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Effect of hypoxia on glutamate efflux and synaptic transmission in the guinea pig hippocampus
Brain Research, 620 (1993) 301-304
301
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25753
Effect of hypoxia on glutamate effiux and synaptic transmission in the guinea pig hippocampus * Donald H. Penning a Joanne W. Goh b, Hossam E1-Beheiry c and James F. Brien
b
" Department of Anesthesia, University of Iowa, Iowa City, I 0 52242 (USA) and Department of b Pharmacology and Toxicology and CAnaesthesia, Queen's University, Kingston, Ont. (Canada)
(Accepted 27 April 1993)
Key words: Hippocampus; Hypoxia; Synaptic transmission; Glutamate
Simultaneous assessment of synaptic activity and glutamate efflux in guinea pig hippocampal brain slices was made before, during and after a 10-min period of hypoxia. Spontaneous glutamate efflux was assessed by determining glutamate concentration in the superfusion medium at discrete times using high performance liquid chromatography (HPLC). Synaptic activity was assessed using extracellular recording of the evoked population spike in the CA l region following stimulation of the Schaffer collateral pathway. Hypoxia decreased (P < 0.05) the amplitude of the population spike by 3 min and abolished it by 5 min. This was accompanied by an increase (P < 0.05) in glutamate concentration in the superfusate at 10 min. Following re oxygenation, the population spike returned to baseline amplitude by 5 min and was greater (P < 0.05) than baseline at 10 and 20 min of recovery. Glutamate concentration returned to baseline levels by 1 min of recovery. This experimental preparation can be used to explore the temporal relationship between glutamate efflux and synaptic activity during hypoxia. The results of this study indicate that, in the hippocampal CA1 region, post-synaptic elements are more sensitive than their presynaptic counterparts to hypoxia.
T h e r e l e a s e o f excitatory a m i n o acids ( E A A ) , such as g l u t a m a t e , in the b r a i n is i m p l i c a t e d as a causative a g e n t in n e u r o n a l cell d e a t h following h y p o x i a / i s c h e m i a 9'1°. R e l e a s e o f g l u t a m a t e in h i p p o c a m p a l neurons d u r i n g h y p o x i a / i s c h e m i a has b e e n d e m o n s t r a t e d in vivo 2'4 a n d in vitro 5. G l u t a m a t e 1° o r o n e o f its r e c e p t o r a n a l o g s 13 i n j e c t e d into t h e b r a i n is neurotoxic, a n d this effect can b e a t t e n u a t e d by E A A r e c e p t o r a n t a g o n i s t s 3. H y p o x i a / i s c h e m i a r a p i d l y i n t e r f e r e s with synaptic transmission. In t h e h u m a n , a d e c r e a s e in c e r e b r a l cortical b l o o d flow to less t h a n 1 6 - 2 0 m l / 1 0 0 g b r a i n / m i n is a s s o c i a t e d with a loss o f cortical e v o k e d p o t e n t i a l s 14. S h o r t p e r i o d s o f h y p o x i a can reversibly abolish t h e p o p u l a t i o n spike r e c o r d e d in t h e C A l region following s t i m u l a t i o n o f t h e S c h a f f e r c o l l a t e r a l p a t h w a y in h i p p o c a m p a l slices, a n d t h e r e is an enh a n c e d a m p l i t u d e o f t h e p o p u l a t i o n spike d u r i n g recovery f r o m hypoxia 11'12. T h e p r e s e n t study was u n d e r t a k e n in g u i n e a pig
h i p p o c a m p a l slices to d e t e r m i n e the effect of an in vitro e p i s o d e o f m o d e r a t e hypoxia on t h e e x t r a c e l l u l a r p o p u l a t i o n spike o f C A 1 p y r a m i d a l n e u r o n s , following s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m , a n d on t h e efflux o f g l u t a m a t e a n d to d e t e r m i n e t h e t e m p o r a l r e l a t i o n ship b e t w e e n t h e s e two variables. T r a n s v e r s e l y s e c t i o n e d h i p p o c a m p a l slices (500 tzm thick) w e r e o b t a i n e d f r o m a d u l t m a l e D u n k i n - H a r t l e y g u i n e a pigs. P r i o r to sacrifice, t h e a n i m a l s w e r e p l a c e d on an ice p a c k for 30 m i n while b e i n g a n a e s t h e t i z e d with 2 % h a l o t h a n e . B o d y t e m p e r a t u r e was not m e a sured. T h e b r a i n was t h e n r a p i d l y r e m o v e d a n d b o t h h i p p o c a m p i w e r e d i s s e c t e d o u t for slice p r e p a r a t i o n . Slices w e r e cut using a M c l l w a i n tissue c h o p p e r a n d p l a c e d in ice-cold artificial c e r e b r o s p i n a l fluid ( A C S F ) c o n t a i n i n g (mM): NaC1 120, KC1 3.1, N a H 2 P O 4 1.3, N a H C O 3 26, CaC12 2, MgC12 1.5, a n d d e x t r o s e 10; p H 7.4 a n d g a s s e d with 95% 0 2, 5 % C O 2. T h e p r o c e d u r e f r o m sacrifice of t h e a n i m a l to p l a c e m e n t of slices in the A C S F s u p e r f u s i o n m e d i u m r e q u i r e d less t h a n 2
Correspondence: D.H. Penning, The University of Iowa, Department of Anesthesia, 200 Hawkins Drive SE618, Iowa City, IO 52242-1079, USA.
