
that SST and glutamate have a synergistic interaction at cortical neurons [5, 10]. Male rats weighing 80-200 g were anaesthetized with urethane (1 g/kg, i.p.). The skull was rapidly opened and the brain removed. The brainstem was blocked and transversely sliced on a vibratome (350/tm) at 0-4°C in modified artificial cerebrospinal fluid (ACSF), Following 1 h recovery at room temperature in modified ACSF, slices were transferred into a submerged type recording chamber and perfused (2 ml/min) at a temperature of 33-34°C with normal ACSF. Normal ACSF consisted of (mM): NaCl 126, KCI 3, CaC12 2, MgC12 2, KH2PO4 1.2, NaHCO3 26, and glucose 10. In modified ACSF, the NaC1 was replaced by iso-osmotic sucrose (252 raM) [1]. For intracellular recordings, glass microelectrodes filled with 3 M KCI (60-100 MI2) were used. Neurons were voltage-clamped by means of a single electrode voltage clamp amplifier (Axoclamp II) at a switching frequency of 3-4 kHz as described elsewhere [19]. In brainstem slices the AMBc was readily visualized by its translucence and position with respect to the facial nucleus [see also ref. 19]. Drugs were pressure-ejected in aqueous solutions from a multibarreled pipette connected to a Picospritzer unit, in volumes of approximately 20-100 pl or were added by perfusion. Somatostatin was obtained from Sigma Chemical Co. (Lot No. 77F-00572). Data were obtained from 21 AMBe neurons which had resting membrane potentials of - 5 0 to - 8 6 mV ( - 67 _ 9 mV; mean + S.D.) and were capable of generating Na+-dependent spikes. As reported [19], pneumophoretic applications of ACh elicited a rapid depolarizing re-
237
sponse in all neurons tested which was reversibly inhibited by bath applied 10/tM D-tubocurarine, a nicotinic cholinoceptor antagonist, (n=4; Fig. 1A). Ejection of SST evoked simple, complex or no changes in resting membrane potential. Simple responses consisted of either a fast or a slow depolarization or a slow hyperpolarization. Complex responses consisted of combinations of these three components (e.g. Fig. 1B, middle), which varied from cell to cell but were relatively consistent in a given cell. Regardless of its effect on membrane poten-
,
tial SST applied prior to ACh reversibly inhibited the ACh-evoked depolarization (Fig. 1B). The effect was maximal within 15-30 s, the amplitude of membrane depolarizations induced by ACh being decreased to 43_+ 19% (n= 10) of control, with 0.3-0.5 pmol of SST. Although not studied systematically, the inhibition exhibited dose dependence in that increasing or decreasing SST pulse duration or number resulted in a corresponding change in the magnitude of the inhibition. Complete recovery occurred within 3-10 min. In addition, SST
O-TO
,-4-"--
A
5min 6 min
C
'
'
A
A
A
15s
A
2.5 min
E A
A
1.5 mJn
A
8 min
Fig. 1. Inhibition by somatostatin (SST) of nicotinic cholinoceptor-mediated excitation in ambigual motoneurons. A C are intracellular records from the same neuron with a resting membrane potential of - 6 1 mV and D and E are from two other neurons which have a resting membrane potential of - 67 and - 57 mV, respectively. Control responses are shown on the left. Time intervals refer to time of SST application. Small arrowheads mark A C h (5-8 pmol) applications, the bold arrows that of SST (0.3~).5 pmol). A: ACh-evoked depolarization is inhibited by 10/tM Dtubocurarine (D-TC) bath-applied for 6 rnin. The response progressively recovers at 8 and 12 min after wash-out. B: A C h depolarization is inhibited following a pulse of SST and recovers after 5 min. Note triphasic complex response to SST. C: current record corresponding to sequence shown in B demonstrates attenuation of ACh-evoked inward current by SST at a holding potential of - 6 1 inV. The antagonism of SST towards A C h was evident in M n 2+ 5 m M (D) and TTX l /zM (E) applied by perfusion. Calibration: horizontal bar 15 s, vertical bar 15 mV in A,B,D,E and 0.6 nA in C.
