- Email: [email protected]
Presynaptic suppression of excitatory postsynaptic potentials in rat ventral horn neurons by muscarinic agonists
182
Bram Research; :';~ ~ (1980) 182- l,St~
BRE2t736
Presynaptic suppression of excitatory postsynaptic potentials in rat ventral horn neurons by muscarinic agonists Z.G. JIANG* and N.J. DUN Department of Pharmacology, Loyola University Medical Center. Maywood. IL 60153, U.S.A.. (Accepted May 6th. 19861 Key words: ventral horn neuron
motoneuron
muscannic agonist - - presynaptic inhibition
In addition to depolarizing the ventral horn cells including antidromically identified motoneurons in thin transverse neonatal rat spinal cord slice preparations, exogenously applied acetylcholine (ACh) suppressed the amplitude of excitatory postsynaptic potentials (EPSPs) either occurring spontaneously or elicited by stimulation of dorsal rootlets. A reduction of EPSPs could still be detected when the ACh-induced depolarization was nullified by hyperpolarizing current. Atropine but not D-tubocurarine effectively antagonized the depolarization and synaptic depression caused by ACh. While depressing theEPSPs. ACh hadno appreciable effect on membrane depolarizations elicited by glutamate. Methacholine mimicked the depolarizing and synaptic depressant effects of ACh. The results suggest that muscarinic agonists inhibit synaptic transmission of ventral horn neurons including motoneurons by a presynaptic mechanism in reducing the output of excitatory transmitters.
T h e r e is evidence of the presence of muscarinic receptors in autonomic nerve endings where their activation depresses synaptic transmission in the autonomic ganglia and neuroeffector junctions by reducing the release of transmitters 8'15. F o r example, muscarinic agonists diminish nicotinic transmission in the sympathetic ganglia 1°, m y e n t e r i c and submucous ganglia 13't4. On the o t h e r hand. a suppression of synaptic transmission by muscarinic agonists was demonstrated only with respect to a few central synapses. for instance, d e n t a t e gyrus ~6 and h i p p o c a m p a t CA1 neurons 5. W e r e p o r t here that transmission in m o t o neurons and o t h e r ventral horn neurons is a t t e n u a t e d by A C h and o t h e r muscarinic agonists and that a presynaptic mechanism m a y underlie this inhibitory action. S p r a g u e - D a w l e y rats aged 10-15 days were used in this study. The p r o c e d u r e s used for obtaining thin (400-500 # m ) transverse slices of neonatal rat thoracolumbar spinal cord were previously described IRA2. One slice with c o r r e s p o n d i n g ventral and dorsal rootlets was transferred to the recording c h a m b e r and su-
perfused continuously with a modified Krebs sotutlon saturated with 95% 0 2 / 5 % CO2: the t e m p e r a t u r e of the solution reaching the specimen was kept a t 34 _ 0.5 °C. lntracellular recordings were m a d e from neurons in the ventral horn by means of glass microelectrodes filled with 3 M potassium acetate, with an impedance of 3 0 - 8 0 Mr2. Ventral h o r n neurons were identified as m o t o n e u r o n s by the a p p e a r a n c e of an all-or-none spike or initial segment action potential following stimulation of ventral rootlets. Electrical stimulation of ventral and dorsal rootlets was accomplished via concentric bipolar electrodes (Fredrick Haer Co. ) positioned close to the respective rootlets. Signals amplified via a W P I 707A preamplifier were either displayed and s t o r e d in a Nicolet Digital Oscilloscope or r e c o r d e d on a G o u l d pen recorder. Signals stored in the Nicolet were later retrieved, processed and plotted on a H e w l e t t P a c k a r d plotter. A C h and other muscarinic agonists were applied to the motoneurons either by superfusion in known concentrations or by pressure e j e c u o n (Picospritzer) from a drug-containing micropipette positioned a b o v e the
* Present address: Department of Physiology, Wannan Medical College, Wannan, People's Republic of China. Correspondence: N.J. Dun. Department of Pharmacology, Loyola University Medical Center. 2160 S. First Avenue. Mavwood. IL 60153, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
