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Impaired hyperpolarising inhibition during insulin hypoglycaemia and fluoroacetate poisoning
164
Brai,7 Research, 69 (1974) 164-169
(~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
Impaired hyperpolarising inhibition during insulin hypoglycaemia and fluoroacetate poisoning CLEMENS LORACHER AND H. D. LUX Max-Planck-hzstitute.lbr P.s3'chiatr3", 8000 Munk'h 40 ( G.tE:R.)
(Accepted November 30th, 1973)
Convulsive activity elicited by insulin-induced hypoglycaemia (IIH) or fluorofatty acid (FFA) intoxication is characterlsed by profound shifts of cerebral metabolite concentrations in the preconvulsive state la. While IIH lowers glycogen, glucose, lactate, glutamine and glutamate levels, it increases strikingly the cerebral ammonia concentration from values of 0.25 ,umole/g control to 2.5 ftmole/g le,la, lnhibitors of the citric acid cycle, such as FFA, raise glucose and glycogen levels as well as cerebral anamonia (up to 1.5 ¢~nlole/g) in the preconvulsive state x,la. At the beginning of convulsive activity, normal concentrations of creatine phosphate (CP) and ATP are observed simultaneously with an increased turnover of energy-rich phosphates. Therefore. it appears questionable whether the lack of energyrich compounds caused by glucose deficiency or by the action of FFA is the general causative factor for the initiation of seizures s. The elevated ammonia levels merit consideration since convulsive activity is induced by' a rise in cerebral NHa to colnparable levels by infusion of ammonium salts
SHORT COMMUNICATIONS
] 65
discontinued after an initial pentobarbital dose. In these experiments recordings were begun more than 3 h after termination of the anaesthesia. Animals used for the experiments with insul in were deprived of dry food for e ne day. Insulin in high dosage (60-100 I.U./kg of body weight) as well as Na-fluoroacetate (0.1 ~ solution; l mg/kg of body weight) was applied intravenously by means of an infusion p u m p with a rate of 0.2q3.8 ml/min. To terminate hypoglycaemia. 0.5-2.0 g isotonic glucose solution was also applied intravenously at a rate of 1.0 ml/min. Before each infusion, a 15 min control period was introduced for the determination of membrane potentials, in order to exclude artifacts resulting from possible cell damage. Determination of blood glucose levels was achieved semlquantitatively by the dextrostix method (determination of blood glucose by means of glucose oxidase impregnated indicator paper. M E R C K AG, Darmstadt G F R , No. 447116), as other methods requiring more extensive manipulations resulted regularly in loss of the observed cells. Recording devices were used as described earlier 6. IIH and glucose application. Eighteen motoneurones were studied. In 6 cells the complete time course of the effects of the hypoglycaemia and of its removal by glucose infusion on the motoneuronal membrane potential could be followed. In the remaining experiments the cells under observation were lost at different times after starting the infusions and recording was continued with a second motoneurone. Within 5-30 min after insulin infusion, a resting membrane depolarisation in the range between 3-10 mV appeared in all motoneurones. Fourteen of 18 motoneurones spontaneously approached control resting membrane potentials within 30-60 rain after the initial depolarisation. Neurones with higher (5 mV) initial depolarising shifts in resting potential showed transient reductions of 5 - t 0 mV in the amplitude of the action potentials also with a time course of 30-60 min. Changes in the amplitude or rune
mV 35] 25
.
