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An intracortical cholinergic inhibitory synapse
Vol . 7, pp . 65-69, 1968 . Life Sciences Printed in Great Britain .
Pergamon Press
AN INTRACORTICAL CHOLINERGIC INHIBITORY SYNAPSE J . W. Phillis & D, H . York Department of Physiology, Monash University, Clayton, Victoria 3168, Australia
(Received 23 October 1967)
Evidence was presented in a previous report (1) to show that the inhibition of cerebral cortical neurones by stimulation of the ipsilateral medullary pyramidal tract (PT) or mesencephalic reticular formation (RF) was mediated by acetylcholine (ACh) .
The observations on PT stimulation suggested that
such inhibitions were a result of excitation of cholinergic inhibitory inter neurones by PT axon collaterals .
The presence of local cholinergic circuits
in the cerebral cortex had previously been inferred from studies in the choline acetyltransferase content of intact and undercut cerebral cortices (2, 3) .
Further pharmacological studies on cortical inhibitions evoked by
direct cortical stimulation and lateral hypothalamic stimulation are described in this report . IVY ethod s Cats were anaesthetised with an intravenous injection of thiopentone sodium (Intraval Sodium, A-iay & Baker), to a gas anaesthetic machine .
set up in a stereotaxic frame and connected
Anaesthesia was subsequently maintained
with a mixture of nitrous oxide, oxygen and methoxyflurane (Penthrane, Abbott) or halothane (Fluothane,
I. C . I . ) .
Drugs were applied iontophoretical-
ly from five barrelled micropipettes, the central barrel of which was used for recording extra-cellular spike potentials of cortical neurones .
A Hewlett-
Packard ~214I . electronic counter coupled to an ink recorder and digital recorder was used to analyse the spontaneous or L-glutamate evoked discharges of cortical neurones .
L-glutamate was usually applied by current
pulses of ~- 10 sec duration repeated at regular intervals .
The counter
incorporated a standard modification (H36) which enabled it to be used with remote stop-start control .
It could be gated by a pulse from a Grass S8 dual
channel stimulator to count spikes evoked from sensitive neurones by brief
65
Vol . 7, No . 1
INHIBITORY SYNAPSE
66
A
ACM !0
ACM
LMS
LM! 100
b0
0 YIN
ss
bb
Ac~ :o
ACM
ss
J MIN
100
N N
!0
W
R F t
0
A & B. Records from precruciate cortical neurones illustrating the depressant effect of ACh, lateral hypothalamic (LHS) and direct cortical (SS) stimulation on L-glutamate evoked firing. Periods of drug application or stimulation are indicated by the horizontal bare . Atropine abolished the inhibitory effects of both ACh and nervous stimulation. C. Comparable record from poet-cruciate neurone in an acutely isolated cortical slab . The vertical scale on the right of each record indicates actual rate of discharge.
Vol . 7, No .
67
INHIBITORY SYNAPSE
1
(10 - 30 msec) applications of L-glutamate . Inhibition was manifested as a reduction in the frequency of spontaneous or L-glutamate evoked firing during and after the period of stimulation .
An
ipsilateral coaxial stimulating electrode was placed in the lateral hypothalamus (LH, All . 5, L4 . 5, D-3) and a fine bipolar coaxial stimulating electrode was inserted directly into the pre- or post-cruciate cerebral cortex to a depth of up to 1 mm . Results Repetitive stimulation in the lateral hypothalamus evoked short latency negative focal potentials in the peri-cruciate neocortex, with monosynaptic firing of some cortical neurones and polysynaptic excitatory responses of others .
Also associated with LH stimulation were short latency inhibitions
of 100-150 msec duration and, less frequently, (in 22 of the 91 cells tested) long duration inhibitions which persisted for up to 1 min . The s}iort latency inhibition evoked by LH stimulation was often observed with ACh- insensitive neurones and was resistant to atropine, hyoscine and strychnine .
Long duration inhibitions of the type illustrated in Fig . lA, were
associated with ACh-sensitivity and were abolished by atropine, hyoscine and strychnine . The inhibitory effects of direct cortical stimulation have previously been described (4, 5) .
Many of the animals used in this earlier survey were how-
ever, anaesthetised with a diallyl-harbituric acid - urethane mixture and single shocks were frequently used .
