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2-Amino-5-phosphonovalerate attenuates the severe hypoglycemia-induced loss of perforant path-evoked field potentials in the rat hippocampus
296
,\cur~J.s~ Wm'e Le//cr.~. ::~ I 19~ 7 t 2t)(~ ~)'! lilsc~ier Scienlific Publishers lrelumt lid
NSL 04579
2-Amino-5-phosphonovalerate attenuates the severe hypoglycemia-induced loss of perforant path-evoked field potentials in the rat hippocampus S.P. Butcher, I. J a c o b s o n , M. Sandberg, H, H a g b e r g a n d A. H a m b e r g e r Institute ~,}/"Neurobiology, University ~?f Goteborg. Goteborg (Sweden)
(Received t 1 November 1986; Reviscd version received 24 December 1986: Accepted 26 December 1986) Key words:
Severe hypoglycemia; Dentate gyrus; Field potential; 2-Amino-5-phosphonovaterate; Rat
The effects of severe hypoglycemia on perforant path-evoked field potentials were examined in the ral hippocampus. Although a complete loss of this response was noted when blood glucose concentration fell below 1 raM, this occurred before cessation of electroencephalogram (EEG) activity, Both spontaneous and evoked respon,ses recovered partially following glucose readministration. 1~-2-Amino-5-phosphonovalerate, an NMDA-sensitive acidic amino acid receptor antagonist, facilitated this recovery from the hypoglycemic challenge when administered via a dialysis probe.
Severe hypoglycemia leads to a gross disruption in brain function, characterised by cessation of electrical activity and alterations in tissue levels of energy substrates and amino acids [2, 6, 12, 17]. This condition is also associated with an overflow of neuroactive amino acids into the extracellular compartment of the brain [8, 20. 21]. Since neuroexcitatory amino acids, notably glutamate and aspartate, are known to be neurotoxic [14, 15], a link between acidic amino acid release and hypoglycemiainduced neuronal damage might be anticipated in view of the results of several recent reports [5, 20, 23, 24]. Dentate gyrus granule cells receive a major glutamatergic/aspartergic innervation from the entorhinal cortex via the perforant pathway [16, 22], and these cells are particularly susceptible to the neurotoxic effects of severe hypoglycemia [4, 5]. In vitro electrophysiological studies have demonstrated that synaptically evoked activity in these cells is severely curtailed under mildly hypoglycemic conditions [9]. In contrast, in vivo somatosensory and auditory evoked responses may persist even during severe hypoglycemia [10]. In the present study we have examined the synaptically evoked response of granule cells in vivo during severe hypoglycemia and after readministration of glucose. In addition, we have investigated the effects of the putative neuro-
Correspondence." S.P. Butcher. Present address: Department of Pharmacology, University of Edinburgh, Edinburgh, U.K.
0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
297 protective NMDA-sensitive acidic amino acid receptor antagonist, D-2-amino-5phosphonovalerate (D-APV) on this response. A detailed description of the methodologies used in microdialysis studies and for induction of severe hypoglycemia has been published previously [3, 20]. Briefly, Spraguc Dawley rats (240 280 g; either sex) were anaesthetized with methohexital (Brietal; Lilly, 75 nlg,/kg) and tracheotomised. Anaesthesia was then maintained using halothane ( < 1.5%). Arterial blood glucose levels were monitored using a hypoglycemia reflotest (Clinicon; F.R.G.). Hypoglycemia was induced by intraperitoneal injection of insulin (40 IU/kg: Actrapid; Nova), and when the blood glucose concentration fell below 2 mM, the animal was ventilated mechanically, Cessation of