:
=
=
=
=
~
_
=
=
=
:
:
-
-
-
-
-
o
AP
J
A
0 II
40
80
120 r r m
N a - ~ e lmg/kg Bw
mv
:
~
-
:
oAP
25 J
B
0 Insut~ 6 O I . U / k g B w
40
80
120
rain
Glucose l g
Fig. 1. Time course of changes in potentials of two biceps-semitendinosus motoneurons. A: after infusion of Na-fluoroacetate. B: after intravenous insulin application mad termination of IlH by intravenous glucose
166
SHORT COMMUNICATIONS
constants o f EPSPs produced by subthreshold afferent muscle nerve stimulation were not observed. If hypoglycaemia was maintained longer than about 1.5 h from the onset of insulin infusion, a gradual depolarisation o f the resting membrane potential developed concomitantly with a gradual rise in the threshold o f all afferent nerves to elicit EPSPs as well as 1PSPs. This was followed by increase in the threshold o f a n t i dromic stimulation until the cell's axon became completely inexcitable. Twelve to 15 rain after insulin application a reduction o f the IPSP and o f its reversal potential were detected. With the exception of one animal in which blood glucose level did not decline below 40 m g % within 2 h (in this animal, during recording from a single motoneurone for 2 h, neither changes o f RP, AP, EPSPs, IPSPs nor o f thresholds for ortho- and antidromic nerve stimulation were obvious), the IPSPs as well as E~psp.~ decreased about 40-70 min (m ::~ 62 rain) after insulin application, the latter coming very close to the resting membrane potentials. This was valid for both direct and polysynaptic I PSPs. No obvious changes in membrane time constants and in cell input conditions were observed during the infusion periods and thereafter. However, when IPSPamplitudes and rise times were plotted against the membrane potential (in approximate steady state) during varied current steps, it was apparent that the IPSP/membrane potential slopes became considerably reduced during long lasting ( - 80 rain) hypoglycaemia. Also, EPSPs became much smaller. This effect proceeded continuously until virtual disappearance o f inhibitory actions. The reduced dependence o f IPSP amplitudes on membrane potential was observed at considerable times after the Ell,s~, had shifted towards the resting membrane potential, i.e. at times always longer than I h after insulin infusion (m 98 min). After glucose application o f 500 mg/kg the amplitude o f IPSPs and the E~l,sl' returned constantly and quite rapidly to control levels with half times o f recovery between 7-10 rain. Exceptions were only seen in 3 deteriorated cells with low resting
a ..~.___
b
c
j
20 msec
,-'~
' ,,:--'k
.../I'
mV
r~---~.~
~
~--..~I 20 msec
C
20]
-'<--:--'e,~
~
L
- : " - - ~ ..... ~ ~ . :
'"--'--.... -" "
20 msec
Fig. 2. Series of IPSPs at different nlembrane potentials, which were varied by current steps. Dots indicate E rpsi,. A;~:control; A~,: 70 min after insu fin application ; A,. : 15 rain after termination of I I H by intravenous glucose solution. Ba: control; Br,: 60 min after insulin application; B,: 110 rain after insulin application. C,~: control; Ct,: 90 rain after fluoroacetate application; C¢,: 105 rain after fluoroacetate application.
SHORT COMMUNICATIONS
167
membrane potentials (50 mV) in which the equilibration of the EII,Sp with the resting potential lasted for more than 20 min. Within 1.5 h after glucose application no return of En~sP to control levels could be observed in these cells. Neither RPs, APs nor EPSPs showed consistent changes after glucose application. Fluctuations of 2-4 mV of the resting potential were frequently seen during infusion and within about 20 min thereafter. The potential instabilities during infusion are probably of artifactual nature and due to blood pressure variations. Na-fluoroacetate. Measurements were made on 15 motoneurones, of which ~ cells were observed up to 120 rain. In the remaining experiments the cells under observation were lost at different times after starting the infusion and recording was continued with a second motoneurone. The alterations of neuronal potentials produced by fluoroacetate are irreversible within observation times of up to 5 h. All motoneurones showed