238
A
25s
3min
...•110my
_o,
I
B
4min
25s
130mV
-72 mV
J
L_
[__
_
[.. I 0"6nA 50ms
Fig. 2. Increased responsiveness to glutamate and depolarizing current injection induced by SST. Control responses with resting membrane potentials are shown on left; middle and right records were taken at times indicated after SST application. A: glutamate pressure-ejections (6 pmol; small arrowheads) evoke depolarization which is enhanced following SST pulse (0.5 pmol; bold arrow). B: a depolarizing current pulse (lower trace) elicits 2 spikes during control but 3 spikes following SST pulse. Note decreased latency to first spike following SST. In the same cell a slight enhancement of the glutamate response was observed (not illustrated).
slowed the rising phase of the ACh-evoked depolarization. Under voltage clamp, ACh evoked an inward current which was inhibited by a prepulse of SST with a time course similar to that revealed under current clamp (Fig. 1C). Thus, the SST antagonism towards ACh could not be attributed to a change of membrane potential induced by SST. Furthermore, the inhibitory effect of SST on the ACh-evoked response persisted in the presence of Mn 2+ (5 raM; n=2; Fig. ID) or TTX (1 #M; n = 2; Fig. 1E), suggesting a direct mechanism at the level of the postsynaptic membrane. In contrast to the response induced by ACh, the glutamate-evoked depolarization was not inhibited but was, to some extent, enhanced (40 + 21%) by prepulses of SST (0.3--0.5 pmol) in 7 out of 10 neurons (Fig. 2A), in analogy to the observed effect of SST on cultured cortical neurons [5]. In the remaining 3 cells, SST did not measurably alter the size and shape of the depolarization induced by glutamate. SST also increased the membrane responsiveness of neurons to current injection (0.6 nA, 150 ms in duration; n = 3). Thus the number of spikes was increased and the latency to the first spike was reduced (Fig. 2B). Therefore, SST did not inhibit ACh, evoked excitation by reducing the overall excitability of these neurons. In conclusion, our findings suggest that SST is capable of modulating the membrane response to nicotinic cholinoceptor activation of AMBc neurons. The underlying
mechanism could involve an allosteric interaction with the nicotinic cholinoceptor itself or physiological antagonism mediated by a separate receptor. In these motoneurons, the interaction between SST and ACh entails inhibition of an inward current, whereas in other CNS neurons under muscarinic cholinoceptor control the action of SST would be to increase outward M,current [11, 13, 20]. The common result in both situations would therefore be decreased responsiveness to cholinergic input. Thus, the question arises as to whether this peptide serves as an endogenous inhibitory modulator of neuronal nicotinic cholinoceptor-opcrated cation channels, in analogy to its proposed function as endogenous 'agonist' at the M-current generating K +-channel [I 3, 20]. We thank Mrs Janet Robinson for excellent technical assistance. Supported by MRC (Canada). Y.T,W. is a recipient of a Memorial University Fellowship. 1 Aghajanian, G.K. and Rasmussen, K., Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices, Synapse, 3 (1989) 33 t-338. 2 Bieger, D., Musearinic activation of rhombencophalic neurones controlling oesophagcal peristalsis in the rat, Neurophammcology, 23 (1984) 1451-1464. 3 Biegcr, D. and Hopkins, D.A., Visecrotopic representation of the upper alimentary tract in the medulla oblonpta in the. rat: the nucleus ambiguus, J. Comp. Neurol., 262 (1987) 546-562. 4 Cunningham, E:T., Jr. and Sawchenko, P.E., A circumscribed projection from the nucleus of the solitary tract t O the nucleus ambiguns in the rat: anatomical evidence for somatostatin-28-immuno-
239
5
6 7
8
9
10
11
12