spontaneous electrical activity usually occurred at a glucose concentration of 0.5 0.8 raM. Glucose (50% w/v) was readministered via an intravenous cannula 3(t rain later. Electrical recordings (electroencephalogram (EEG) and field potential) were made from the hilus of the dentate gyrus (AP -3.8: ML +2: [18]) using a tungsten electrode glued to the side of a dialysis probe (for a detailed description, see ref. 20). Evoked potentials and EEG activity were amplified using a Digitimer "Neurolog" system and field potentials were stored and averaged using an ABC microcomputer [1 I]. The dialysis probe was perfused continuously with oxygenated, glucose-free Krebs Ringer bicarbonate buffer at 1.25 id/rnin. D-APV (final concentration, 1 raM: Tocris Chemicals, U.K.) was included in the perfusion medium throughout the experiments indicated in the text. The perforant path was stimulated (1 mA intensity: 0.1 ms duration: 1 Hz) using a concentric steel electrode (Clark Biomedical Supplies, U.K.) positioned in the angular bundle (AP-8.0; ML +-4.5; [18]). The effects of severe hypoglycemia and glucose readministration on the perforant path-evoked field potential are shown in Fig. 1. Peak amplitude was 4.6_+0.3 mV under normal conditions. This was not altered until blood glucose fell below 0.8 I raM, even though a series of characteristic changes in EEG activity were noted [13, 19, 20], In control animals the evoked field potential then rapidly declined and was eventually lost. This occurred during the 'early isoelectric' phase of EEG activity (low frequency, low amplitude activity interspersed with isoelectric periods). EEG activity did not cease until approximately 20 30 rain later (glucose below 0.6 mM). It should be noted that granule cell excitability was increased as blood glucose fell (Fig. 1B: note the enlarged population spike), although population spike amplitude declined in parallel with peak amplitude at low blood glucose levels. Although the precise mechanism(s) underlying this increase in population spike amplitude remains uncertain, a presynaptic action due to increased transmitter release is unlikely since extracellular glutamate/aspartate levels are unaffected by hypoglycemia until onset of the isoelectric period [8, 20]. Following readministration of glucose, the evoked potential recovered partially (20~40% of control) over 30 40 minutes, after which time no further changes were noted (Fig. 2). No population spikes were observed during this period (data not shown). A fairly similar pattern of events was observed in the presence of D-APV (1 mM). In this case the loss of evoked potential appeared to be more resistant to glucose de-
298
A
B
.i
C
I
D
Fig. 1. Perforant pathway-evoked field potentials in the rat dentate gyrus during progressive insulin hypoglycemia and subsequent recovery following glucose administration. The perforant path was stimulated using square wave pulses of 1 mA intensity and 0.1 msec duration at 1 Hz. Bars: 1 mV and 10 ms. A: control (blood glucose = 6.0 raM). B: pre isoelectric (blood glucose = 1.6 mM). C: isoelectric (blood glucose = 0.6 raM). D: recovery (60 rain) (blood glucose =6.0 mM).