resting membrane depolarisations in the range o | 2-8 mV, which developed slowly within 10-30 min after infusion. Five motoneurones spontaneously regained almost control resting membrane potential 35-45 min after infusion. Action potentials always showed comparable changes in amplitudes, A depolarising shift of the Elesl~ towards the resting membrane potential became obvious after 60-110 min (m -- 87 rain) from starting the infusions, The difference between EiPsl, and the resting membrane potential was reduced to less than 10 ~o of the controls. Within 2 h loss of hyperpolarising inhibition was almost complete in all cells. ElesV being now very near the resting potential. The effectiveness of excitatory synaptic transmission initially appeared unimpaired. Monosynaptic EPSPs elicited by threshold stimulation of IA muscle afferents usually showed no changes within an observation period of 100-120 rain after infusion. However. intoxication for longer periods was found to elevate the stimulus threshold for all excitatory as well as inhibitory actions. Some time after ElesP had shifted towards resting membrane potential. i.e. at times always longer than 105 min after fluoroacetate application, a reduced dependency of IPSP amplitudes on membrane potential was observed (m ~ 120 mini. This was similar to the results during long lasting hypoglycaemia. Since the cell input conductance as well as the resting membrane time constant remained unchanged throughout the observation period, these effects cannot be attributed to changes in the electric properties o f the postsynaptic nonjunctional membrane. The possibility o f impaired presynaptic conduction or impaired transmitter release cannot be ruled out. The changes in resti ng potential, action potential and EPSP seen during FFA intoxication and IIH were somewhat variable, but only minimal in those cells in which greater movement artifacts could be excluded. Furthermore, the cells recovered from these changes at times at which the effects on the IPSP were just developing. It appears then, that the initial changes in resting membrane potential are not significant for the central nervous disturbances which are induced by IIH or F F A intoxication, With increasing intoxication time a depolarising shift of the ElrsP occurred bringing it toward but not positive to the prevailing resting potential. This results in a decreased efficiency of inhibitory synaptic transmission. The equilibration of the EiPsP with the resting potential indicates that in IlH and FFA intoxication the electro-
168
SHORT COMMUNICATIONS ~|00 10
A
-80 "~
-60
"'..
0
-I00
mV MP •
-I0
-100
0 ~'-o
mV IPSP
-80
"',
-60 rnV MP
10 4
i
or
-80
-I0
mV IPSP
"~-6]
EIpsp
mV -2
• \,
"'\,
",. % " \,
=.,. ~o,, "\
"
~/ ~ ""%'~""'" ,f
°~.a ........ %'o .~.x.a~.~¢jol 1
- I 0 ] mV IPSP
mV MP \
t'2
"'.. "'4.'<,-<
-60
lO - " ' ' . < ' ~ ' ~ _ .
°.i °~
D
/" . . / " " -10 ] <2-~2.~ ~' 0
.... . . . . 40
80
120 rain
Fig. 3. A-C show size and sign of the IPSPs plotted against membrane potential, Arrows indicate resting membrane potential. Points of intersection of curves with zero line indicate reversal potentials (Elvs~,) of IPSPs. In B and C, change in inclination of curves indicates change in conductance of IPSPs. A: before (0), 70 rain ( ~ ) after insulin application and 25 min ( ) after termination of IIH by glucose solution. B: before (O) 60 min ( ~ ) 100 min (~) and 120 min ( : ) after insulin application. C: before (O) 90 min ( ~ ) 110 min (Lq) and 130 min ( : ) after fluoroacetate application. D: mean En,sp values of 5 motoneurons after insulin application (- ,: ) and of 7 motoneurons after fluoroacetate application (- • -). Dotted line (- - , - -) indicates changes of mean Eu,se values of 3 cells after termination of IlH by glucose application, whereas line with squares ( ~ ) mean En,sP values of two motoneurons in which IlH was continued. Ewsp values are plotted from resting membrane potentials (RP). Infusions started at 0 and were terminated at the times indicated by arrows (~ for insulin, ~ for fluoroacetate). Dotted arrows (:, ~') indicate glucose infusion for termination of IIH.