reactive interneurons subserving reflex control of esophageal motility, J. Neurosci., 9 (1989) 1668-1682. Dichter, M.A. and Delfs, J.R., Somatostatin and cortical neurons in cell culture, Adv. Biochem. Psychopharmacol., 28 (1981) 145 157. Dodd, J. and Kelly, J.S., Is somatostatin an excitatory transmitter in the hippocampus?, Nature, 273 (1978) 674~575. Epelbaum, J., Somatostatin in the central nervous system: physiology and pathological modifications, Prog. Neurobiol., 27 (1986) 63-100. Gray, D.B., Pilar, G.R. and Ford, M.J., Opiate and peptide inhibition of transmitter release in parasympathetic terminals, J. Neurosci., 9 (1989) 1683-1692. Gray, D.B., Zelazny, D., Manthay, N. and Pilar, G., Endogenous modulation of ACh release by somatostatin and the differential roles of Ca z÷ channels, J. Neurosci., 10 (1990) 2687-2696. Ioffe, S., Havlicek, V., Friesen, H. and Chernick, V., Effect of somatostatin (SRIF) and L-glutamate on neurons of the sensorimotor cortex in awake habituated rabbits, Brain Res., 153 (1978) 414~,18. Jacquin, J., Champagnat, J., Madamba, S., Denavit-Saubi6, M. and Siggins, G.R., Somatostatin depresses excitability in neurons of the solitary tract complex through hyperpolarization and augmentation of Im, a non-inactivating voltage-dependent outward current blocked by muscarinic agonists, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 948 952. Mancillas, J.R., Siggins, G.R. and Bloom, F.E., Somatostatin
selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 75187521. 13 Moore, S.D., Madamba, S.G., Joels, M. and Siggins, G.R., Somatostatin augments the M-current in hippocampal neurons, Science, 239 (1988) 278-280. 14 Mueller, A.L., Kunkel, D.D. and Schwartzkroin, P.A,, Electrophysiological actions of somatostatin (SRIF) in hippocampus: an in vitro study, Cell. Mol. Neurobiol., 6 (1986) 363-379. 15 Pittman, Q.J. and Siggins, G.R., Somatostatin hyperpolarizes hippocampal pyramidal cells in vitro, Brain Res., 221 (1981) 402-408. 16 Scharfman, H.E. and Schwartzkroin, P.A., Further studies of the effects of somatostatin and related peptides in area CAI of rabbit hippocampus, Cell. Mot. Neurobiol., 8 (1988)411 429. 17 Vyas, D., Wang, Y.T. and Bieger, D., Nucleus reticularis intermedialis (NIR) - - possible cholinergic source subserving oesophageal peristalsis in the rat, Soc. Neurosci. Abstr., 16 (1990) 303.11. 18 Wang, Y.T., Neuman, R.S. and Bieger, D., Excitatory action of somatostatin (SST) on rat ambigual motoneurons in vitro, Soc. Neurosci. Abstr., 16 (1990) 422.6. 19 Wang, Y.T., Neuman, R.S. and Bieger, D., Nicotinic cholinoceptor-mediated excitation in ambigual motoneurones of the rat, Neuroscience, in press. 20 Watson, T,W. and Pittman, Q.J., Pharmacological evidence that somatostatin activates the m-current in hippocampal pyramidal neurons, Neurosci. Lett., 91 (1988) 172 176.
A'eurosciencc Lci,'er;, i 2~ ( 19~)1~2~0 239 t;~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940 91,$ 03.50 d DONIS 030439409100137R
NSL 07574
Somatostatin inhibits nicotinic cholinoceptor Mediated-excitation in rat ambigual motoneurons in vitro Yu Tian Wang, Richard S. N e u m a n and Detlef Bieger Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland (Canada) (Received 14 August 1990; Revised version received 23 October 1990; Accepted 21 November 1990)
Key words: Somatostatin; Acetylcholine; Nicotinic receptor; Nucleus ambiguus; Brainstem slice; Intracellular recording The interaction between somatostatin and acetylcholine, two putative transmitters in the nucleus ambiguus, was investigated on single ambigual neurons in a brainstem slice preparation. Somatostatin reversibly inhibited the nicotinic cholinoceptor-mediated depolarization and inward current induced by acetylcholine. This inhibition persisted in the presence of tetrodotoxin (TTX) or Mn 2+. In contrast, somatostatin enhanced both the glutamate-evoked depolarization and spiking discharges generated by current injection. These results suggest that somatostatin exerts a differential action in modulating excitatory inputs to the nucleus ambiguus at the level of postsynaptic receptors.