pletion since this response was not lost until 5 15 min before the isoelectric period. A n increase in granule cell excitability was again noted as glucose levels fell (data not shown). Following glucose infusion the recovery o f the evoked field potential response was more p r o n o u n c e d (50-65% o f control) on the D - A P V treated side (Fig. 2). Thus, D - A P V would a p p e a r to protect the perforant path evoked response from the adverse effects o f hypoglycemia. This might reflect a neuroprotective effect o f DA P V [24], since an increase in the n u m b e r o f viable granule cells in the dentate gyrus would explain these data. This suggests that hypoglycemic cell injury may be mediated by endogenous excitotoxins [5, 20], and that measurement o f synaptically evoked potentials might therefore provide a convenient functional method for screening potentially therapeutic drugs. In contrast to these data, Deutsch et al. [10] have reported that somatosensory and auditory evoked responses are unaffected during severe hypoglycemia. This might be
299
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TIME Fig. 2. Effects of D-APV ( 1 mM) on the recovery of evoked field potcntials following scverc hypoglycemia. Glucose was administered by intravenous infusion following 30 rain of isoelectric EEG activity. Results (means÷: S.E.M.: n 4) are expressed as a % of the control response against time tk)llowing glucose injection (Q control: • APV treated). Statistical differences betwecn 60-min values were assessed using a Walsh text (*P
explained if non-vulnerable neuronal populations were involved in the latter responses. However, other groups have demonstrated a loss of somatosensory responses during severe hypoglycemia [I, 7] and the present finding that the evoked response is lost before the cessation of EEG activity is in agreement with the results of these groups. It should also be noted that the in vitro perforant path-evoked response [9] disappears when glucose levels fell below 2 mM (at which concentration the in vivo response is facilitated). A more efficient use of alternative energy substrates in vivo may explain this difference. This work is supported by a Swedish Medical Research Council Grant (C12X00164). S.P.B. is supported by a research fellowship awarded by the Royal Society and M.S. by the Swedish Natural Science Research Council (1905). I Agardh, ('.-D. nnd Rosen, L., Neurophysiological recovery afler hypoglycemic coma in the rat: correlation with cerebral metabolism, J. Cereb. Blood Flow Metab., 3 (1983) 78 85. 2 Agardh, C'.-D.. Folbcrgrowi, J. and Siesio, B.K., Cerebral metabolic changes in profound, insulin induced hypoglycemia, and in the recovery period folowing glucose administration, ,I. Neurochenl. 31 (197811135 1142. 3 Agardh. C.-D., C h a p m a n , A.G., Nilsson. B. and Siesjo. B.K., Endogenous substrates utilized b5 rat brain in severe insulin-induced hypoglycemia, J. Neurochem. 36 ( 1981 ) 490 500.
300 4 Auer, R.N., Wieloch, T., Olsson. M.Y. and Siesjo, B.K.. The distribution oF hypoglycemia brain damage, ActaNeurophathol..64(1984) 177 191. 5 Auer, R.N., Kalimo, H., Olsson, Y. and Wieloch. T., The dentate gyrus m hypoglycemia: pathoIog} implicating excitotoxin mediated neuronal necrosis, Acla Neuropathol., 67 (1985) 279 288. 6 Behar, KL., Den Hollander, J.A, Petrofl', A.C., ttetherington, tt.P., Prichard, J.W. and Shuhnan. R.G., EtTec! of hypoglycemic enccphalopathy upon amino acids, high energy phosphates and ptti m the rat brain in vivo: detection by scqucntial ql and ~P NMR spectroscopy, J. Neurochem.. 44 (19851 1045 1055. 7 Brierley, J.B., Brown, A.W. and Meldrum, B.S., The nature and time course of neuronal alterations resulting from oligaemia and hypoglycemia in the brain of Macaca mulatta, Brain Res., 25 (19711483 199. 8 Butcher. S.P., Sandberg, M.. |-|agberg, H. and Hamberger, A., On the origins of neuroactive amim~ acids released during severe hypoglycemia, J. Neurochem., (1987), in press. 9 Cox, D.W.G. and Bachelard, M.S.. Attenuation of evoked field potential from dentate granule cells by low glucose, pyruvate and malate~ and sodium fluoride, Brain Res.~ 239 (1982) 527 534. 