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Influence of benzodiazepines on GABA-evoked responses of amphibian brain and spinal neurones in vitro
Neuropharmacology
Vol. 23, No. 7B. pp. 851-852, Printed in Great Britain
1984
00X-3908,84 S3.00 + 0.00 Pergamon Press Ltd
1NFLUENCEOF BENZODIAZEPINES ON GABA-EVOKEDRESPONSES OF AMPHIBIAN BRAIN AND SPINAL NEURONESIN VITRO
A. Nistri Department
of
Pharmacology,
St.
and C. Berti
Bartholomew’s
Hospital
Medical
College,
London
EClM 6BQ
U.K.
Summary Responses to bath-applications of GABA were electrophysiologically recorded from in vitro spinal and brain preparations of the frog. Nanomolar concentrations of the novel benzodiazepine midarolam enhanced the amplitude of GABA effects on dorsal afferent fibres in the spinal cord and on tectal neurones in the brain. These results suggest that benrodiazepines are powerful modulators of GABA actions at different sites in the amphibian central nervous system. It is well established that the GABA receptor complex found in the vertebrate central nervous system frequently includes a benzodiazepine binding sub-unit (Olsen, 1982). Electrophysiological studies have shown that benzodiazepines enhance a variety of GABA-mediated postsynaptic responses (Haefely, 1983) although this effect has often been noted using relatively high concentrations of benzodiarepines. The purpose of the present study was to examine the effectiveness of a novel and watersoluble benzodiazepine, midarolam, on GABA-evoked responses. In particular, it was deemed useful to compare the influence of midazolam on GABA responses from spinal neurones with those from brain neurones. It is in fact conceivable that GABA receptors might differ in their sensitivity to benzodiazeprnes depending on the area of the central nervous system studied. In order to avoid possible interferences by anaesthetics and blood pressure changes we decided to investigate electrophysiologically GABA-evoked responses of in vitro neurones. Hence we used an in vitro slice 1983) and in parallel studies an preparation of the frog spinal cord (Nistri and Berti, in vitro brain preparation of the same animal species. With spinal slices GABA responses were recorded from afferent fibres which appear to possess GABA-operated synapses on their terminals (Nistri, 1983). With in vitro brain preparations GABA responses were recorded from tectal neurones following optic nerve stimulation. Experimental techniques Froos (Rana temooraria or esculenta) were decerebrated orior to dissectino out the soinal cord or anaesthetized with tricaine (0.1%) for removal of the brain. Longitudinal sbinal cord slices or whole brain preparations were bathed in oxygenated Ringer solution at 4OC and all drugs were applied via the bathing solution (Nistri and Berti, 1983). With spinal slices recordings were carried out via a miniature calomel electrode placed close to the entry of a lumbar dorsal root into the cord while electrical stimuli were applied to adjacent dorsal or ventral roots. With brain preparations a 3M NaCl-filled glass microelectrode was superficially placed in the optic tectum while the contralateral optic nerve was electrically stimulated. 2
Results and Discussion On soinal slices GABA elicited dose-deoendent and reversible deoolarizations recorded from afferent fibres. These responses had a rapid onset and displayed fading following sustained tissue exposure to GABA. By plotting GABA dose depolarization curves, it was log possible to calculate an apparent ED50 of 0.85 mM for the amino acid and a limiting Constructing GABA dose depolarization concentration - log response slope close to one. curves in the presence of 1 mM nipecotic acid (a GABA uptake blocker) gave identical data. Midazolam, in a concentration range of 0.5 - 25 nM, increased the amplitude of GABA responses (without affecting their fading) and shifted GABA dose response curves to the left with no change in their maximum. The full enhancement by midazolam consisted of halving value for GABA. The effect of midazolam was prevented or reversed by 1 - 25 nM Ro the 14ED?B37, a benzodiazepine receptor antagonist. As midazolam did not enhance spinal cord a specific potentiation responses to glycine, glutamate or 12 mM K+, these data indicate by midazolam of GABA response amplitude. In the optic tectum of the frog brain 1 mM GABA produced a transient enhancement (Fig. lb) in the superficially-recorded negative field potentials (see GrUsser and GrUsser-Cornehls, Sustained application of GABA trans1976) evoked by controlateral optic nerve stimuli. formed this enhancement into a large and reversible depression of these potentials (Fig. lc). Midazolam This biphasic action of GABA was not apparently altered by 1 mM nipecotic acid. (lo-25 nM) per se induced a small increase in the postsynaptic potential waves. When GABA the early enhancement by GABA of the field potenwas tested in the presence of midazolam, tials was increased but particularly striking was the delay in the onset of the GABA851
852
(b)
c
Y
II
O.ZmV
20 ms Fig. 1. Effect of GABA (I mMI on i'ield potentrals recorded from the frog optic tectunl m w. Responses were elrcited by electrical stimulation of the contralateral optic nerve (cl.2 Hz; 1 ms; supramaxrmal voltage). a: several superimposed oscilloscope tracinqs show in control Ringer solution three components which follow 0 (stimulus artefactl, namely t.he presynaptic volley (A) and the two postsynaptic waves (R and f'). h: rn the presence oi GABA (30s application 0 enhai~cemer~t of- pastsynaptic waves. c: sustained (90s) applicatiorl of GABA producing depression of i3 and C waves. induced
depression.