Somatostatin (SST) is regarded as a neurotransmitter or neuromodulator in the peripheral and central nervous system (CNS) [7], although the nature of its synaptic action is still unclear. Electrophysiological evidence regarding the effects of SST on CNS neurons has been equivocal, both inhibitory and excitatory effects having been observed in hippocampal slices [6, 14-16]. Since the inhibitory effects of SST were abolished by muscarinic agonists in hippocampal neurons [13, 20], excitatory effects of SST have been attributed by some investigators to disinhibition elicited by the presence of exogenous or endogenous acetylcholine (ACh) [12, 13]. SST also reportedly decreases ACh release [8, 9]. Recent immunohistochemical studies have demonstrated that a group of motoneurons forming the compact formation of the nucleus ambiguus (AMB¢) in the rat [3] receives SST and ACh inputs from the subnucleus centralis of the solitary complex [4] and the nucleus reticularis parvicetlularis intermedialis of the medulla oblongata [17], respectively. Electrophysiological and pharmacological studies have revealed ACh-evoked excitations in AMBc neurons, which are mediated by nicotinic cholinoceptors [2, 19]. In addition, preliminary work has demonstrated a fast depolarizing response to SST in AMBc neurons [18]. These findings prompted us to investigate the possible interaction between SST and ACh in brainstem transverse slice preparations. Furthermore, the interaction with glutamate was examined in light of the observation Correspondence: D. Bieger, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6.
that SST and glutamate have a synergistic interaction at cortical neurons [5, 10]. Male rats weighing 80-200 g were anaesthetized with urethane (1 g/kg, i.p.). The skull was rapidly opened and the brain removed. The brainstem was blocked and transversely sliced on a vibratome (350/tm) at 0-4°C in modified artificial cerebrospinal fluid (ACSF), Following 1 h recovery at room temperature in modified ACSF, slices were transferred into a submerged type recording chamber and perfused (2 ml/min) at a temperature of 33-34°C with normal ACSF. Normal ACSF consisted of (mM): NaCl 126, KCI 3, CaC12 2, MgC12 2, KH2PO4 1.2, NaHCO3 26, and glucose 10. In modified ACSF, the NaC1 was replaced by iso-osmotic sucrose (252 raM) [1]. For intracellular recordings, glass microelectrodes filled with 3 M KCI (60-100 MI2) were used. Neurons were voltage-clamped by means of a single electrode voltage clamp amplifier (Axoclamp II) at a switching frequency of 3-4 kHz as described elsewhere [19]. In brainstem slices the AMBc was readily visualized by its translucence and position with respect to the facial nucleus [see also ref. 19]. Drugs were pressure-ejected in aqueous solutions from a multibarreled pipette connected to a Picospritzer unit, in volumes of approximately 20-100 pl or were added by perfusion. Somatostatin was obtained from Sigma Chemical Co. (Lot No. 77F-00572). Data were obtained from 21 AMBe neurons which had resting membrane potentials of - 5 0 to - 8 6 mV ( - 67 _ 9 mV; mean + S.D.) and were capable of generating Na+-dependent spikes. As reported [19], pneumophoretic applications of ACh elicited a rapid depolarizing re-
237
sponse in all neurons tested which was reversibly inhibited by bath applied 10/tM D-tubocurarine, a nicotinic cholinoceptor antagonist, (n=4; Fig. 1A). Ejection of SST evoked simple, complex or no changes in resting membrane potential. Simple responses consisted of either a fast or a slow depolarization or a slow hyperpolarization. Complex responses consisted of combinations of these three components (e.g. Fig. 1B, middle), which varied from cell to cell but were relatively consistent in a given cell. Regardless of its effect on membrane poten-
,
