19
J. Physiol. (1979), 291, pp. 19-28 With 4 text-figure Printed in Great Britain
THE EXCITATION AND DEPRESSION OF SPINAL NEURONES BY IBOTENIC ACID BY D. R. CURTIS, D. LODGE AND H. McLENNAN* From the Department of Pharmacology, John Curtin School of Medical Research, P.O. Box 334, Canberra City, ACT, 2601, Australia
(Received 2 October 1978) SUMMARY
1. The firing of spinal interneurones and Renshaw cells by microelectrophoretic (± )-ibotenate, which was approximately eight times more active as an excitant than L-glutamate, was followed by prolonged depression of the sensitivity of the neurones to excitant amino acids and acetylcholine. 2. The depression, which lasted for 15-30 min when ibotenate was ejected for 3-6 min, was blocked by the GABA-antagonist bicuculline methochloride, and was independent of prior firing since it occurred with subthreshold concentrations of ibotenate and when ibotenate firing had been blocked by DL-a-aminoadipate. 3. When administered electrophoretically for 5 min, muscimol, a potent GABA agonist, reduced neuronal excitability for prolonged periods and this effect was also prevented by bicuculline methochloride. 4. The depression of neuronal excitability produced by GABA, taurine, isoguvacine or 3-aminopropane sulphonate, ejected for periods of 5-6 min, recovered rapidly. 5. It is suggested that ibotenate is converted in vivo to muscimol or a related compound which has a prolonged, bicuculline-sensitive depressant action on the excitability of neurones. INTRODUCTION
Electrophoretically administered (± )-ibotenate, like many other acidic amino acids, excites mammalian central neurones (Johnston, Curtis, de Groat & Duggan, 1968; Johnston, Curtis, Davies & McCulloch, 1974; McLennan & Wheal, 1978). MacDonald & Nistri (1978) have recently reported, however, that the excitation of feline spinal interneurones is followed by reversible depression of neuronal excitability, particularly after relatively prolonged administration of ibotenate. The duration of the depression, not observed with other excitant amino acids, lasted many minutes depending on the period over which ibotenate was ejected near the neurone. The depression was characterized by a fading response to ibotenate itself, reduced sensitivity to glutamate, and a reduction in both spontaneous and orthodromic synaptic excitation. Furthermore, extracellularly recorded action potentials * Permanent address: Department of Physiology, Faculty of Medicine, The University of British Columbia, Vancouver, Canada.
0022-3751/79/3410-0761 $01.50 © 1979 The Physiological Society
D. R. CURTIS, D. LODGE AND H. McLENNAN 20 were increased in amplitude, suggesting that the neurones may have been hyperpolarized during the phase of depression. Using bicuculline, picrotoxin and strychnine MacDonald & Nistri (1978) considered that the activation of either GABA or glycine receptors was unlikely to account for the reduced cell excitability associated with excitation by ibotenate. On the other hand an interaction with excitant amino acid receptors was not excluded, and a further investigation has been made of the effects of antagonists of both excitatory and inhibitory amino acids on the actions of ibotenate on spinal neurones of the cat. METHODS
The experiments were performed on unidentified dorsal horn interneurones and Renshaw cells of cats anaesthetized with pentobarbitone sodium (35 mg kg-' i.P., supplemented as required). The spinal cord was exposed and cut at the thoraco-lumbar junction, and the L7 and SI ventral roots were sectioned distally and mounted for stimulation. Body temperature was maintained at 37-38 'C, and experiments were terminated if the systolic blood pressure fell below 100 mmHg. Substances were administered electrophoretically from seven-barrel glass micropipettes of tip diameter 5-8,sm, the central 3*6M-NaCl-containing barrels of which were used to record extracellular action potentials of single neurones. The firing rates of these cells were plotted continuously on a pen recorder. The following solutions were used in the outer barrels: acetylcholine bromide (ACh, 250 mM); y-aminobutyrate (GABA, 200 mm, pH 3); muscimol hydrobromide, isoguvacine hydrobromide (both 100 mM); 3-aminopropane sulphonate (50 mm in 165 mM-NaCl, pH 3); (±)-muscazone (approx. 50 mm, pH 7.5); (+ )-bicuculline methochloride (10 mM in 165 mM-NaCl); L-glutamate, L-homocysteate, DL-a-aminoadipate, taurine (all 200 mM, pH 7 5); D-homocysteate, (± )-ibotenate (50 mm in 165 mM-NaCl, pH 7*5); N-methyl-D-aspartate (NMDA, 10 mm in 165 mM-NaCl, pH 7.5); kainate and quisqualate (5 mm in 165 mM-NaCl, pH 7.5). All of the excitant amino acids, taurine and DL-a-aminoadipate were in solution as sodium salts. The ibotenate used in this study was that isolated from Amanita parntherina (Chilton & Ott, 1976), whereas that used earlier (Johnston et al. 1968, 1974), and by MacDonald & Nistri (1978), originated from Amanita mu8caria (Eugster, 1969). Using thin-layer chromatography in a butanol-acetic acid-water system (4:1:1, v/v), fresh solutions of ibotenate appeared not to be contaminated with muscimol or other ultraviolet- or ninhydrin-sensitive substances. Solutions of ibotenate from micropipettes used experimentally and stored in the dark for more than 2 weeks at room temperature contained small amounts of ninhydrin-sensitive contaminants. The possibility that ibotenate might interact with central GABA receptors was studied by examining its influence on the sodium independent binding of [3H]GABA (4.6 x 10-9M) by rat brain membranes (Enna & Snyder, 1975). I.v.