Fax: (1) (319) 356-2940. * Presented in part at the Annual Meeting of the Canadian Anaesthetists' Society, Toronto, June 1992.
302 min. 12-15 slices were sandwiched between two nylon nets stretched over Plexiglas rings of diameter 1" o.d., 7 / 8 " i.d. and 7 / 8 " o.d., 3 / 4 " i.d.. The tissue was then allowed to equilibrate at room temperature for 2 h before experiments were initiated. At the end of the equilibration period, the sandwiched slices were placed in a Plexiglas tissue chamber recessed into an aluminum block. Slices were completely submerged in the ACSF. A series of resistors located within the block allowed for variable adjustment of the bath temperature using an external controller. ACSF solution was gassed with 95% 0 2, 5% CO 2 and was delivered to the chamber at a flow rate of 2.5 ml/min. To ensure efficient superfusion of the hippocampal slices, the inlet for the ACSF medium was located below the slices, and superfusate was directed through a channel before being removed by vacuum suction. An additional gas line for dispensing 95% 0 2 / 5 % CO 2 was placed directly over the submerged slices to ensure maximal oxygenation of the tissue, and the temperature of the superfusion medium was maintained at 35 _+ 0.2°C. The condition of hypoxia was produced by using 95% N 2, 5% CO 2 as the gas blown over the slices and bubbled through the ACSF solution. In a separate series of experiments (n = 5),the pH and gas tensions (_+ S.D.) of the tissue bath ACSF were measured during control and hypoxic conditions. The pH, pO 2 and pCO 2 values for control were 7.28_+ 0.02, 630_+ 17, and 47.1 _+ 1.6, respectively, and for hypoxic conditions were 7.25 _+ 0.01, 64 _+ 2, and 49.9 _+ 0.1, respectively. CA~ neuronal population spikes were recorded in the pyramidal cell body layer using extracellular recording electrodes (borosilicate glass, resistance 2-3 MO, filled with 4 M NaCI) pulled on a F l a m i n g / B r o w n microelectrode puller (Sutter Instruments). These near-maximal synaptic responses were evoked by stimulation of the stratum radiatum utilizing negative pulses
CONTROL
ti 0
q
5 MIN HYPOXIA
l -~t
delivered through concentric bipolar electrodes (SNEX-100, Rhodes Electronics) connected to a Grass S-88 stimulator and photoelectric constant current stimulus isolation unit (PSIU6). Stimulation frequency was once every 10 s. Signals were detected through an Axoclamp-2A amplifier and displayed on an oscilloscope. Responses were monitored for at least 15 min prior to experimental manipulation to ensure stability and for at least 20 min after the hypoxic episode. Following stabilization of the hippocampal CA~ electrophysiological signal, ACSF sampling for the determination of glutamate concentration as an index of spontaneous glutamate effiux and population spike recordings were made before, during and after a 10-min period of hypoxia. Sampling of the superfusate (0.6-ml samples taken over a 30-s period) and collection of electrophysiological signals occurred simultaneously at 1, 3, 5, and 10 min during hypoxia and 1, 3, 5, 10, and 20 min post-hypoxia. Quantitation of glutamate in the superfusate samples collected at discrete experimental times was conducted by an established procedure involving reversephase h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC) with fluorescence detection of the thio-substituted isoindole derivative formed by precolumn derivatization with o - p h t h a l a l d e h y d e and /3mercaptoethanol 6'7. The lower limit of quantitative sensitivity and the within-day coefficient of variation of the method were determined previously and were 9 pmol, and < 10%, respectively. Five consecutive traces of the evoked population spikes were averaged at various times using pClamp software (Axon Instruments Inc.) on an IBM-AT clone microcomputer. Data were also stored in its entirety on VHS videotape following digitization by a VR-10A Digital Data Recorder (Instrutech Corp.). Data analysis consisted of an average of the downward and up-
10 MIN HYPOXIA
t
10MIN RECOVERY
4 1
Fig. 1. Effect of hypoxia on the CA 1 population spike. Responses were evoked by stimulation of the stratum radiatum input and recorded in the CA t pyramidal cell body layer. Records shown are an average of 5 consecutive sweeps each. Stimulation rate was once every 10 s and duration of hypoxia (95% N 2, 5% C O ~ ) w a s 10 min.