10 Deutsch, E.. Freeman, S., Sohmer. H. and Gafori, M., The persistence of somatosensory and auditor 5 pathway evoked potentials in severe hypoglycemia in the cat, Electroencephatogr. Clin. Ncurophysiol.. 61(1985) 161 164. 11 Jacobson, l., Butcher, S.P. and Hamberger, A., An analysis of the effects of excitatory amino acid receptor antagonists on evoked field potentials in the olfactory bulb, Neuroscience, 19 (1986) 267-273. 12 Lewis, L.D., Ljunggren, B., Norberg, K. and Sicsjo, B.K., Change in carbohydrate substrates, amino acids and ammonia in the brain during insulin induced hypoglycemia, J. Neurochem., 23 (1974a) 659 671. 13 Lewis, L.D., Ljunggren, B., Ratcheson, R.A. and Siesjo, B.K., Cerebral energy state in insulin-induced hypoglycemia, related to blood glucose and to EEG, J. Neurochem., 23 (1974b) 673 679. 14 Mangano, R.M. and Schwarcz, R., Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus, Brain Res, Bull., 10 (1983) 47 51. 15 McBean, G.J. and Roberts, P.J., Chronic infusion of L-glutamate causes neurotoxicity in rat striatum. Brain Res., 290(1984) 372 375. 16 Nadler, J.V., White, W.F., Vaca. K.W., Pery, B.W, and Cotman, C.W.. Biochemical correlates of transmission mediated by glutamate and aspartate, J. Neurochem.. 31 (1978) 147-155. 17 Norberg, K. and Siesjo, B.K., Oxidative metabolism in the cerebral cortex of the rat in severe insulininduced hypoglycemia, J. Neurochem., 26 (1976) 345 352. 18 Paxinos, G. and Watsom C., The Rat Brain in Stereotaxic Co-ordinates. Academic, New York, 1982. 19 Ratcheson, R.A., Blank, A.C. and Ferrendelli, J.A., Regionally selective metabolic effects of hypoglycemia, Brain, 36 (1981) 1962 1958. 20 Sandberg, M., Butcher, S.P. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe hypoglycemia: in vivo dialysis of the rat hippocampus, J. Neurochem., 47 (1986) 178- 184. 21 Tossman, U., Wieloch, T. and Ungerstedt, U., ;,-Aminobutyric acid and taurine release in striatum of the rat during hypoglycemic coma, studied by microdialysis, Neurosci. Lett., 62 (1985) 231 235. 22 White, W.F., Nadler, J.V., Hamberger, A., Cotman, C.W. and Cummins, J.T., Glutamate as transmit ter of hippocampal perforant path, Nature (London), 270 (1977) 356 357. 23 Wieloch, T., Hypoglycemia induced neuronal damage prevented by an N-methyl-D-aspartate receptor antagonist, Science, 230 (1985) 681 682. 24 Wicloch, T., Engelsen, B., Westerberg, E. and Auer, R., Lesions of the glutamatergic cortico-striatal projections in the rat ameliorate hypoglycemic brain damage in the striatum, Neurosci. Lett., 58 (1985) 25 30.
,\cur~J.s~ Wm'e Le//cr.~. ::~ I 19~ 7 t 2t)(~ ~)'! lilsc~ier Scienlific Publishers lrelumt lid
NSL 04579
2-Amino-5-phosphonovalerate attenuates the severe hypoglycemia-induced loss of perforant path-evoked field potentials in the rat hippocampus S.P. Butcher, I. J a c o b s o n , M. Sandberg, H, H a g b e r g a n d A. H a m b e r g e r Institute ~,}/"Neurobiology, University ~?f Goteborg. Goteborg (Sweden)
(Received t 1 November 1986; Reviscd version received 24 December 1986: Accepted 26 December 1986) Key words:
Severe hypoglycemia; Dentate gyrus; Field potential; 2-Amino-5-phosphonovaterate; Rat
The effects of severe hypoglycemia on perforant path-evoked field potentials were examined in the ral hippocampus. Although a complete loss of this response was noted when blood glucose concentration fell below 1 raM, this occurred before cessation of electroencephalogram (EEG) activity, Both spontaneous and evoked respon,ses recovered partially following glucose readministration. 1~-2-Amino-5-phosphonovalerate, an NMDA-sensitive acidic amino acid receptor antagonist, facilitated this recovery from the hypoglycemic challenge when administered via a dialysis probe.