183 recording neuron. The antagonists were applied to the slices by superfusion only. The results were expressed as mean +_ S.D. The results are based on recordings from 22 motoneurons identified antidromically by stimulation of ventral rootlets and 20 unidentified ventral horn neurons that could not be activated antidromically. In the latter case, these neurons could be either interneurons or m o t o n e u r o n s whose axons were d a m a g e d during sectioning process. The mean resting membrane potential, input resistance and m e m b r a n e time constant were - 6 4 . 5 + 6.1 mV, 44 _+ 32 M Q and 5.0 _+ 1.9 ms for m o t o n e u r o n s , and - 6 1 . 3 _+ 9.3 mV, 88 + 39 M Q and 7.1 + 2.0 ms for unidentified ventral horn neurons, respectively. The higher m e m b r a n e resistance and longer time constant of the unidentified ventral horn neurons may reflect the values of smaller d i a m e t e r interneurons. W h e n m e a s u r e d at bath t e m p e r a t u r e of 34 _+ 0.5 °C, the m e a n conduction velocity of m o t o r axons was 34 + 11 m/s (n = 9). Application by bath (10-100 ktM) or by pressure ejection ( A C h = 0.1 M, 40 psi, 5 - 2 0 ms pulse duration), A C h caused two conspicuous and consistent effects on m o t o n e u r o n s and unidentified ventral horn cells. First, A C h caused a slow depolarization in 15 of the 22 identified m o t o n e u r o n s and 16 of the 20 unidentified ventral horn cells tested (see for example Figs. 1 and 2); only in two unidentified neurons where A C h elicited a hyperpolarization (Fig. 3). The electrophysiological and pharmacological characteristics of A C h - i n d u c e d depolarizations in ventral horn neurons were described elsewhere 3. The second major action of A C h , which was the main concern of this study, was a depression of excitatory postsynaptic potentials (EPSPs) e v o k e d by stimulations of dorsal rootlets or occurring spontaneously. Exogenous A C h a t t e n u a t e d the amplitude of EPSPs in half of the 42 ventral horn neurons sampled (10 of 22 m o t o n e u r o n s , and 12 of 20 unidentified ventral horn cells); this effect was observed irrespective of the m o d e of A C h application. As the responses p r o d u c e d by pressure ejection were m o r e rapid in onset and fast in recovery (Figs. 1-3), the 'puff' m e t h o d was used extensively in this study. A representative experiment in which pressure ejection of A C h caused a slow depolarization and a depression of EPSPs in a m o t o n e u r o n is shown in Fig. 1. The depolarizations induced by A C h were generally associated with a
small to m o d e r a t e increase in m e m b r a n e resistance (18 _ 8 % , n = 10); the latter could be m o r e readily d e m o n s t r a t e d when the depolarization was nullified by hyperpolarizing current as illustrated in Fig. l B . With respect to EPSPs, a noticeable depression could be detected at the onset of m e m b r a n e depolarization or shortly thereafter (Figs. 1 and 2). The depression however consistently outlasted the depolarization by seconds to minutes (Figs. 1 and 2). F u r t h e r m o r e , returning the m e m b r a n e potential to the original level by passage of hyperpolarizing current did not restore the EPSPs to their control level in all 7 neurons investigated (Fig. 1B). F o r a quantitative comparison, the mean reduction in the amplitude of EPSPs in 5 neu-
~
c
ioo, 15my
~ ' <-2 °~l'°~ ATROPINE
B ~
~ A ACh
I~M
¢ ][[li~lllllIL [:[ lli]~TTr 1Ch
A ACh
-~;I,0mv
[[llllLIlllll]ll]lll]ll]lll[i[I ACh 5 r a m
A GLtl
WASH
A
A
Fig. 1. Depolarization and suppression of EPSPs by ACh in a rat motoneuron. A: individual EPSPs evoked by dorsal rootlet stimulation (arrowhead) before (a) and after (b) pressure ejection of ACh; a and b correspond to the time indicated on recording B. Two hyperpolarizing electrotonic potentials immediately following a and b are shown to the right. B: slow chart recording depicting EPSPs (upward deflections) and hyperpolarizing electrotonic potentials (downward deflections) induced by hyperpolarizing current pulses which are represented by downward deflections of lower tracing. ACh applied by pressure ejection (arrowheads, 15 ms pulse duration, 40 psi) evoked a slow depolarization and a concomitant decrease of EPSPs. A depression of EPSPs could still be demonstrated when the muscarinic depolarization was nullified by returning the membrane potential to the resting level (recording shown in the middle). Atropine effectively antagonized both the depolarization and synaptic depression induced by ACh. C: antidromic spike evoked by stimulation of ventral rootlets. D: membrane depolarizations induced by pressure ejection of glutamate (arrowheads, 100 mM, 100 ms pulse duration, 40 psi) in a motoneuron. The glutamate response was not affected by ACh (0.5 mM) applied by superfusion. Recordings in A, B and C were taken from the same motoneuron, whereas recordings in D were from a different motoneuron.