Most of these findings have been confirmed
in the present investigation which has revealed in addition, a long duration inhibitory effect of repetitive surface stimulation . Short duration inhibitory effects of surface stimulation were observed on most of the neurones tested in the vicinity of the stimulating electrode .
These
effects have been shown to correspond to hyperpolarisations of the neurones involved (4, 5) .
With repetitive stimulation, a long lasting (up to 1 min)
inhibition of the glutamate firing of some cells was evident (in 33 of the 78 cells tested) .
Ninety-one percent of these cells were also depressed by ACh
(see Fig. 1B) and the effects of both electrical stimulation and ACh were abolished or reduced by atropine, hyoscine and strychnine . cells were found in cortical layers 11, 111 and IV . were also well developed in isolated cortical slabs .
Most of these
Long duration inhibitions Two acute slabs were
Vol . 7, No . 1
INHIBITORY SYNAPSE
68
prepared by extensive undercutting of the entire area of the pericruciate cortex .
The records in Fig. 1C were obtained from one of these slabs and
illustrate a well developed long duration inhibitory effect of surface stimulation on an ACh-sensitive neurone. Discussion The results ôbtained in this survey cast further doubt on the general applicability of conclusions derived from barbiturate anaesthetised animals. Depression of the excitant actions of ACh and the inhibitory actions of noradrenaline on thalamic neurones by small doses of pentobarbitone sodium, in the absence of any marked alterations in cell excitability, has already been described (6) .
The fact that volatile anaesthetics were used in the
present study also provides an adequate explanation for the failure of previous investigators to observe comparable atropine-labile inhibitions in the cerebral cortex (7) and may help to explain many of the conflicting reports about the ability of strychnine to block cortical inhibitions (8, 9, 10) . The finding that long duration cholinergic cortical inhibitions can be evoked by stimulation of such different structures as the pyramidal tracts, mesencephalic reticular formation and lateral hypothalamus indicated that a cholinergic inhibitory interneurone might be present in the cerebral cortex . This hypothesis has been strengthened by the observation that similar inhibitions can be evoked by direct stimulation of isolated cortical slabs. The long duration of these inhibitory effects may be a result of several factors, including repetitive firing by cortical interneurones or a prolonged action of the synaptically released ACh.
Long duration hyperpolarising
inhibitions have recently been described in mammalian ganglia (11) and in certain cells of Aplysia californicans (12) .
In the latter, the long duration
inhibition is supplementary to rapidly decaying IPSPs and requires repeated stimulation for its generation .
The situation in the cerebral cortex appears
to be analogous in many respects . Summary Evidence is presented for the presence of a cholinergic inhibitory synapse
in the cerebral cortex .
The failure of previous investigators to observe
cholinergic inhibitory effects of direct stimulation of the cortex may have been due to an action of barbiturates .
Vol . 7, No . 1
INHIBITORY SYNAPSE References
1. 2.
J. W, PHILLIS and D, H . YORK, Brain Res . 5, 517 (1967) . -_ i C.O, HEBB, K, KRNJEVIC and A, SILVER, Nature, 198, 692 (1963) .
3.
J . W, PHILLIS and G . C, CHONG, Nature , 207, 1253 (1965) .
4.
K. KRNJEVIC, M, RANDIC and D. W. STRAUGHAN, J. Physiol. 184, 16 (1966) .
5.
/ / K, KRNJEVIC, M, RANDIC and D, W, STRAUGHAN, J . Physiol. 184, 49 (1966) .
6.
J . W, PHILLIS and A, K. TEBECIS, Life Sci, 6, 1621 (1967) .
7.
K, KRNJEVIC, M, RANDIC and D. W, STRAUGHAN, J, Physiol. 184, 7a
8.
(lsss) .
J . M. CRAWFORD, D, R, CURTIS, P, E, VOORHOEVE and V,J . WILSON, Nature , 200, 845 (1963) .
9.
V, B, BROOKS and H, ASANUMA, Am . J . Physiol. 208, 674 (1965) .
10 .
A4 . SAWA, N, bTARL'YAMA, S. KAJI and K, NAKAMURA, Jap. J. Physiol. 16, 126 (1966) .
11 .
B, LIBET, J, Neurophysiol . 30, 494 (1967),
12 .