RESULTS
Ibotenate, ejected as an anion for periods of 30-40sec, excited spinal interneurones and Renshaw cells, and was approximately eight times more potent than L-glutamate and of similar potency to N-methyl-D-aspartate. The rate of onset and recovery of excitation was much slower, however, than that of glutamate. When repeated at intervals of 2-4min during the cycling of a series of excitant amino acids, these effects of ibotenate were very reproducible over long periods of time. In such experiments, firing by ibotenate has been found to be reduced by DL-a-aminoadipate to much the same extent as that produced by N-methyl-D-aspartate, and to be relatively insensitive to antagonism by L-glutamate diethylester (McLennan & Lodge, 1979), As described by MacDonald & Nistri (1978) the administration of ibotenate near a
IBOTENATE EXCITATION AND DEPRESSION 21 neurone for 3-6 min resulted in a gradual 'fade' in the excitatory response and a subsequent period of depressed excitability lasting up to 30 min (seventeenneurones). During this period responses to other excitatory amino acids (D- and L-homocysteate, quisqualate, NMDA and L-glutamate) were greatly reduced or abolished, although as noted by the previous authors, larger ejecting currents of an excitant could overcome the depression. The extracellularly recorded action potentials were not significantly changed in magnitude during the subsequent period of reduced excitability (see Fig. 1 C). A
B
100-
o
o
D
Ibotenate 24
I
OX-oX-o xe
x
°
80
0
~-60-~
4
40-
7
.1
X~~~0--XI,
7nA I \E
It
/x °O
/
x
20 -
u
X L7 VR o ACh14nA18sec
0
LH26nA12sec
* IBOT24fnA
/x A I
I
I
I
I
0
5
10
15
20
min B
D
C ACh
ILA
I
500
gV ]
'
i
,44 r..
.
I
I
Ir
I
I
I 10 msec
Fig. 1. Time course of the depression produced by microelectrophoretically administered ibotenate of the excitation of a Renshaw cell by ventral root L7 stimulation (@*), electrophoretic acetylcholine (ACh, 0) and L-homnocysteate (LH, x). In A is plotted the rate of firing (spikes sec-1) induced by ibotenate (IBOT, *), ejected for 5 min with a current of 24 nA and the effects of ventral root stimulation (total number of action potentials evoked), ACh and LH (maximum rate of firing attained) as percentage of the control pre-ibotenate values. The upper records of B, C and D are ratemeter tracings of the firing induced by ACh and LH, the lower records are responses induced by ventral root stimulation before, approximately 1.5 and 15 min after the ejection of ibotenate as indicated by the vertical arrows in A.