184 rons to which ACh (50 and 100/~M) was applied by superfusion amounted to 43 + 7%. While depressing the EPSPs evoked synaptically, ACh was found to have little or no depressant effect on membrane depolarizations evoked by glutamate, a putative transmitter in the spinal cord 2'4. Glutamate was applied to the ventral horn neurons by pressure ejection from a drug-containing micropipette (0.1 M, 5-200 ms pulse duration, 40 psi). Depolarizations induced by glutamate in 3 motoneurons and 2 unidentified ventral horn neurons were not depressed by superfusion of ACh (0.1 and 0.5 mM) to the slices. The mean amplitude of glutamate induced-depolarizations before and after ACh superfusion was 11 + 4 mV and 12 + 6 mV, respectively. One typical experiment is illustrated in Fig. 1D. Pharmacological studies revealed that both the depolarization and synaptic depression caused by ACh were muscarinic in nature as they were effectively antagonized by muscarinic (atropine, 1 btM) but not by nicotinic (D-tubocurarine, 10-50 ktM, or hexamethonium, 50-100/~M) receptor blockers in all 8 ventral horn neurons investigated (Fig. 1B). Moreover, methacholine applied either by bath (10 and 50/~M, n = 2) or by pressure ejection (n = 3) mimicked the depolarizing as well as the synaptic depressant effect of ACh. Again, atropine was effective in blocking the methacholine-induced depolarization and synaptic depression in these neurons. It is of interest to note that ACh suppressed spontaneously occurring EPSPs as well. Spontaneous EPSPs such as those illustrated in Fig. 2 were re-
A 10ms [5mV
B "
- - -
".
i
ACh
.
.
.
.
.
.
.
.
.
.
3
i
ACh
Fig. 2. Depolarization and suppression of spontaneous EPSPs by ACh in an unidentified ventral horn neuron. A: fast recording showing individual spontaneous EPSPs. B: slow chart recordings depicting membrane depolarization and suppression of spontaneous EPSPs by pressure ejection of A C h (arrowheads, 5 ms pulse duration, 40 psi).
corded in 8 ventral horn neurons (2 motoneurons and 6 unidentified cells); spontaneous EPSPs in some incidences, reached threshold and discharged action potentials. ACh diminished the spontaneously occurring EPSPs in 5 of the 8 ventral horn cells examined. In the experiment shown in Fig. 2. ACh elicited a small depolarization and markedly decreased the ongoing EPSPs in this unidentified ventral horn cell The last series of experiments was carried out to evaluate the possibility that the depression caused by ACh was secondary to the release of an inhibitory transmitter(sl. In this case. the effects of strychnine on ACh-induced responses were analyzed. Strychnine was chosen as it antagonized the action of glycine which is thought to be the principal inhibitory transmitter in the spinal cord 7. Pretreating the slices with strychnine (0.1-1 uM) did not appreciably alter the ACh-induced depolarization and synaptic depression in 3 motoneurons and 2 unidentified ventral horn neurons investigated. On the other hand. interesting results were obtained with respect to strychnine in two unidentified ventral horn neurons where ACh produced a hyperpolarization and a concomitant depression of EPSPs. In this case. strychnine markedly attenuated the hyperpolarizing response without appreciably affecting the synaptic depression induced by ACh (Fig. 3). A similar result was obtained in the other unidentified ventral horn neuron. Our results show that exogenous ACh produced two prominent effects on ventral horn cells including motoneurons: a slow depolarization and a decrease of evoked or spontaneous EPSPs. As the responses were sensitive to atropine but not to O-tubocurarine and hexamethonium, they were both mediated by muscarmic receptors. In an earlier study, exogenous ACh was found to depolarize motoneurons by stimulating directly postsynaptic muscarinic receptors 3. As to the depressant action of ACh. our results indicate that it may be acting at a site different from that generating the depolarization First. the synaptic depression and depolarization induced by ACh exhibited different time course in the same neurons, i.e.. the depression being longer lasting than the depolarization. Second, returning the membrane potential to the resting level by hyperpotarizing current did not restore EPSPs to their original level. For these reasons, the muscarimc sites responsible for ACh induced-depolarization on
185 CONTROL
i ACh
i
Strychnine I ~
I0"
c
ii!~ll/I~:ll:~]lTTlrllllIl[ll ACh '
'
305
'1 I° v
10ms
I 5mV
Fig. 3. Hyperpolarization and suppression of evoked EPSPs by ACh in an unidentified ventral horn neuron. Upper and lower recordings to the left are slow chart paper recordings. Small upward deflections represent EPSPs evoked by stimulation of dorsal rootlets. Downward deflections represent hyperpolarizing electrotonic potentials elicited by constant current pulses (not shown). Pressure ejection of ACh (arrowheads, 10 ms pulse duration, 40 psi) in this case produced a hyperpolarization and a marked decrease of EPSPs. Individual EPSPs and hyperpolarizing electrotonic potentials taken at the time marked by a and b are shown to the right. A 60c/r reduction could be detected between recordings a and b, whereas the electrotonic potentials remained the same. After superfusing thc slice with strychnine ( 1/~M) for 10 min, pressure ejection of ACh which now caused a much smaller hyperpolarization, remained effective in suppressing the EPSPs. Individual EPSPs and hyperpolarizing electrotonic potentials taken at the time marked by' c and d arc shown to the right.