chemical gradient of the ion species (chloride) carrying the inhibitory postsynaptic current is determined by the resting m e m b r a n e potential. A similar reduction of the electromotive force o f the IPSP has been observed with a m m o n i u m salt intoxicationS, 6. Since cerebral a m m o n i a levels in IIH and F F A intoxications are comparable to those in a m m o n i a salt intoxication la it is likely that the depolarising shift o f the EIPSP with IIH and F F A intoxication results from increased cerebral a m m o n i a levels. This likelihood receives further support from the similarity in the time course of recovery of the iPSP (20 min) after application of glucose in the case o f 11H, with the time course with which a m m o n i a is eliminated after application of glucose (15 rain) in the case of increased cerebral NHa levels due to IIH l'-',la. It is noteworthy that convulsions are reported to occur within I-2 I1 after infusion o f F F A 1,l:3, which appears to agree well with the time course of the observed changes of the EIPSP. In the case of long lasting l l H and of F F A intoxication, there may be a correlation between the reduction of synaptic excitatory as well as inhibitory efficacy and the decreasing cerebral A T P and CP levels s. The i m p o r t a n c e o f diminished inhibitory conductance as a causative factor for convulsive activity in i l H and ftuoroacetate poisoning is deduced as: (a) changes o f inhibitory as well as excitatory synaptic transmission appear considerable times (IlH m = 98 rain, fluoroacetate m = 120 min) after EIPSP has shifted towards resting m e m b r a n e potential (IlH m = 62 min, fluoroacetate m = 87 rain); (b) these potential
SHORT COMMUNICATIONS
I 69
reductions affect inhibitory and excitatory synaptic transmission to an equal extent. Studies on the interactions of EPSPs and IPSPs indicate that almost linear summation of potentials is as frequent as a partial shunt of the EPSP by the conductance increase during the IPSP 3,~°. From this it is obvious that the loss of hyperpolarising postsynaptic inhibition, due to changes in the electrochemical gradient of chloride ions during IIH and fluoroacetate poisoning should result in a considerable decrease of inhibitory potency. Similar changes after ammonia intoxication have also been observed in neocortical nerve cells 9. if the results apply in general to CNS neurone~, ammonium salts as well as IIH and I-FA intoxication may distort the balance ol excitatory drive by postsynaptic inhibition. It is already known from poisons which directly interfere with inhibitory synaptic transmission (strychnine, picrotoxin, bicuculline), that this imbalance can lead to convulsive activity2, El. Decreased inhibitor~ action due to a loss of hyperpolarising e.m.f, could similarly be an essential factor for other metabolically induced convulsive activities. We thank Professor Roger Eckert tot reading the manuscipt.
1 BENITEZ, D., PSCHEII)T, G. R., AND STONE, W. E., Formation of ammonia ion in the cerebrum in fluoracetate poisoning, Amer. J. Physiol., 176 (1954) 488-492. 2 CURTIS, D. R., DUGGAN, A. W., FELIX, D., AND JOHNSI-ON, G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222--1224. 3 CURTIS, D. R., AND ECt:LLS, J. C., The time course of excitatory and inhibitory synaptic actions. J. Physiol. (Lond.), 145 (1959) 529-546. 4 GASTAUT, H., SAIEI~,J., MAN(), T., SANTOS, D., ,~,',:t) LVAc;OUl~l,S., Generalized epileptic seizures induced by nonconvulsant substances: Experimental study with special reference to ammonium chloride, Epilepsia (ArosE.), 9 (1968) 317.-327. 5 Lt~x, H. D., Ammonium and chloride extrusion: Hyperpolarizing synaptic inhibition in spinal motoneurons, Science, 173 (1971) 555 557. 6 l,ux, H. D., LORACHtTr,C., ANt) NEH~R, E., The action of ammonium on postsynaptic inhibition of cat spinal motoneurons, Exp. Brain Res., 11 (1970)431-447. 7 Lux, H. D., AND SCIlURr:RT, P., Postsynaptic inhibition: Intracellular effects of various ions iil spinal motoneurons, Science, 166 (1969) 625-626. 8 PASSONNEAU,J. V., Discussion: Energy metabolites in experimental seizures. In H. H. JASt'~R (Ed.), Basic Mechanisms of the Epilepsies, Little, Brown, Boston, Mass., 1969, pp. 98-103. 9 RAABI:,W., Lt:x, H. D., GUMMIT, R. J., AND AVAI n, G. F., Disinhibition in cat motor cortex by ammonia, Fed. Proc., 32 (1973) 419 (Abs.). 