The excitation of Renshaw cells by acetylcholine was also reduced following ibotenate. With all three neurones tested both the response to electrophoretic acetylcholine and that to ventral root stimulation were reduced more or less in parallel with those to an excitant amino acid. An example of the effect of ibotenate on a Renshaw cell is illustrated in Fig. 1. Fig. 1A plots the firing induced by acetylcholine, L-homocysteate and a supramaximal ventral root stimulus, as a percentage
D. R. CURTIS, D. LODGE AND H. McLENNAN of the control observations before ibotenate, together with the slow onset, and the subsequent fade, of the firing induced by ibotenate (24 nA for 5 min, peak rate 80 spikes sec-1). Figs. 1 B, C and D illustrate rate-meter records (upper) of the firing by acetylcholine and L-homocysteate, and filmed records (lower) of the ventral root response, at the times indicated by the appropriate arrows in Fig. 1 A. 22
B
A
IBOT 75 100 80-
4
IBOT 50
X-x
xlX-
J. Physiol. (1979), 291, pp. 19-28 With 4 text-figure Printed in Great Britain
THE EXCITATION AND DEPRESSION OF SPINAL NEURONES BY IBOTENIC ACID BY D. R. CURTIS, D. LODGE AND H. McLENNAN* From the Department of Pharmacology, John Curtin School of Medical Research, P.O. Box 334, Canberra City, ACT, 2601, Australia
(Received 2 October 1978) SUMMARY
1. The firing of spinal interneurones and Renshaw cells by microelectrophoretic (± )-ibotenate, which was approximately eight times more active as an excitant than L-glutamate, was followed by prolonged depression of the sensitivity of the neurones to excitant amino acids and acetylcholine. 2. The depression, which lasted for 15-30 min when ibotenate was ejected for 3-6 min, was blocked by the GABA-antagonist bicuculline methochloride, and was independent of prior firing since it occurred with subthreshold concentrations of ibotenate and when ibotenate firing had been blocked by DL-a-aminoadipate. 3. When administered electrophoretically for 5 min, muscimol, a potent GABA agonist, reduced neuronal excitability for prolonged periods and this effect was also prevented by bicuculline methochloride. 4. The depression of neuronal excitability produced by GABA, taurine, isoguvacine or 3-aminopropane sulphonate, ejected for periods of 5-6 min, recovered rapidly. 5. It is suggested that ibotenate is converted in vivo to muscimol or a related compound which has a prolonged, bicuculline-sensitive depressant action on the excitability of neurones. INTRODUCTION
Electrophoretically administered (± )-ibotenate, like many other acidic amino acids, excites mammalian central neurones (Johnston, Curtis, de Groat & Duggan, 1968; Johnston, Curtis, Davies & McCulloch, 1974; McLennan & Wheal, 1978). MacDonald & Nistri (1978) have recently reported, however, that the excitation of feline spinal interneurones is followed by reversible depression of neuronal excitability, particularly after relatively prolonged administration of ibotenate. The duration of the depression, not observed with other excitant amino acids, lasted many minutes depending on the period over which ibotenate was ejected near the neurone. The depression was characterized by a fading response to ibotenate itself, reduced sensitivity to glutamate, and a reduction in both spontaneous and orthodromic synaptic excitation. Furthermore, extracellularly recorded action potentials * Permanent address: Department of Physiology, Faculty of Medicine, The University of British Columbia, Vancouver, Canada.
0022-3751/79/3410-0761 $01.50 © 1979 The Physiological Society
D. R. CURTIS, D. LODGE AND H. McLENNAN 20 were increased in amplitude, suggesting that the neurones may have been hyperpolarized during the phase of depression. Using bicuculline, picrotoxin and strychnine MacDonald & Nistri (1978) considered that the activation of either GABA or glycine receptors was unlikely to account for the reduced cell excitability associated with excitation by ibotenate. On the other hand an interaction with excitant amino acid receptors was not excluded, and a further investigation has been made of the effects of antagonists of both excitatory and inhibitory amino acids on the actions of ibotenate on spinal neurones of the cat. METHODS
The experiments were performed on unidentified dorsal horn interneurones and Renshaw cells of cats anaesthetized with pentobarbitone sodium (35 mg kg-' i.P., supplemented as required). The spinal cord was exposed and cut at the thoraco-lumbar junction, and the L7 and SI ventral roots were sectioned distally and mounted for stimulation. Body temperature was maintained at 37-38 'C, and experiments were terminated if the systolic blood pressure fell below 100 mmHg. Substances were administered electrophoretically from seven-barrel glass micropipettes of tip diameter 5-8,sm, the central 3*6M-NaCl-containing barrels of which were used to record extracellular action potentials of single neurones. The firing rates of these cells were plotted continuously on a pen recorder. The following solutions were used in the outer barrels: acetylcholine bromide (ACh, 250 mM); y-aminobutyrate (GABA, 200 mm, pH 3); muscimol hydrobromide, isoguvacine hydrobromide (both 100 mM); 3-aminopropane sulphonate (50 mm in 165 mM-NaCl, pH 3); (±)-muscazone (approx. 50 mm, pH 7.5); (+ )-bicuculline methochloride (10 mM in 165 mM-NaCl); L-glutamate, L-homocysteate, DL-a-aminoadipate, taurine (all 200 mM, pH 7 5); D-homocysteate, (± )-ibotenate (50 mm in 165 mM-NaCl, pH 7*5); N-methyl-D-aspartate (NMDA, 10 mm in 165 mM-NaCl, pH 7.5); kainate and quisqualate (5 mm in 165 mM-NaCl, pH 7.5). All of the excitant amino acids, taurine and DL-a-aminoadipate were in solution as sodium salts. The ibotenate used in this study was that isolated from Amanita parntherina (Chilton & Ott, 1976), whereas that used earlier (Johnston et al. 1968, 1974), and by MacDonald & Nistri (1978), originated from Amanita mu8caria (Eugster, 1969). Using thin-layer chromatography in a butanol-acetic acid-water system (4:1:1, v/v), fresh solutions of ibotenate appeared not to be contaminated with muscimol or other ultraviolet- or ninhydrin-sensitive substances. Solutions of ibotenate from micropipettes used experimentally and stored in the dark for more than 2 weeks at room temperature contained small amounts of ninhydrin-sensitive contaminants. The possibility that ibotenate might interact with central GABA receptors was studied by examining its influence on the sodium independent binding of [3H]GABA (4.6 x 10-9M) by rat brain membranes (Enna & Snyder, 1975). I.v.