tial SST applied prior to ACh reversibly inhibited the ACh-evoked depolarization (Fig. 1B). The effect was maximal within 15-30 s, the amplitude of membrane depolarizations induced by ACh being decreased to 43_+ 19% (n= 10) of control, with 0.3-0.5 pmol of SST. Although not studied systematically, the inhibition exhibited dose dependence in that increasing or decreasing SST pulse duration or number resulted in a corresponding change in the magnitude of the inhibition. Complete recovery occurred within 3-10 min. In addition, SST
O-TO
,-4-"--
A
5min 6 min
C
'
'
A
A
A
15s
A
2.5 min
E A
A
1.5 mJn
A
8 min
Fig. 1. Inhibition by somatostatin (SST) of nicotinic cholinoceptor-mediated excitation in ambigual motoneurons. A C are intracellular records from the same neuron with a resting membrane potential of - 6 1 mV and D and E are from two other neurons which have a resting membrane potential of - 67 and - 57 mV, respectively. Control responses are shown on the left. Time intervals refer to time of SST application. Small arrowheads mark A C h (5-8 pmol) applications, the bold arrows that of SST (0.3~).5 pmol). A: ACh-evoked depolarization is inhibited by 10/tM Dtubocurarine (D-TC) bath-applied for 6 rnin. The response progressively recovers at 8 and 12 min after wash-out. B: A C h depolarization is inhibited following a pulse of SST and recovers after 5 min. Note triphasic complex response to SST. C: current record corresponding to sequence shown in B demonstrates attenuation of ACh-evoked inward current by SST at a holding potential of - 6 1 inV. The antagonism of SST towards A C h was evident in M n 2+ 5 m M (D) and TTX l /zM (E) applied by perfusion. Calibration: horizontal bar 15 s, vertical bar 15 mV in A,B,D,E and 0.6 nA in C.
238
A
25s
3min
...•110my
_o,
I
B
4min
25s
130mV
-72 mV
J
L_
[__
_
[.. I 0"6nA 50ms
Fig. 2. Increased responsiveness to glutamate and depolarizing current injection induced by SST. Control responses with resting membrane potentials are shown on left; middle and right records were taken at times indicated after SST application. A: glutamate pressure-ejections (6 pmol; small arrowheads) evoke depolarization which is enhanced following SST pulse (0.5 pmol; bold arrow). B: a depolarizing current pulse (lower trace) elicits 2 spikes during control but 3 spikes following SST pulse. Note decreased latency to first spike following SST. In the same cell a slight enhancement of the glutamate response was observed (not illustrated).
slowed the rising phase of the ACh-evoked depolarization. Under voltage clamp, ACh evoked an inward current which was inhibited by a prepulse of SST with a time course similar to that revealed under current clamp (Fig. 1C). Thus, the SST antagonism towards ACh could not be attributed to a change of membrane potential induced by SST. Furthermore, the inhibitory effect of SST on the ACh-evoked response persisted in the presence of Mn 2+ (5 raM; n=2; Fig. ID) or TTX (1 #M; n = 2; Fig. 1E), suggesting a direct mechanism at the level of the postsynaptic membrane. In contrast to the response induced by ACh, the glutamate-evoked depolarization was not inhibited but was, to some extent, enhanced (40 + 21%) by prepulses of SST (0.3--0.5 pmol) in 7 out of 10 neurons (Fig. 2A), in analogy to the observed effect of SST on cultured cortical neurons [5]. In the remaining 3 cells, SST did not measurably alter the size and shape of the depolarization induced by glutamate. SST also increased the membrane responsiveness of neurons to current injection (0.6 nA, 150 ms in duration; n = 3). Thus the number of spikes was increased and the latency to the first spike was reduced (Fig. 2B). Therefore, SST did not inhibit ACh, evoked excitation by reducing the overall excitability of these neurons. In conclusion, our findings suggest that SST is capable of modulating the membrane response to nicotinic cholinoceptor activation of AMBc neurons. The underlying