J, KFIIOE, ?~'ature, 215, 1503 (1967),
69
Pergamon Press
AN INTRACORTICAL CHOLINERGIC INHIBITORY SYNAPSE J . W. Phillis & D, H . York Department of Physiology, Monash University, Clayton, Victoria 3168, Australia
(Received 23 October 1967)
Evidence was presented in a previous report (1) to show that the inhibition of cerebral cortical neurones by stimulation of the ipsilateral medullary pyramidal tract (PT) or mesencephalic reticular formation (RF) was mediated by acetylcholine (ACh) .
The observations on PT stimulation suggested that
such inhibitions were a result of excitation of cholinergic inhibitory inter neurones by PT axon collaterals .
The presence of local cholinergic circuits
in the cerebral cortex had previously been inferred from studies in the choline acetyltransferase content of intact and undercut cerebral cortices (2, 3) .
Further pharmacological studies on cortical inhibitions evoked by
direct cortical stimulation and lateral hypothalamic stimulation are described in this report . IVY ethod s Cats were anaesthetised with an intravenous injection of thiopentone sodium (Intraval Sodium, A-iay & Baker), to a gas anaesthetic machine .
set up in a stereotaxic frame and connected
Anaesthesia was subsequently maintained
with a mixture of nitrous oxide, oxygen and methoxyflurane (Penthrane, Abbott) or halothane (Fluothane,
I. C . I . ) .
Drugs were applied iontophoretical-
ly from five barrelled micropipettes, the central barrel of which was used for recording extra-cellular spike potentials of cortical neurones .
A Hewlett-
Packard ~214I . electronic counter coupled to an ink recorder and digital recorder was used to analyse the spontaneous or L-glutamate evoked discharges of cortical neurones .
L-glutamate was usually applied by current
pulses of ~- 10 sec duration repeated at regular intervals .
The counter
incorporated a standard modification (H36) which enabled it to be used with remote stop-start control .
It could be gated by a pulse from a Grass S8 dual
channel stimulator to count spikes evoked from sensitive neurones by brief
65
Vol . 7, No . 1
INHIBITORY SYNAPSE
66
A
ACM !0
ACM
LMS
LM! 100
b0
0 YIN
ss
bb
Ac~ :o
ACM
ss
J MIN
100
N N
!0
W
R F t
0
A & B. Records from precruciate cortical neurones illustrating the depressant effect of ACh, lateral hypothalamic (LHS) and direct cortical (SS) stimulation on L-glutamate evoked firing. Periods of drug application or stimulation are indicated by the horizontal bare . Atropine abolished the inhibitory effects of both ACh and nervous stimulation. C. Comparable record from poet-cruciate neurone in an acutely isolated cortical slab . The vertical scale on the right of each record indicates actual rate of discharge.
Vol . 7, No .
67
INHIBITORY SYNAPSE
1
(10 - 30 msec) applications of L-glutamate . Inhibition was manifested as a reduction in the frequency of spontaneous or L-glutamate evoked firing during and after the period of stimulation .
An
ipsilateral coaxial stimulating electrode was placed in the lateral hypothalamus (LH, All . 5, L4 . 5, D-3) and a fine bipolar coaxial stimulating electrode was inserted directly into the pre- or post-cruciate cerebral cortex to a depth of up to 1 mm . Results Repetitive stimulation in the lateral hypothalamus evoked short latency negative focal potentials in the peri-cruciate neocortex, with monosynaptic firing of some cortical neurones and polysynaptic excitatory responses of others .
Also associated with LH stimulation were short latency inhibitions
of 100-150 msec duration and, less frequently, (in 22 of the 91 cells tested) long duration inhibitions which persisted for up to 1 min . The s}iort latency inhibition evoked by LH stimulation was often observed with ACh- insensitive neurones and was resistant to atropine, hyoscine and strychnine .
Long duration inhibitions of the type illustrated in Fig . lA, were
associated with ACh-sensitivity and were abolished by atropine, hyoscine and strychnine . The inhibitory effects of direct cortical stimulation have previously been described (4, 5) .
Many of the animals used in this earlier survey were how-
ever, anaesthetised with a diallyl-harbituric acid - urethane mixture and single shocks were frequently used .