Our data suggest that midarolam potently augments postsynaptic responses to GABA irr the brain and spinal cord OF the frog. Since only one component of the biphasic GABA action on tectal neurones was enhanced by midazolam, the tectum might perhaps represent an interestlng system to inuestlqate the nature and extent of physrolaqical coupling between GARA arltl benzodiarepine receptors. Acknoaled~ments. Midazolam and Ro 14-7437 were donated by Hoffman-la --. a fellow of the European Molecular Ricloqy Orqaniration (EMBO\I.
Roche
Wr Co.
(‘.I!!. tiles
REFEREWES Griisser, 0.J. and GrUsser-Cornchls,, il. (197h), The neurnphysio~ogy of the in: Frog Neurobiology, !Ll~nas. R. and Precht, ld. Eds.1, pp. 335-350. Haefely, LJ. (lYW>, IS: 19-3Y. Nistri, A. (19H31, in : Handbook New York.
The
biological
Sprnal of the
basis
of benrodrazepine
actions,
tectum
Springer.
J. Psychoactive
cord pharmacology of GABA and chemically-related Spinal Co_rg, (Davidoff, R.A. Cd.), Vnl. 1. pp.
ammo 45-109.
Nistri, A. and tlerti, C. (198i!, Cafferne-ilndured potentiation of GABA effects spinal cord: an electrophysioloqicai study, Brain Res., 258: 261-270. ___R.lnJ. (1782), Drug interactions Pharmac. loxicol. 22: 245-277. --
OlSWl,
at
the
GARA
receptor-ionnphore
complex,
optlcum,
Berlin Drug:;,
acids, Dekker,
on
Ann.
irnq
Rev.
Vol. 23, No. 7B. pp. 851-852, Printed in Great Britain
1984
00X-3908,84 S3.00 + 0.00 Pergamon Press Ltd
1NFLUENCEOF BENZODIAZEPINES ON GABA-EVOKEDRESPONSES OF AMPHIBIAN BRAIN AND SPINAL NEURONESIN VITRO
A. Nistri Department
of
Pharmacology,
St.
and C. Berti
Bartholomew’s
Hospital
Medical
College,
London
EClM 6BQ
U.K.
Summary Responses to bath-applications of GABA were electrophysiologically recorded from in vitro spinal and brain preparations of the frog. Nanomolar concentrations of the novel benzodiazepine midarolam enhanced the amplitude of GABA effects on dorsal afferent fibres in the spinal cord and on tectal neurones in the brain. These results suggest that benrodiazepines are powerful modulators of GABA actions at different sites in the amphibian central nervous system. It is well established that the GABA receptor complex found in the vertebrate central nervous system frequently includes a benzodiazepine binding sub-unit (Olsen, 1982). Electrophysiological studies have shown that benzodiazepines enhance a variety of GABA-mediated postsynaptic responses (Haefely, 1983) although this effect has often been noted using relatively high concentrations of benzodiarepines. The purpose of the present study was to examine the effectiveness of a novel and watersoluble benzodiazepine, midarolam, on GABA-evoked responses. In particular, it was deemed useful to compare the influence of midazolam on GABA responses from spinal neurones with those from brain neurones. It is in fact conceivable that GABA receptors might differ in their sensitivity to benzodiazeprnes depending on the area of the central nervous system studied. In order to avoid possible interferences by anaesthetics and blood pressure changes we decided to investigate electrophysiologically GABA-evoked responses of in vitro neurones. Hence we used an in vitro slice 1983) and in parallel studies an preparation of the frog spinal cord (Nistri and Berti, in vitro brain preparation of the same animal species. With spinal slices GABA responses were recorded from afferent fibres which appear to possess GABA-operated synapses on their terminals (Nistri, 1983). With in vitro brain preparations GABA responses were recorded from tectal neurones following optic nerve stimulation. Experimental techniques Froos (Rana temooraria or esculenta) were decerebrated orior to dissectino out the soinal cord or anaesthetized with tricaine (0.1%) for removal of the brain. Longitudinal sbinal cord slices or whole brain preparations were bathed in oxygenated Ringer solution at 4OC and all drugs were applied via the bathing solution (Nistri and Berti, 1983). With spinal slices recordings were carried out via a miniature calomel electrode placed close to the entry of a lumbar dorsal root into the cord while electrical stimuli were applied to adjacent dorsal or ventral roots. With brain preparations a 3M NaCl-filled glass microelectrode was superficially placed in the optic tectum while the contralateral optic nerve was electrically stimulated. 2