mechanism could involve an allosteric interaction with the nicotinic cholinoceptor itself or physiological antagonism mediated by a separate receptor. In these motoneurons, the interaction between SST and ACh entails inhibition of an inward current, whereas in other CNS neurons under muscarinic cholinoceptor control the action of SST would be to increase outward M,current [11, 13, 20]. The common result in both situations would therefore be decreased responsiveness to cholinergic input. Thus, the question arises as to whether this peptide serves as an endogenous inhibitory modulator of neuronal nicotinic cholinoceptor-opcrated cation channels, in analogy to its proposed function as endogenous 'agonist' at the M-current generating K +-channel [I 3, 20]. We thank Mrs Janet Robinson for excellent technical assistance. Supported by MRC (Canada). Y.T,W. is a recipient of a Memorial University Fellowship. 1 Aghajanian, G.K. and Rasmussen, K., Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices, Synapse, 3 (1989) 33 t-338. 2 Bieger, D., Musearinic activation of rhombencophalic neurones controlling oesophagcal peristalsis in the rat, Neurophammcology, 23 (1984) 1451-1464. 3 Biegcr, D. and Hopkins, D.A., Visecrotopic representation of the upper alimentary tract in the medulla oblonpta in the. rat: the nucleus ambiguus, J. Comp. Neurol., 262 (1987) 546-562. 4 Cunningham, E:T., Jr. and Sawchenko, P.E., A circumscribed projection from the nucleus of the solitary tract t O the nucleus ambiguns in the rat: anatomical evidence for somatostatin-28-immuno-
239
5
6 7
8
9
10
11
12
reactive interneurons subserving reflex control of esophageal motility, J. Neurosci., 9 (1989) 1668-1682. Dichter, M.A. and Delfs, J.R., Somatostatin and cortical neurons in cell culture, Adv. Biochem. Psychopharmacol., 28 (1981) 145 157. Dodd, J. and Kelly, J.S., Is somatostatin an excitatory transmitter in the hippocampus?, Nature, 273 (1978) 674~575. Epelbaum, J., Somatostatin in the central nervous system: physiology and pathological modifications, Prog. Neurobiol., 27 (1986) 63-100. Gray, D.B., Pilar, G.R. and Ford, M.J., Opiate and peptide inhibition of transmitter release in parasympathetic terminals, J. Neurosci., 9 (1989) 1683-1692. Gray, D.B., Zelazny, D., Manthay, N. and Pilar, G., Endogenous modulation of ACh release by somatostatin and the differential roles of Ca z÷ channels, J. Neurosci., 10 (1990) 2687-2696. Ioffe, S., Havlicek, V., Friesen, H. and Chernick, V., Effect of somatostatin (SRIF) and L-glutamate on neurons of the sensorimotor cortex in awake habituated rabbits, Brain Res., 153 (1978) 414~,18. Jacquin, J., Champagnat, J., Madamba, S., Denavit-Saubi6, M. and Siggins, G.R., Somatostatin depresses excitability in neurons of the solitary tract complex through hyperpolarization and augmentation of Im, a non-inactivating voltage-dependent outward current blocked by muscarinic agonists, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 948 952. Mancillas, J.R., Siggins, G.R. and Bloom, F.E., Somatostatin
selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 75187521. 13 Moore, S.D., Madamba, S.G., Joels, M. and Siggins, G.R., Somatostatin augments the M-current in hippocampal neurons, Science, 239 (1988) 278-280. 14 Mueller, A.L., Kunkel, D.D. and Schwartzkroin, P.A,, Electrophysiological actions of somatostatin (SRIF) in hippocampus: an in vitro study, Cell. Mol. Neurobiol., 6 (1986) 363-379. 15 Pittman, Q.J. and Siggins, G.R., Somatostatin hyperpolarizes hippocampal pyramidal cells in vitro, Brain Res., 221 (1981) 402-408. 16 Scharfman, H.E. and Schwartzkroin, P.A., Further studies of the effects of somatostatin and related peptides in area CAI of rabbit hippocampus, Cell. Mot. Neurobiol., 8 (1988)411 429. 17 Vyas, D., Wang, Y.T. and Bieger, D., Nucleus reticularis intermedialis (NIR) - - possible cholinergic source subserving oesophageal peristalsis in the rat, Soc. Neurosci. Abstr., 16 (1990) 303.11. 18 Wang, Y.T., Neuman, R.S. and Bieger, D., Excitatory action of somatostatin (SST) on rat ambigual motoneurons in vitro, Soc. Neurosci. Abstr., 16 (1990) 422.6. 19 Wang, Y.T., Neuman, R.S. and Bieger, D., Nicotinic cholinoceptor-mediated excitation in ambigual motoneurones of the rat, Neuroscience, in press. 20 Watson, T,W. and Pittman, Q.J., Pharmacological evidence that somatostatin activates the m-current in hippocampal pyramidal neurons, Neurosci. Lett., 91 (1988) 172 176.