Severe hypoglycemia leads to a gross disruption in brain function, characterised by cessation of electrical activity and alterations in tissue levels of energy substrates and amino acids [2, 6, 12, 17]. This condition is also associated with an overflow of neuroactive amino acids into the extracellular compartment of the brain [8, 20. 21]. Since neuroexcitatory amino acids, notably glutamate and aspartate, are known to be neurotoxic [14, 15], a link between acidic amino acid release and hypoglycemiainduced neuronal damage might be anticipated in view of the results of several recent reports [5, 20, 23, 24]. Dentate gyrus granule cells receive a major glutamatergic/aspartergic innervation from the entorhinal cortex via the perforant pathway [16, 22], and these cells are particularly susceptible to the neurotoxic effects of severe hypoglycemia [4, 5]. In vitro electrophysiological studies have demonstrated that synaptically evoked activity in these cells is severely curtailed under mildly hypoglycemic conditions [9]. In contrast, in vivo somatosensory and auditory evoked responses may persist even during severe hypoglycemia [10]. In the present study we have examined the synaptically evoked response of granule cells in vivo during severe hypoglycemia and after readministration of glucose. In addition, we have investigated the effects of the putative neuro-
Correspondence." S.P. Butcher. Present address: Department of Pharmacology, University of Edinburgh, Edinburgh, U.K.
0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
297 protective NMDA-sensitive acidic amino acid receptor antagonist, D-2-amino-5phosphonovalerate (D-APV) on this response. A detailed description of the methodologies used in microdialysis studies and for induction of severe hypoglycemia has been published previously [3, 20]. Briefly, Spraguc Dawley rats (240 280 g; either sex) were anaesthetized with methohexital (Brietal; Lilly, 75 nlg,/kg) and tracheotomised. Anaesthesia was then maintained using halothane ( < 1.5%). Arterial blood glucose levels were monitored using a hypoglycemia reflotest (Clinicon; F.R.G.). Hypoglycemia was induced by intraperitoneal injection of insulin (40 IU/kg: Actrapid; Nova), and when the blood glucose concentration fell below 2 mM, the animal was ventilated mechanically, Cessation of spontaneous electrical activity usually occurred at a glucose concentration of 0.5 0.8 raM. Glucose (50% w/v) was readministered via an intravenous cannula 3(t rain later. Electrical recordings (electroencephalogram (EEG) and field potential) were made from the hilus of the dentate gyrus (AP -3.8: ML +2: [18]) using a tungsten electrode glued to the side of a dialysis probe (for a detailed description, see ref. 20). Evoked potentials and EEG activity were amplified using a Digitimer "Neurolog" system and field potentials were stored and averaged using an ABC microcomputer [1 I]. The dialysis probe was perfused continuously with oxygenated, glucose-free Krebs Ringer bicarbonate buffer at 1.25 id/rnin. D-APV (final concentration, 1 raM: Tocris Chemicals, U.K.) was included in the perfusion medium throughout the experiments indicated in the text. The perforant path was stimulated (1 mA intensity: 0.1 ms duration: 1 Hz) using a concentric steel electrode (Clark Biomedical Supplies, U.K.) positioned in the angular bundle (AP-8.0; ML +-4.5; [18]). The effects of severe hypoglycemia and glucose readministration on the perforant path-evoked field potential are shown in Fig. 1. Peak amplitude was 4.6_+0.3 mV under normal conditions. This was not altered until blood glucose fell below 0.8 I raM, even though a series of characteristic changes in EEG activity were noted [13, 19, 20], In control animals the evoked field potential then rapidly declined and was eventually lost. This occurred during the 'early isoelectric' phase of EEG activity (low frequency, low amplitude activity interspersed with isoelectric periods). EEG activity did not cease until approximately 20 30 rain later (glucose below 0.6 mM). It should be noted that granule cell excitability was increased as blood glucose fell (Fig. 1B: note the enlarged population spike), although population spike amplitude declined in parallel with peak amplitude at low blood glucose levels. Although the precise mechanism(s) underlying this increase in population spike amplitude remains uncertain, a presynaptic action due to increased transmitter release is unlikely since extracellular glutamate/aspartate levels are unaffected by hypoglycemia until onset of the isoelectric period [8, 20]. Following readministration of glucose, the evoked potential recovered partially (20~40% of control) over 30 40 minutes, after which time no further changes were noted (Fig. 2). No population spikes were observed during this period (data not shown). A fairly similar pattern of events was observed in the presence of D-APV (1 mM). In this case the loss of evoked potential appeared to be more resistant to glucose de-
298
A
B
.i
C
I
D
Fig. 1. Perforant pathway-evoked field potentials in the rat dentate gyrus during progressive insulin hypoglycemia and subsequent recovery following glucose administration. The perforant path was stimulated using square wave pulses of 1 mA intensity and 0.1 msec duration at 1 Hz. Bars: 1 mV and 10 ms. A: control (blood glucose = 6.0 raM). B: pre isoelectric (blood glucose = 1.6 mM). C: isoelectric (blood glucose = 0.6 raM). D: recovery (60 rain) (blood glucose =6.0 mM).