10 HALt., W., BURKE, R. E., SMITH, T. G., NH_SON. P. G., AND FRANK, K., Dendritic location of synapses and possible mechanisms for the monosynaptic EPSP in motoneurons, J. Neurophysiol., 30 (1967) 1169 1193. 11 ROPER, S., AND DIAMOND, J., Does strychnine block inhibition postsynaptically? Nature (Lond.). 223 (1969) 1168-1169. 12 SHAVe, R. K., AND HEINE, J. D., Effect of insulin o n nitrous constituents of rat brain, ./. ,Vem'~chem., 12 (1965) 527-532. 13 T~:ws, J. K., AND Slo~I, W. E., Free amino acids and related compounds in brain and other tissues: Effects of convulsant drugs. In W. A. HIMwl('rt Ar~lJJ. P. Sc~lAnl~(Eds.), Horizons ill Neuropsvchopharmacology, Progress in Brain Research, Vol. 16, Elsevier, Amsterdam, 1965, pp. 135--163. 14 TREr~DELI~i~BURC;, P., Ammoniak und Ammoniaksalze. In A. HEErrER (Ed.), Handbuch der Experhnentellen Pharmakologie, Fol. I, Springer, Berlin, 1923, pp. 470--503.
Brai,7 Research, 69 (1974) 164-169
(~ Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
Impaired hyperpolarising inhibition during insulin hypoglycaemia and fluoroacetate poisoning CLEMENS LORACHER AND H. D. LUX Max-Planck-hzstitute.lbr P.s3'chiatr3", 8000 Munk'h 40 ( G.tE:R.)
(Accepted November 30th, 1973)
Convulsive activity elicited by insulin-induced hypoglycaemia (IIH) or fluorofatty acid (FFA) intoxication is characterlsed by profound shifts of cerebral metabolite concentrations in the preconvulsive state la. While IIH lowers glycogen, glucose, lactate, glutamine and glutamate levels, it increases strikingly the cerebral ammonia concentration from values of 0.25 ,umole/g control to 2.5 ftmole/g le,la, lnhibitors of the citric acid cycle, such as FFA, raise glucose and glycogen levels as well as cerebral anamonia (up to 1.5 ¢~nlole/g) in the preconvulsive state x,la. At the beginning of convulsive activity, normal concentrations of creatine phosphate (CP) and ATP are observed simultaneously with an increased turnover of energy-rich phosphates. Therefore. it appears questionable whether the lack of energyrich compounds caused by glucose deficiency or by the action of FFA is the general causative factor for the initiation of seizures s. The elevated ammonia levels merit consideration since convulsive activity is induced by' a rise in cerebral NHa to colnparable levels by infusion of ammonium salts
SHORT COMMUNICATIONS
] 65
discontinued after an initial pentobarbital dose. In these experiments recordings were begun more than 3 h after termination of the anaesthesia. Animals used for the experiments with insul in were deprived of dry food for e ne day. Insulin in high dosage (60-100 I.U./kg of body weight) as well as Na-fluoroacetate (0.1 ~ solution; l mg/kg of body weight) was applied intravenously by means of an infusion p u m p with a rate of 0.2q3.8 ml/min. To terminate hypoglycaemia. 0.5-2.0 g isotonic glucose solution was also applied intravenously at a rate of 1.0 ml/min. Before each infusion, a 15 min control period was introduced for the determination of membrane potentials, in order to exclude artifacts resulting from possible cell damage. Determination of blood glucose levels was achieved semlquantitatively by the dextrostix method (determination of blood glucose by means of glucose oxidase impregnated indicator paper. M E R C K AG, Darmstadt G F R , No. 447116), as other methods requiring more extensive manipulations resulted regularly in loss of the observed cells. Recording devices were used as described earlier 6. IIH and glucose application. Eighteen motoneurones were studied. In 6 cells the complete time course of the effects of the hypoglycaemia and of its removal by glucose infusion on the motoneuronal membrane potential could be followed. In the remaining experiments the cells under observation were lost at different times after starting the infusions and recording was continued with a second motoneurone. Within 5-30 min after insulin infusion, a resting membrane depolarisation in the range between 3-10 mV appeared in all motoneurones. Fourteen of 18 motoneurones spontaneously approached control resting membrane potentials within 30-60 rain after the initial depolarisation. Neurones with higher (5 mV) initial depolarising shifts in resting potential showed transient reductions of 5 - t 0 mV in the amplitude of the action potentials also with a time course of 30-60 min. Changes in the amplitude or rune
mV 35] 25
.