one hand and synaptic depression on the other appear to be distinct. In this connection, the possibility that A C h inhibits synaptic transmission by diminishing the postsynaptic chemosensitivity to excitatory transmitters should be considered. The precise nature of excitatory transmitter(s) acting on motoneurons and ventral horn cells remains to be established, the amino acid glutamate has been widely acknowledged as one of the possible candidates 2,4. The observation that while depressing the EPSPs, A C h was without significant effect on m e m b r a n e depolarizations induced by glutamate suggests that the postsynaptic chemosensitivity to glutamate was not affected by ACh. Could the synaptic inhibition involve the release of an inhibitory transmitter(s) from spinal interneurons activated by A C h ? The interesting finding that strychnine abolished the hyperpolarization induced by A C h but not the synaptic depression argued against the involvement of an inhibitory transmitter, in so far as glycine is concerned. On the other hand, glycine a p p e a r e d to be responsible for the ACh-induced hyperpolarization as the latter was antago-
nized by strychnine. The negative results obtained with respect to strychnine and glutamate raise the possibility that A C h depresses transmission in ventral horn neurons by a presynaptic mechanism whereby the activation of muscarinic receptors situated on presynaptic nerve endings inhibits transmitter liberation. In this respect, the hypothesis of presynaptic regulation of transmitter release by muscarinic receptors has received strong support from studies carried out in the peripheral nervous tissues (see ref. 8 for review). It is tempting to propose that an inhibitory muscarinic mechanism analogous to that described in the peripheral tissues may exist in nerve endings presynaptic to motoneurons and ventral horn cells. In this connection, the present findings in conjunction with the recent demonstration of choline acetyltransferase containing fibers and interneurons in the ventral horn 16,~ raise the interesting possibility that A C h when released from collateral fibers of m o t o n e u r o n s or from cholinergic interneurons may, in addition to excite directly m o t o n e u r o n s and interneurons by interacting with postsynaptic muscarinic receptors, regu-
186 late i m p u l s e t r a n s m i s s i o n t o m o t o n e u r o n s
or mter-
T h i s s t u d y was s u p p o r t e d b y G r a n t N S 18711).
n e u r o n s via a p r e s y n a p t i c m e c h a n i s m .
1 Borges, L.F. and Iversen, S.D., Topography of choline acetyltransferase immunoreactive neurons and fibers in the rat spinal cord, Brain Research, 362 (1986) 140-148. 2 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the mammalian central nervous system. Rev. Physiol. Biochem. Exp. Pharmacol., 69 (1978) 97-188. 3 Dun, N.J. and Jiang, Z.G., An intracellular study of the effects of acetylcholine on motoneurons in a neonatal rat spinal cord slice preparation, Soc. Neurosci. Abstr. I I (1985) 12.3. 4 Fagg, G.E. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 5 Hounsgaard, J., Presynaptic inhibitory action of acetylcholine in area CAI of the hippocampus, Exp. NeuroL. 62 (1978) 787-797. 6 Houser, C.R., Crawford, G.D., Barber, R.P.. Salvaterra. P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Research, 266 (1983) 97-119. 7 Johnston, G.A.R., Neuropharmacoiogy of amino acid inhibitory transmitters, Annu. Rev. Pharmacol. Toxicol. 18 (1978) 269-289. 8 Kilbinger, H., Presynaptic muscarine receptors modulating acetylcholine release, Trends Pharmacol. Sci.. 5 11984) 103-105. 9 Kimura, H., McGeer, P.L., Peng, J.H. and McGeer. E.G..
10
11
12
13
14
15
16
The central cholinergic system studied bv choline acetvltransferase immunohistochemistry in tbc rat. J. Comp. Neurol.. 200 (19811 151-201. Koketsu. K. and Yamada. M.. Presynaptic muscarmic receptors inhibiting active acetylcholine release in the bullfrog sympathetic ganglion Br. J. Pharrnacol.. 77 (19827 83-88. Ma. R.C. and Dun. N.J.. Vasopressm depolarizes lateral horn ceils of the neonatal rat spinal cord in vitro. Brain Research. 348 11985) 36-42. Ma. R.C. and Dun. N.J.. Excitation of the lateral horn cells of the neonatal rat spinal cord by 5-hydroxytryptamine. Dev. Brain Res.. 24 C1986J 89-98 Morita. K.. North. R.A. and Tokimasa. I'.. Musearmic presynaptic inhibition of synaptic transmission in myenteric plexus of guinea-pig ileum. I. Physiol. ~LondonL 333 11982) 141-149. North. R.A.. Slack. B.E. and Surprenant. A.. Muscarinic M~ and M 2 receptors mediate depolarization and presynaptic inhibition in guinea-pig enteric nervous system. J, Physiol. (London). 368 (19851 435-452. Starke. K.. Regulation of noradrenaline release by presynaptic receptor systems. Rev. Physiol. Biochem. Pharmacol.. 77 f1977) 1-124. Yamamoto. C. and Kawai. K Presyuaptic action of acetvlcholine in thin sections from the guinea pig dentate gyrus in vitro. Exp. Neurol.. 19 I1967] 176-t87
Bram Research; :';~ ~ (1980) 182- l,St~
BRE2t736
Presynaptic suppression of excitatory postsynaptic potentials in rat ventral horn neurons by muscarinic agonists Z.G. JIANG* and N.J. DUN Department of Pharmacology, Loyola University Medical Center. Maywood. IL 60153, U.S.A.. (Accepted May 6th. 19861 Key words: ventral horn neuron
motoneuron
muscannic agonist - - presynaptic inhibition
In addition to depolarizing the ventral horn cells including antidromically identified motoneurons in thin transverse neonatal rat spinal cord slice preparations, exogenously applied acetylcholine (ACh) suppressed the amplitude of excitatory postsynaptic potentials (EPSPs) either occurring spontaneously or elicited by stimulation of dorsal rootlets. A reduction of EPSPs could still be detected when the ACh-induced depolarization was nullified by hyperpolarizing current. Atropine but not D-tubocurarine effectively antagonized the depolarization and synaptic depression caused by ACh. While depressing theEPSPs. ACh hadno appreciable effect on membrane depolarizations elicited by glutamate. Methacholine mimicked the depolarizing and synaptic depressant effects of ACh. The results suggest that muscarinic agonists inhibit synaptic transmission of ventral horn neurons including motoneurons by a presynaptic mechanism in reducing the output of excitatory transmitters.