RESULTS
Ibotenate, ejected as an anion for periods of 30-40sec, excited spinal interneurones and Renshaw cells, and was approximately eight times more potent than L-glutamate and of similar potency to N-methyl-D-aspartate. The rate of onset and recovery of excitation was much slower, however, than that of glutamate. When repeated at intervals of 2-4min during the cycling of a series of excitant amino acids, these effects of ibotenate were very reproducible over long periods of time. In such experiments, firing by ibotenate has been found to be reduced by DL-a-aminoadipate to much the same extent as that produced by N-methyl-D-aspartate, and to be relatively insensitive to antagonism by L-glutamate diethylester (McLennan & Lodge, 1979), As described by MacDonald & Nistri (1978) the administration of ibotenate near a
IBOTENATE EXCITATION AND DEPRESSION 21 neurone for 3-6 min resulted in a gradual 'fade' in the excitatory response and a subsequent period of depressed excitability lasting up to 30 min (seventeenneurones). During this period responses to other excitatory amino acids (D- and L-homocysteate, quisqualate, NMDA and L-glutamate) were greatly reduced or abolished, although as noted by the previous authors, larger ejecting currents of an excitant could overcome the depression. The extracellularly recorded action potentials were not significantly changed in magnitude during the subsequent period of reduced excitability (see Fig. 1 C). A
B
100-
o
o
D
Ibotenate 24
I
OX-oX-o xe
x
°
80
0
~-60-~
4
40-
7
.1
X~~~0--XI,
7nA I \E
It
/x °O
/
x
20 -
u
X L7 VR o ACh14nA18sec
0
LH26nA12sec
* IBOT24fnA
/x A I
I
I
I
I
0
5
10
15
20
min B
D
C ACh
ILA
I
500
gV ]
'
i
,44 r..
.
I
I
Ir
I
I
I 10 msec
Fig. 1. Time course of the depression produced by microelectrophoretically administered ibotenate of the excitation of a Renshaw cell by ventral root L7 stimulation (@*), electrophoretic acetylcholine (ACh, 0) and L-homnocysteate (LH, x). In A is plotted the rate of firing (spikes sec-1) induced by ibotenate (IBOT, *), ejected for 5 min with a current of 24 nA and the effects of ventral root stimulation (total number of action potentials evoked), ACh and LH (maximum rate of firing attained) as percentage of the control pre-ibotenate values. The upper records of B, C and D are ratemeter tracings of the firing induced by ACh and LH, the lower records are responses induced by ventral root stimulation before, approximately 1.5 and 15 min after the ejection of ibotenate as indicated by the vertical arrows in A.
The excitation of Renshaw cells by acetylcholine was also reduced following ibotenate. With all three neurones tested both the response to electrophoretic acetylcholine and that to ventral root stimulation were reduced more or less in parallel with those to an excitant amino acid. An example of the effect of ibotenate on a Renshaw cell is illustrated in Fig. 1. Fig. 1A plots the firing induced by acetylcholine, L-homocysteate and a supramaximal ventral root stimulus, as a percentage
D. R. CURTIS, D. LODGE AND H. McLENNAN of the control observations before ibotenate, together with the slow onset, and the subsequent fade, of the firing induced by ibotenate (24 nA for 5 min, peak rate 80 spikes sec-1). Figs. 1 B, C and D illustrate rate-meter records (upper) of the firing by acetylcholine and L-homocysteate, and filmed records (lower) of the ventral root response, at the times indicated by the appropriate arrows in Fig. 1 A. 22
B
A
IBOT 75 100 80-
4
IBOT 50
X-x
xlX-