:
=
=
=
=
~
_
=
=
=
:
:
-
-
-
-
-
o
AP
J
A
0 II
40
80
120 r r m
N a - ~ e lmg/kg Bw
mv
:
~
-
:
oAP
25 J
B
0 Insut~ 6 O I . U / k g B w
40
80
120
rain
Glucose l g
Fig. 1. Time course of changes in potentials of two biceps-semitendinosus motoneurons. A: after infusion of Na-fluoroacetate. B: after intravenous insulin application mad termination of IlH by intravenous glucose
166
SHORT COMMUNICATIONS
constants o f EPSPs produced by subthreshold afferent muscle nerve stimulation were not observed. If hypoglycaemia was maintained longer than about 1.5 h from the onset of insulin infusion, a gradual depolarisation o f the resting membrane potential developed concomitantly with a gradual rise in the threshold o f all afferent nerves to elicit EPSPs as well as 1PSPs. This was followed by increase in the threshold o f a n t i dromic stimulation until the cell's axon became completely inexcitable. Twelve to 15 rain after insulin application a reduction o f the IPSP and o f its reversal potential were detected. With the exception of one animal in which blood glucose level did not decline below 40 m g % within 2 h (in this animal, during recording from a single motoneurone for 2 h, neither changes o f RP, AP, EPSPs, IPSPs nor o f thresholds for ortho- and antidromic nerve stimulation were obvious), the IPSPs as well as E~psp.~ decreased about 40-70 min (m ::~ 62 rain) after insulin application, the latter coming very close to the resting membrane potentials. This was valid for both direct and polysynaptic I PSPs. No obvious changes in membrane time constants and in cell input conditions were observed during the infusion periods and thereafter. However, when IPSPamplitudes and rise times were plotted against the membrane potential (in approximate steady state) during varied current steps, it was apparent that the IPSP/membrane potential slopes became considerably reduced during long lasting ( - 80 rain) hypoglycaemia. Also, EPSPs became much smaller. This effect proceeded continuously until virtual disappearance o f inhibitory actions. The reduced dependence o f IPSP amplitudes on membrane potential was observed at considerable times after the Ell,s~, had shifted towards the resting membrane potential, i.e. at times always longer than I h after insulin infusion (m 98 min). After glucose application o f 500 mg/kg the amplitude o f IPSPs and the E~l,sl' returned constantly and quite rapidly to control levels with half times o f recovery between 7-10 rain. Exceptions were only seen in 3 deteriorated cells with low resting
a ..~.___
b
c
j
20 msec
,-'~
' ,,:--'k
.../I'
mV
r~---~.~
~
~--..~I 20 msec
C
20]
-'<--:--'e,~
~
L
- : " - - ~ ..... ~ ~ . :
'"--'--.... -" "
20 msec
Fig. 2. Series of IPSPs at different nlembrane potentials, which were varied by current steps. Dots indicate E rpsi,. A;~:control; A~,: 70 min after insu fin application ; A,. : 15 rain after termination of I I H by intravenous glucose solution. Ba: control; Br,: 60 min after insulin application; B,: 110 rain after insulin application. C,~: control; Ct,: 90 rain after fluoroacetate application; C¢,: 105 rain after fluoroacetate application.