T h e r e is evidence of the presence of muscarinic receptors in autonomic nerve endings where their activation depresses synaptic transmission in the autonomic ganglia and neuroeffector junctions by reducing the release of transmitters 8'15. F o r example, muscarinic agonists diminish nicotinic transmission in the sympathetic ganglia 1°, m y e n t e r i c and submucous ganglia 13't4. On the o t h e r hand. a suppression of synaptic transmission by muscarinic agonists was demonstrated only with respect to a few central synapses. for instance, d e n t a t e gyrus ~6 and h i p p o c a m p a t CA1 neurons 5. W e r e p o r t here that transmission in m o t o neurons and o t h e r ventral horn neurons is a t t e n u a t e d by A C h and o t h e r muscarinic agonists and that a presynaptic mechanism m a y underlie this inhibitory action. S p r a g u e - D a w l e y rats aged 10-15 days were used in this study. The p r o c e d u r e s used for obtaining thin (400-500 # m ) transverse slices of neonatal rat thoracolumbar spinal cord were previously described IRA2. One slice with c o r r e s p o n d i n g ventral and dorsal rootlets was transferred to the recording c h a m b e r and su-
perfused continuously with a modified Krebs sotutlon saturated with 95% 0 2 / 5 % CO2: the t e m p e r a t u r e of the solution reaching the specimen was kept a t 34 _ 0.5 °C. lntracellular recordings were m a d e from neurons in the ventral horn by means of glass microelectrodes filled with 3 M potassium acetate, with an impedance of 3 0 - 8 0 Mr2. Ventral h o r n neurons were identified as m o t o n e u r o n s by the a p p e a r a n c e of an all-or-none spike or initial segment action potential following stimulation of ventral rootlets. Electrical stimulation of ventral and dorsal rootlets was accomplished via concentric bipolar electrodes (Fredrick Haer Co. ) positioned close to the respective rootlets. Signals amplified via a W P I 707A preamplifier were either displayed and s t o r e d in a Nicolet Digital Oscilloscope or r e c o r d e d on a G o u l d pen recorder. Signals stored in the Nicolet were later retrieved, processed and plotted on a H e w l e t t P a c k a r d plotter. A C h and other muscarinic agonists were applied to the motoneurons either by superfusion in known concentrations or by pressure e j e c u o n (Picospritzer) from a drug-containing micropipette positioned a b o v e the
* Present address: Department of Physiology, Wannan Medical College, Wannan, People's Republic of China. Correspondence: N.J. Dun. Department of Pharmacology, Loyola University Medical Center. 2160 S. First Avenue. Mavwood. IL 60153, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
183 recording neuron. The antagonists were applied to the slices by superfusion only. The results were expressed as mean +_ S.D. The results are based on recordings from 22 motoneurons identified antidromically by stimulation of ventral rootlets and 20 unidentified ventral horn neurons that could not be activated antidromically. In the latter case, these neurons could be either interneurons or m o t o n e u r o n s whose axons were d a m a g e d during sectioning process. The mean resting membrane potential, input resistance and m e m b r a n e time constant were - 6 4 . 5 + 6.1 mV, 44 _+ 32 M Q and 5.0 _+ 1.9 ms for m o t o n e u r o n s , and - 6 1 . 