Results and Discussion On soinal slices GABA elicited dose-deoendent and reversible deoolarizations recorded from afferent fibres. These responses had a rapid onset and displayed fading following sustained tissue exposure to GABA. By plotting GABA dose depolarization curves, it was log possible to calculate an apparent ED50 of 0.85 mM for the amino acid and a limiting Constructing GABA dose depolarization concentration - log response slope close to one. curves in the presence of 1 mM nipecotic acid (a GABA uptake blocker) gave identical data. Midazolam, in a concentration range of 0.5 - 25 nM, increased the amplitude of GABA responses (without affecting their fading) and shifted GABA dose response curves to the left with no change in their maximum. The full enhancement by midazolam consisted of halving value for GABA. The effect of midazolam was prevented or reversed by 1 - 25 nM Ro the 14ED?B37, a benzodiazepine receptor antagonist. As midazolam did not enhance spinal cord a specific potentiation responses to glycine, glutamate or 12 mM K+, these data indicate by midazolam of GABA response amplitude. In the optic tectum of the frog brain 1 mM GABA produced a transient enhancement (Fig. lb) in the superficially-recorded negative field potentials (see GrUsser and GrUsser-Cornehls, Sustained application of GABA trans1976) evoked by controlateral optic nerve stimuli. formed this enhancement into a large and reversible depression of these potentials (Fig. lc). Midazolam This biphasic action of GABA was not apparently altered by 1 mM nipecotic acid. (lo-25 nM) per se induced a small increase in the postsynaptic potential waves. When GABA the early enhancement by GABA of the field potenwas tested in the presence of midazolam, tials was increased but particularly striking was the delay in the onset of the GABA851
852
(b)
c
Y
II
O.ZmV
20 ms Fig. 1. Effect of GABA (I mMI on i'ield potentrals recorded from the frog optic tectunl m w. Responses were elrcited by electrical stimulation of the contralateral optic nerve (cl.2 Hz; 1 ms; supramaxrmal voltage). a: several superimposed oscilloscope tracinqs show in control Ringer solution three components which follow 0 (stimulus artefactl, namely t.he presynaptic volley (A) and the two postsynaptic waves (R and f'). h: rn the presence oi GABA (30s application 0 enhai~cemer~t of- pastsynaptic waves. c: sustained (90s) applicatiorl of GABA producing depression of i3 and C waves. induced
depression.
Our data suggest that midarolam potently augments postsynaptic responses to GABA irr the brain and spinal cord OF the frog. Since only one component of the biphasic GABA action on tectal neurones was enhanced by midazolam, the tectum might perhaps represent an interestlng system to inuestlqate the nature and extent of physrolaqical coupling between GARA arltl benzodiarepine receptors. Acknoaled~ments. Midazolam and Ro 14-7437 were donated by Hoffman-la --. a fellow of the European Molecular Ricloqy Orqaniration (EMBO\I.
Roche
Wr Co.
(‘.I!!. tiles
REFEREWES Griisser, 0.J. and GrUsser-Cornchls,, il. (197h), The neurnphysio~ogy of the in: Frog Neurobiology, !Ll~nas. R. and Precht, ld. Eds.1, pp. 335-350. Haefely, LJ. (lYW>, IS: 19-3Y. Nistri, A. (19H31, in : Handbook New York.
The
biological
Sprnal of the
basis
of benrodrazepine
actions,
tectum
Springer.
J. Psychoactive
cord pharmacology of GABA and chemically-related Spinal Co_rg, (Davidoff, R.A. Cd.), Vnl. 1. pp.
ammo 45-109.
Nistri, A. and tlerti, C. (198i!, Cafferne-ilndured potentiation of GABA effects spinal cord: an electrophysioloqicai study, Brain Res., 258: 261-270. ___R.lnJ. (1782), Drug interactions Pharmac. loxicol. 22: 245-277. --
OlSWl,
at
the
GARA
receptor-ionnphore
complex,
optlcum,
Berlin Drug:;,
acids, Dekker,
on
Ann.
irnq
Rev.