SHORT COMMUNICATIONS
167
membrane potentials (50 mV) in which the equilibration of the EII,Sp with the resting potential lasted for more than 20 min. Within 1.5 h after glucose application no return of En~sP to control levels could be observed in these cells. Neither RPs, APs nor EPSPs showed consistent changes after glucose application. Fluctuations of 2-4 mV of the resting potential were frequently seen during infusion and within about 20 min thereafter. The potential instabilities during infusion are probably of artifactual nature and due to blood pressure variations. Na-fluoroacetate. Measurements were made on 15 motoneurones, of which ~ cells were observed up to 120 rain. In the remaining experiments the cells under observation were lost at different times after starting the infusion and recording was continued with a second motoneurone. The alterations of neuronal potentials produced by fluoroacetate are irreversible within observation times of up to 5 h. All motoneurones showed resting membrane depolarisations in the range o | 2-8 mV, which developed slowly within 10-30 min after infusion. Five motoneurones spontaneously regained almost control resting membrane potential 35-45 min after infusion. Action potentials always showed comparable changes in amplitudes, A depolarising shift of the Elesl~ towards the resting membrane potential became obvious after 60-110 min (m -- 87 rain) from starting the infusions, The difference between EiPsl, and the resting membrane potential was reduced to less than 10 ~o of the controls. Within 2 h loss of hyperpolarising inhibition was almost complete in all cells. ElesV being now very near the resting potential. The effectiveness of excitatory synaptic transmission initially appeared unimpaired. Monosynaptic EPSPs elicited by threshold stimulation of IA muscle afferents usually showed no changes within an observation period of 100-120 rain after infusion. However. intoxication for longer periods was found to elevate the stimulus threshold for all excitatory as well as inhibitory actions. Some time after ElesP had shifted towards resting membrane potential. i.e. at times always longer than 105 min after fluoroacetate application, a reduced dependency of IPSP amplitudes on membrane potential was observed (m ~ 120 mini. This was similar to the results during long lasting hypoglycaemia. Since the cell input conductance as well as the resting membrane time constant remained unchanged throughout the observation period, these effects cannot be attributed to changes in the electric properties o f the postsynaptic nonjunctional membrane. The possibility o f impaired presynaptic conduction or impaired transmitter release cannot be ruled out. The changes in resti ng potential, action potential and EPSP seen during FFA intoxication and IIH were somewhat variable, but only minimal in those cells in which greater movement artifacts could be excluded. Furthermore, the cells recovered from these changes at times at which the effects on the IPSP were just developing. It appears then, that the initial changes in resting membrane potential are not significant for the central nervous disturbances which are induced by IIH or F F A intoxication, With increasing intoxication time a depolarising shift of the ElrsP occurred bringing it toward but not positive to the prevailing resting potential. This results in a decreased efficiency of inhibitory synaptic transmission. The equilibration of the EiPsP with the resting potential indicates that in IlH and FFA intoxication the electro-
168
SHORT COMMUNICATIONS ~|00 10
A
-80 "~
-60
"'..
0
-I00
mV MP •
-I0
-100
0 ~'-o
mV IPSP
-80
"',
-60 rnV MP
10 4
i
or
-80
-I0
mV IPSP
"~-6]
EIpsp
mV -2
• \,
"'\,
",. % " \,
=.,. ~o,, "\
"
~/ ~ ""%'~""'" ,f
°~.a ........ %'o .~.x.a~.~¢jol 1
- I 0 ] mV IPSP
mV MP \
t'2
"'.. "'4.'<,-<
-60
lO - " ' ' . < ' ~ ' ~ _ .