3 _+ 9.3 mV, 88 + 39 M Q and 7.1 + 2.0 ms for unidentified ventral horn neurons, respectively. The higher m e m b r a n e resistance and longer time constant of the unidentified ventral horn neurons may reflect the values of smaller d i a m e t e r interneurons. W h e n m e a s u r e d at bath t e m p e r a t u r e of 34 _+ 0.5 °C, the m e a n conduction velocity of m o t o r axons was 34 + 11 m/s (n = 9). Application by bath (10-100 ktM) or by pressure ejection ( A C h = 0.1 M, 40 psi, 5 - 2 0 ms pulse duration), A C h caused two conspicuous and consistent effects on m o t o n e u r o n s and unidentified ventral horn cells. First, A C h caused a slow depolarization in 15 of the 22 identified m o t o n e u r o n s and 16 of the 20 unidentified ventral horn cells tested (see for example Figs. 1 and 2); only in two unidentified neurons where A C h elicited a hyperpolarization (Fig. 3). The electrophysiological and pharmacological characteristics of A C h - i n d u c e d depolarizations in ventral horn neurons were described elsewhere 3. The second major action of A C h , which was the main concern of this study, was a depression of excitatory postsynaptic potentials (EPSPs) e v o k e d by stimulations of dorsal rootlets or occurring spontaneously. Exogenous A C h a t t e n u a t e d the amplitude of EPSPs in half of the 42 ventral horn neurons sampled (10 of 22 m o t o n e u r o n s , and 12 of 20 unidentified ventral horn cells); this effect was observed irrespective of the m o d e of A C h application. As the responses p r o d u c e d by pressure ejection were m o r e rapid in onset and fast in recovery (Figs. 1-3), the 'puff' m e t h o d was used extensively in this study. A representative experiment in which pressure ejection of A C h caused a slow depolarization and a depression of EPSPs in a m o t o n e u r o n is shown in Fig. 1. The depolarizations induced by A C h were generally associated with a
small to m o d e r a t e increase in m e m b r a n e resistance (18 _ 8 % , n = 10); the latter could be m o r e readily d e m o n s t r a t e d when the depolarization was nullified by hyperpolarizing current as illustrated in Fig. l B . With respect to EPSPs, a noticeable depression could be detected at the onset of m e m b r a n e depolarization or shortly thereafter (Figs. 1 and 2). The depression however consistently outlasted the depolarization by seconds to minutes (Figs. 1 and 2). F u r t h e r m o r e , returning the m e m b r a n e potential to the original level by passage of hyperpolarizing current did not restore the EPSPs to their control level in all 7 neurons investigated (Fig. 1B). F o r a quantitative comparison, the mean reduction in the amplitude of EPSPs in 5 neu-
~
c
ioo, 15my
~ ' <-2 °~l'°~ ATROPINE
B ~
~ A ACh
I~M
¢ ][[li~lllllIL [:[ lli]~TTr 1Ch
A ACh
-~;I,0mv
[[llllLIlllll]ll]lll]ll]lll[i[I ACh 5 r a m
A GLtl
WASH
A
A
Fig. 1. Depolarization and suppression of EPSPs by ACh in a rat motoneuron. A: individual EPSPs evoked by dorsal rootlet stimulation (arrowhead) before (a) and after (b) pressure ejection of ACh; a and b correspond to the time indicated on recording B. Two hyperpolarizing electrotonic potentials immediately following a and b are shown to the right. B: slow chart recording depicting EPSPs (upward deflections) and hyperpolarizing electrotonic potentials (downward deflections) induced by hyperpolarizing current pulses which are represented by downward deflections of lower tracing. ACh applied by pressure ejection (arrowheads, 15 ms pulse duration, 40 psi) evoked a slow depolarization and a concomitant decrease of EPSPs. A depression of EPSPs could still be demonstrated when the muscarinic depolarization was nullified by returning the membrane potential to the resting level (recording shown in the middle). Atropine effectively antagonized both the depolarization and synaptic depression induced by ACh. C: antidromic spike evoked by stimulation of ventral rootlets. D: membrane depolarizations induced by pressure ejection of glutamate (arrowheads, 100 mM, 100 ms pulse duration, 40 psi) in a motoneuron. The glutamate response was not affected by ACh (0.5 mM) applied by superfusion. Recordings in A, B and C were taken from the same motoneuron, whereas recordings in D were from a different motoneuron.