pletion since this response was not lost until 5 15 min before the isoelectric period. A n increase in granule cell excitability was again noted as glucose levels fell (data not shown). Following glucose infusion the recovery o f the evoked field potential response was more p r o n o u n c e d (50-65% o f control) on the D - A P V treated side (Fig. 2). Thus, D - A P V would a p p e a r to protect the perforant path evoked response from the adverse effects o f hypoglycemia. This might reflect a neuroprotective effect o f DA P V [24], since an increase in the n u m b e r o f viable granule cells in the dentate gyrus would explain these data. This suggests that hypoglycemic cell injury may be mediated by endogenous excitotoxins [5, 20], and that measurement o f synaptically evoked potentials might therefore provide a convenient functional method for screening potentially therapeutic drugs. In contrast to these data, Deutsch et al. [10] have reported that somatosensory and auditory evoked responses are unaffected during severe hypoglycemia. This might be
299
60UI ¢0 Ul t._
40
!
0
I
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20 GII
0 0
=
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TIME Fig. 2. Effects of D-APV ( 1 mM) on the recovery of evoked field potcntials following scverc hypoglycemia. Glucose was administered by intravenous infusion following 30 rain of isoelectric EEG activity. Results (means÷: S.E.M.: n 4) are expressed as a % of the control response against time tk)llowing glucose injection (Q control: • APV treated). Statistical differences betwecn 60-min values were assessed using a Walsh text (*P
explained if non-vulnerable neuronal populations were involved in the latter responses. However, other groups have demonstrated a loss of somatosensory responses during severe hypoglycemia [I, 7] and the present finding that the evoked response is lost before the cessation of EEG activity is in agreement with the results of these groups. It should also be noted that the in vitro perforant path-evoked response [9] disappears when glucose levels fell below 2 mM (at which concentration the in vivo response is facilitated). A more efficient use of alternative energy substrates in vivo may explain this difference. This work is supported by a Swedish Medical Research Council Grant (C12X00164). S.P.B. is supported by a research fellowship awarded by the Royal Society and M.S. by the Swedish Natural Science Research Council (1905). I Agardh, ('.-D. nnd Rosen, L., Neurophysiological recovery afler hypoglycemic coma in the rat: correlation with cerebral metabolism, J. Cereb. Blood Flow Metab., 3 (1983) 78 85. 2 Agardh, C'.-D.. Folbcrgrowi, J. and Siesio, B.K., Cerebral metabolic changes in profound, insulin induced hypoglycemia, and in the recovery period folowing glucose administration, ,I. Neurochenl. 31 (197811135 1142. 3 Agardh. C.-D., C h a p m a n , A.G., Nilsson. B. and Siesjo. B.K., Endogenous substrates utilized b5 rat brain in severe insulin-induced hypoglycemia, J. Neurochem. 36 ( 1981 ) 490 500.