°.i °~
D
/" . . / " " -10 ] <2-~2.~ ~' 0
.... . . . . 40
80
120 rain
Fig. 3. A-C show size and sign of the IPSPs plotted against membrane potential, Arrows indicate resting membrane potential. Points of intersection of curves with zero line indicate reversal potentials (Elvs~,) of IPSPs. In B and C, change in inclination of curves indicates change in conductance of IPSPs. A: before (0), 70 rain ( ~ ) after insulin application and 25 min ( ) after termination of IIH by glucose solution. B: before (O) 60 min ( ~ ) 100 min (~) and 120 min ( : ) after insulin application. C: before (O) 90 min ( ~ ) 110 min (Lq) and 130 min ( : ) after fluoroacetate application. D: mean En,sp values of 5 motoneurons after insulin application (- ,: ) and of 7 motoneurons after fluoroacetate application (- • -). Dotted line (- - , - -) indicates changes of mean Eu,se values of 3 cells after termination of IlH by glucose application, whereas line with squares ( ~ ) mean En,sP values of two motoneurons in which IlH was continued. Ewsp values are plotted from resting membrane potentials (RP). Infusions started at 0 and were terminated at the times indicated by arrows (~ for insulin, ~ for fluoroacetate). Dotted arrows (:, ~') indicate glucose infusion for termination of IIH.
chemical gradient of the ion species (chloride) carrying the inhibitory postsynaptic current is determined by the resting m e m b r a n e potential. A similar reduction of the electromotive force o f the IPSP has been observed with a m m o n i u m salt intoxicationS, 6. Since cerebral a m m o n i a levels in IIH and F F A intoxications are comparable to those in a m m o n i a salt intoxication la it is likely that the depolarising shift o f the EIPSP with IIH and F F A intoxication results from increased cerebral a m m o n i a levels. This likelihood receives further support from the similarity in the time course of recovery of the iPSP (20 min) after application of glucose in the case o f 11H, with the time course with which a m m o n i a is eliminated after application of glucose (15 rain) in the case of increased cerebral NHa levels due to IIH l'-',la. It is noteworthy that convulsions are reported to occur within I-2 I1 after infusion o f F F A 1,l:3, which appears to agree well with the time course of the observed changes of the EIPSP. In the case of long lasting l l H and of F F A intoxication, there may be a correlation between the reduction of synaptic excitatory as well as inhibitory efficacy and the decreasing cerebral A T P and CP levels s. The i m p o r t a n c e o f diminished inhibitory conductance as a causative factor for convulsive activity in i l H and ftuoroacetate poisoning is deduced as: (a) changes o f inhibitory as well as excitatory synaptic transmission appear considerable times (IlH m = 98 rain, fluoroacetate m = 120 min) after EIPSP has shifted towards resting m e m b r a n e potential (IlH m = 62 min, fluoroacetate m = 87 rain); (b) these potential
SHORT COMMUNICATIONS
I 69
reductions affect inhibitory and excitatory synaptic transmission to an equal extent. Studies on the interactions of EPSPs and IPSPs indicate that almost linear summation of potentials is as frequent as a partial shunt of the EPSP by the conductance increase during the IPSP 3,~°. From this it is obvious that the loss of hyperpolarising postsynaptic inhibition, due to changes in the electrochemical gradient of chloride ions during IIH and fluoroacetate poisoning should result in a considerable decrease of inhibitory potency. Similar changes after ammonia intoxication have also been observed in neocortical nerve cells 9. if the results apply in general to CNS neurone~, ammonium salts as well as IIH and I-FA intoxication may distort the balance ol excitatory drive by postsynaptic inhibition. It is already known from poisons which directly interfere with inhibitory synaptic transmission (strychnine, picrotoxin, bicuculline), that this imbalance can lead to convulsive activity2, El. Decreased inhibitor~ action due to a loss of hyperpolarising e.m.f, could similarly be an essential factor for other metabolically induced convulsive activities. We thank Professor Roger Eckert tot reading the manuscipt.
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