184 rons to which ACh (50 and 100/~M) was applied by superfusion amounted to 43 + 7%. While depressing the EPSPs evoked synaptically, ACh was found to have little or no depressant effect on membrane depolarizations evoked by glutamate, a putative transmitter in the spinal cord 2'4. Glutamate was applied to the ventral horn neurons by pressure ejection from a drug-containing micropipette (0.1 M, 5-200 ms pulse duration, 40 psi). Depolarizations induced by glutamate in 3 motoneurons and 2 unidentified ventral horn neurons were not depressed by superfusion of ACh (0.1 and 0.5 mM) to the slices. The mean amplitude of glutamate induced-depolarizations before and after ACh superfusion was 11 + 4 mV and 12 + 6 mV, respectively. One typical experiment is illustrated in Fig. 1D. Pharmacological studies revealed that both the depolarization and synaptic depression caused by ACh were muscarinic in nature as they were effectively antagonized by muscarinic (atropine, 1 btM) but not by nicotinic (D-tubocurarine, 10-50 ktM, or hexamethonium, 50-100/~M) receptor blockers in all 8 ventral horn neurons investigated (Fig. 1B). Moreover, methacholine applied either by bath (10 and 50/~M, n = 2) or by pressure ejection (n = 3) mimicked the depolarizing as well as the synaptic depressant effect of ACh. Again, atropine was effective in blocking the methacholine-induced depolarization and synaptic depression in these neurons. It is of interest to note that ACh suppressed spontaneously occurring EPSPs as well. Spontaneous EPSPs such as those illustrated in Fig. 2 were re-
A 10ms [5mV
B "
- - -
".
i
ACh
.
.
.
.
.
.
.
.
.
.
3
i
ACh
Fig. 2. Depolarization and suppression of spontaneous EPSPs by ACh in an unidentified ventral horn neuron. A: fast recording showing individual spontaneous EPSPs. B: slow chart recordings depicting membrane depolarization and suppression of spontaneous EPSPs by pressure ejection of A C h (arrowheads, 5 ms pulse duration, 40 psi).
corded in 8 ventral horn neurons (2 motoneurons and 6 unidentified cells); spontaneous EPSPs in some incidences, reached threshold and discharged action potentials. ACh diminished the spontaneously occurring EPSPs in 5 of the 8 ventral horn cells examined. In the experiment shown in Fig. 2. ACh elicited a small depolarization and markedly decreased the ongoing EPSPs in this unidentified ventral horn cell The last series of experiments was carried out to evaluate the possibility that the depression caused by ACh was secondary to the release of an inhibitory transmitter(sl. In this case. the effects of strychnine on ACh-induced responses were analyzed. Strychnine was chosen as it antagonized the action of glycine which is thought to be the principal inhibitory transmitter in the spinal cord 7. Pretreating the slices with strychnine (0.1-1 uM) did not appreciably alter the ACh-induced depolarization and synaptic depression in 3 motoneurons and 2 unidentified ventral horn neurons investigated. On the other hand. interesting results were obtained with respect to strychnine in two unidentified ventral horn neurons where ACh produced a hyperpolarization and a concomitant depression of EPSPs. In this case. strychnine markedly attenuated the hyperpolarizing response without appreciably affecting the synaptic depression induced by ACh (Fig. 3). A similar result was obtained in the other unidentified ventral horn neuron. Our results show that exogenous ACh produced two prominent effects on ventral horn cells including motoneurons: a slow depolarization and a decrease of evoked or spontaneous EPSPs. As the responses were sensitive to atropine but not to O-tubocurarine and hexamethonium, they were both mediated by muscarmic receptors. In an earlier study, exogenous ACh was found to depolarize motoneurons by stimulating directly postsynaptic muscarinic receptors 3. As to the depressant action of ACh. our results indicate that it may be acting at a site different from that generating the depolarization First. the synaptic depression and depolarization induced by ACh exhibited different time course in the same neurons, i.e.. the depression being longer lasting than the depolarization. Second, returning the membrane potential to the resting level by hyperpotarizing current did not restore EPSPs to their original level. For these reasons, the muscarimc sites responsible for ACh induced-depolarization on
185 CONTROL
i ACh
i
Strychnine I ~
I0"
c
ii!~ll/I~:ll:~]lTTlrllllIl[ll ACh '
'
305
'1 I° v
10ms
I 5mV
Fig. 3. Hyperpolarization and suppression of evoked EPSPs by ACh in an unidentified ventral horn neuron. Upper and lower recordings to the left are slow chart paper recordings. Small upward deflections represent EPSPs evoked by stimulation of dorsal rootlets. Downward deflections represent hyperpolarizing electrotonic potentials elicited by constant current pulses (not shown). Pressure ejection of ACh (arrowheads, 10 ms pulse duration, 40 psi) in this case produced a hyperpolarization and a marked decrease of EPSPs. Individual EPSPs and hyperpolarizing electrotonic potentials taken at the time marked by a and b are shown to the right. A 60c/r reduction could be detected between recordings a and b, whereas the electrotonic potentials remained the same. After superfusing thc slice with strychnine ( 1/~M) for 10 min, pressure ejection of ACh which now caused a much smaller hyperpolarization, remained effective in suppressing the EPSPs. Individual EPSPs and hyperpolarizing electrotonic potentials taken at the time marked by' c and d arc shown to the right.