300 4 Auer, R.N., Wieloch, T., Olsson. M.Y. and Siesjo, B.K.. The distribution oF hypoglycemia brain damage, ActaNeurophathol..64(1984) 177 191. 5 Auer, R.N., Kalimo, H., Olsson, Y. and Wieloch. T., The dentate gyrus m hypoglycemia: pathoIog} implicating excitotoxin mediated neuronal necrosis, Acla Neuropathol., 67 (1985) 279 288. 6 Behar, KL., Den Hollander, J.A, Petrofl', A.C., ttetherington, tt.P., Prichard, J.W. and Shuhnan. R.G., EtTec! of hypoglycemic enccphalopathy upon amino acids, high energy phosphates and ptti m the rat brain in vivo: detection by scqucntial ql and ~P NMR spectroscopy, J. Neurochem.. 44 (19851 1045 1055. 7 Brierley, J.B., Brown, A.W. and Meldrum, B.S., The nature and time course of neuronal alterations resulting from oligaemia and hypoglycemia in the brain of Macaca mulatta, Brain Res., 25 (19711483 199. 8 Butcher. S.P., Sandberg, M.. |-|agberg, H. and Hamberger, A., On the origins of neuroactive amim~ acids released during severe hypoglycemia, J. Neurochem., (1987), in press. 9 Cox, D.W.G. and Bachelard, M.S.. Attenuation of evoked field potential from dentate granule cells by low glucose, pyruvate and malate~ and sodium fluoride, Brain Res.~ 239 (1982) 527 534. 10 Deutsch, E.. Freeman, S., Sohmer. H. and Gafori, M., The persistence of somatosensory and auditor 5 pathway evoked potentials in severe hypoglycemia in the cat, Electroencephatogr. Clin. Ncurophysiol.. 61(1985) 161 164. 11 Jacobson, l., Butcher, S.P. and Hamberger, A., An analysis of the effects of excitatory amino acid receptor antagonists on evoked field potentials in the olfactory bulb, Neuroscience, 19 (1986) 267-273. 12 Lewis, L.D., Ljunggren, B., Norberg, K. and Sicsjo, B.K., Change in carbohydrate substrates, amino acids and ammonia in the brain during insulin induced hypoglycemia, J. Neurochem., 23 (1974a) 659 671. 13 Lewis, L.D., Ljunggren, B., Ratcheson, R.A. and Siesjo, B.K., Cerebral energy state in insulin-induced hypoglycemia, related to blood glucose and to EEG, J. Neurochem., 23 (1974b) 673 679. 14 Mangano, R.M. and Schwarcz, R., Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus, Brain Res, Bull., 10 (1983) 47 51. 15 McBean, G.J. and Roberts, P.J., Chronic infusion of L-glutamate causes neurotoxicity in rat striatum. Brain Res., 290(1984) 372 375. 16 Nadler, J.V., White, W.F., Vaca. K.W., Pery, B.W, and Cotman, C.W.. Biochemical correlates of transmission mediated by glutamate and aspartate, J. Neurochem.. 31 (1978) 147-155. 17 Norberg, K. and Siesjo, B.K., Oxidative metabolism in the cerebral cortex of the rat in severe insulininduced hypoglycemia, J. Neurochem., 26 (1976) 345 352. 18 Paxinos, G. and Watsom C., The Rat Brain in Stereotaxic Co-ordinates. Academic, New York, 1982. 19 Ratcheson, R.A., Blank, A.C. and Ferrendelli, J.A., Regionally selective metabolic effects of hypoglycemia, Brain, 36 (1981) 1962 1958. 20 Sandberg, M., Butcher, S.P. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe hypoglycemia: in vivo dialysis of the rat hippocampus, J. Neurochem., 47 (1986) 178- 184. 21 Tossman, U., Wieloch, T. and Ungerstedt, U., ;,-Aminobutyric acid and taurine release in striatum of the rat during hypoglycemic coma, studied by microdialysis, Neurosci. Lett., 62 (1985) 231 235. 22 White, W.F., Nadler, J.V., Hamberger, A., Cotman, C.W. and Cummins, J.T., Glutamate as transmit ter of hippocampal perforant path, Nature (London), 270 (1977) 356 357. 23 Wieloch, T., Hypoglycemia induced neuronal damage prevented by an N-methyl-D-aspartate receptor antagonist, Science, 230 (1985) 681 682. 24 Wicloch, T., Engelsen, B., Westerberg, E. and Auer, R., Lesions of the glutamatergic cortico-striatal projections in the rat ameliorate hypoglycemic brain damage in the striatum, Neurosci. Lett., 58 (1985) 25 30.