Most of these findings have been confirmed
in the present investigation which has revealed in addition, a long duration inhibitory effect of repetitive surface stimulation . Short duration inhibitory effects of surface stimulation were observed on most of the neurones tested in the vicinity of the stimulating electrode .
These
effects have been shown to correspond to hyperpolarisations of the neurones involved (4, 5) .
With repetitive stimulation, a long lasting (up to 1 min)
inhibition of the glutamate firing of some cells was evident (in 33 of the 78 cells tested) .
Ninety-one percent of these cells were also depressed by ACh
(see Fig. 1B) and the effects of both electrical stimulation and ACh were abolished or reduced by atropine, hyoscine and strychnine . cells were found in cortical layers 11, 111 and IV . were also well developed in isolated cortical slabs .
Most of these
Long duration inhibitions Two acute slabs were
Vol . 7, No . 1
INHIBITORY SYNAPSE
68
prepared by extensive undercutting of the entire area of the pericruciate cortex .
The records in Fig. 1C were obtained from one of these slabs and
illustrate a well developed long duration inhibitory effect of surface stimulation on an ACh-sensitive neurone. Discussion The results ôbtained in this survey cast further doubt on the general applicability of conclusions derived from barbiturate anaesthetised animals. Depression of the excitant actions of ACh and the inhibitory actions of noradrenaline on thalamic neurones by small doses of pentobarbitone sodium, in the absence of any marked alterations in cell excitability, has already been described (6) .
The fact that volatile anaesthetics were used in the
present study also provides an adequate explanation for the failure of previous investigators to observe comparable atropine-labile inhibitions in the cerebral cortex (7) and may help to explain many of the conflicting reports about the ability of strychnine to block cortical inhibitions (8, 9, 10) . The finding that long duration cholinergic cortical inhibitions can be evoked by stimulation of such different structures as the pyramidal tracts, mesencephalic reticular formation and lateral hypothalamus indicated that a cholinergic inhibitory interneurone might be present in the cerebral cortex . This hypothesis has been strengthened by the observation that similar inhibitions can be evoked by direct stimulation of isolated cortical slabs. The long duration of these inhibitory effects may be a result of several factors, including repetitive firing by cortical interneurones or a prolonged action of the synaptically released ACh.
Long duration hyperpolarising
inhibitions have recently been described in mammalian ganglia (11) and in certain cells of Aplysia californicans (12) .
In the latter, the long duration
inhibition is supplementary to rapidly decaying IPSPs and requires repeated stimulation for its generation .
The situation in the cerebral cortex appears
to be analogous in many respects . Summary Evidence is presented for the presence of a cholinergic inhibitory synapse
in the cerebral cortex .
The failure of previous investigators to observe
cholinergic inhibitory effects of direct stimulation of the cortex may have been due to an action of barbiturates .
Vol . 7, No . 1
INHIBITORY SYNAPSE References
1. 2.
J. W, PHILLIS and D, H . YORK, Brain Res . 5, 517 (1967) . -_ i C.O, HEBB, K, KRNJEVIC and A, SILVER, Nature, 198, 692 (1963) .
3.
J . W, PHILLIS and G . C, CHONG, Nature , 207, 1253 (1965) .
4.
K. KRNJEVIC, M, RANDIC and D. W. STRAUGHAN, J. Physiol. 184, 16 (1966) .
5.
/ / K, KRNJEVIC, M, RANDIC and D, W, STRAUGHAN, J . Physiol. 184, 49 (1966) .
6.
J . W, PHILLIS and A, K. TEBECIS, Life Sci, 6, 1621 (1967) .
7.
K, KRNJEVIC, M, RANDIC and D. W, STRAUGHAN, J, Physiol. 184, 7a
8.
(lsss) .
J . M. CRAWFORD, D, R, CURTIS, P, E, VOORHOEVE and V,J . WILSON, Nature , 200, 845 (1963) .
9.
V, B, BROOKS and H, ASANUMA, Am . J . Physiol. 208, 674 (1965) .
10 .
A4 . SAWA, N, bTARL'YAMA, S. KAJI and K, NAKAMURA, Jap. J. Physiol. 16, 126 (1966) .
11 .
B, LIBET, J, Neurophysiol . 30, 494 (1967),
12 .
J, KFIIOE, ?~'ature, 215, 1503 (1967),
69