one hand and synaptic depression on the other appear to be distinct. In this connection, the possibility that A C h inhibits synaptic transmission by diminishing the postsynaptic chemosensitivity to excitatory transmitters should be considered. The precise nature of excitatory transmitter(s) acting on motoneurons and ventral horn cells remains to be established, the amino acid glutamate has been widely acknowledged as one of the possible candidates 2,4. The observation that while depressing the EPSPs, A C h was without significant effect on m e m b r a n e depolarizations induced by glutamate suggests that the postsynaptic chemosensitivity to glutamate was not affected by ACh. Could the synaptic inhibition involve the release of an inhibitory transmitter(s) from spinal interneurons activated by A C h ? The interesting finding that strychnine abolished the hyperpolarization induced by A C h but not the synaptic depression argued against the involvement of an inhibitory transmitter, in so far as glycine is concerned. On the other hand, glycine a p p e a r e d to be responsible for the ACh-induced hyperpolarization as the latter was antago-
nized by strychnine. The negative results obtained with respect to strychnine and glutamate raise the possibility that A C h depresses transmission in ventral horn neurons by a presynaptic mechanism whereby the activation of muscarinic receptors situated on presynaptic nerve endings inhibits transmitter liberation. In this respect, the hypothesis of presynaptic regulation of transmitter release by muscarinic receptors has received strong support from studies carried out in the peripheral nervous tissues (see ref. 8 for review). It is tempting to propose that an inhibitory muscarinic mechanism analogous to that described in the peripheral tissues may exist in nerve endings presynaptic to motoneurons and ventral horn cells. In this connection, the present findings in conjunction with the recent demonstration of choline acetyltransferase containing fibers and interneurons in the ventral horn 16,~ raise the interesting possibility that A C h when released from collateral fibers of m o t o n e u r o n s or from cholinergic interneurons may, in addition to excite directly m o t o n e u r o n s and interneurons by interacting with postsynaptic muscarinic receptors, regu-
186 late i m p u l s e t r a n s m i s s i o n t o m o t o n e u r o n s
or mter-
T h i s s t u d y was s u p p o r t e d b y G r a n t N S 18711).
n e u r o n s via a p r e s y n a p t i c m e c h a n i s m .
1 Borges, L.F. and Iversen, S.D., Topography of choline acetyltransferase immunoreactive neurons and fibers in the rat spinal cord, Brain Research, 362 (1986) 140-148. 2 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the mammalian central nervous system. Rev. Physiol. Biochem. Exp. Pharmacol., 69 (1978) 97-188. 3 Dun, N.J. and Jiang, Z.G., An intracellular study of the effects of acetylcholine on motoneurons in a neonatal rat spinal cord slice preparation, Soc. Neurosci. Abstr. I I (1985) 12.3. 4 Fagg, G.E. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 5 Hounsgaard, J., Presynaptic inhibitory action of acetylcholine in area CAI of the hippocampus, Exp. NeuroL. 62 (1978) 787-797. 6 Houser, C.R., Crawford, G.D., Barber, R.P.. Salvaterra. P.M. and Vaughn, J.E., Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase, Brain Research, 266 (1983) 97-119. 7 Johnston, G.A.R., Neuropharmacoiogy of amino acid inhibitory transmitters, Annu. Rev. Pharmacol. Toxicol. 18 (1978) 269-289. 8 Kilbinger, H., Presynaptic muscarine receptors modulating acetylcholine release, Trends Pharmacol. Sci.. 5 11984) 103-105. 9 Kimura, H., McGeer, P.L., Peng, J.H. and McGeer. E.G..
10
11
12
13
14
15
16
The central cholinergic system studied bv choline acetvltransferase immunohistochemistry in tbc rat. J. Comp. Neurol.. 200 (19811 151-201. Koketsu. K. and Yamada. M.. Presynaptic muscarmic receptors inhibiting active acetylcholine release in the bullfrog sympathetic ganglion Br. J. Pharrnacol.. 77 (19827 83-88. Ma. R.C. and Dun. N.J.. Vasopressm depolarizes lateral horn ceils of the neonatal rat spinal cord in vitro. Brain Research. 348 11985) 36-42. Ma. R.C. and Dun. N.J.. Excitation of the lateral horn cells of the neonatal rat spinal cord by 5-hydroxytryptamine. Dev. Brain Res.. 24 C1986J 89-98 Morita. K.. North. R.A. and Tokimasa. I'.. Musearmic presynaptic inhibition of synaptic transmission in myenteric plexus of guinea-pig ileum. I. Physiol. ~LondonL 333 11982) 141-149. North. R.A.. Slack. B.E. and Surprenant. A.. Muscarinic M~ and M 2 receptors mediate depolarization and presynaptic inhibition in guinea-pig enteric nervous system. J, Physiol. (London). 368 (19851 435-452. Starke. K.. Regulation of noradrenaline release by presynaptic receptor systems. Rev. Physiol. Biochem. Pharmacol.. 77 f1977) 1-124. Yamamoto. C. and Kawai. K Presyuaptic action of acetvlcholine in thin sections from the guinea pig dentate gyrus in vitro. Exp. Neurol.. 19 I1967] 176-t87