303 ward components of the population spike, and normalization of experimental values to pre-treatment controis. Data for glutamate concentration in the superfusate were expressed as percent of control value. Statistical analysis involved repeated-measures ANOVA followed by Newman-Keul's post-hoc test for a statistically significant (P < 0.05) F statistic. Values reported are mean + S.E.M. (n = 8). The effect of hypoxia on the extracellular recording from the CA 1 region of a hippocampal slice is shown in Fig. 1. There was a complete loss of the population spike after 5 min of hypoxia, which was sustained for the remainder of the 10-min period of hypoxia. There was no change in the presynaptic volley. By 10 min of recovery, the presynaptic volley remained unchanged, but the population spike amplitude was increased. While the magnitude of the change varied between the eight experimental animals, the pattern of the change was always the same. The results for all eight animals are presented in Table I. The population spike amplitude was decreased significantly (P < 0.05) by 3 min of hypoxia, and was essentially abolished at 5 min and remained so at 10 min of hypoxia. During the reoxygenation phase, the population spike recovered to its control amplitude by 5 min and was significantly increased (P < 0.05) over control amplitude at 10 and 20 min. Glutamate concentration in the superfusate was significantly increased ( P < 0.05) at 10 min of hypoxia. During re-oxygenation, glutamate concentration rapidly returned to control value. The loss of the population spike that occurs with hypoxia precedes the appearance of glutamate in the artificial CSF. Our data indicate that the rise in glutamate efflux occurred after the loss in synaptic transmission since glutamate efflux did not change between 3 and 5 min, at which time the population spike had nearly disappeared. The observation that the population spike, the size of which is a reflection of the number of post-synaptic neurons recruited to fire action potentials 1, was markedly reduced prior to any changes in glutamate efflux suggests that the CA l
neurons are more sensitive to hypoxic insult than presynaptic terminals. The rise in glutamate efflux at 10 min of hypoxia may be mediated by depolarization of presynaptic elements through increased extracellular K ÷ accumulation. It has been reported that exposure of hippocampal slices to anoxia produces a gradual rise in extracellular K ÷ concentrations which is maximal at approximately 10 min 8. We do not feel that the delay in appearance of increased glutamate concentrations in the ACSF following the loss of synaptic activity represents the transit-time from the extracellular space to the ACSF since there is almost immediate return to control concentrations of glutamate during the onset of the re-oxygenation phase. Another finding of this study is the increased population spike amplitude during recovery from hypoxia. Recovery of synaptic transmission was seen in each slice following 10 min of hypoxia. The population spike amplitude was back to control values by 5 min and was significantly increased at 10-20 min of recovery. The stimulus characteristics and electrode placement were not altered during the experiment. Evidence for this is the stable baseline between collection time-points during the control period and the constant amplitude of the presynaptic volley. It remains to be determined whether the increase in amplitude of the population spike seen in the present investigation is due to an enhancement of evoked neurotransmitter release (or decreased re-uptake) or an increased post-synaptic responsiveness. Other investigators have measured glutamate release during conditions of hypoxia/ischemia for both in vitro 5 and in vivo experiments 2, and have demonstrated the electrophysiological effects of hypoxia in brain slices 11'12. We believe this is the first report combining these two experimental indices of synaptic activity. Our experimental preparation allows the opportunity to explore the temporal relationship between glutamate release and synaptic activity during hypoxia. The results of our studies indicate that, in the hippocampal CA 1 region, post-synaptic elements are more
TABLE I
Mean population spike amplitude and glutamate efflux from hippocampal slices during and following hypoxia Values are mean ( + S.E.M., n = 8)
Hypoxia (min) % Control Population spike ( + S.E.M.) Glutamate * * ( + S.E.M.)
1 101 (6) 140 (29)
3 28 * (18) 130 (15)
Recovery (min) 5 4 * (4) 98 (8)
10 0 * 149 * (22)
1 8* (5) 106 (14)
3 49 * (21) 103 (9)
5 99 (13) 95 (10)
10 132 * (7) 115 (15)
20 143 * (13) 127 (8)
* Significantly different from pre-hypoxia control ( P <0.05); ** the control value for the glutamate concentration in the superfusate was 24.4 + 6.0 p m o l / m l .
304 sensitive than
their
presynaptic
counterparts
poxia. F u r t h e r , a p o s t - h y p o x i c e n h a n c e m e n t
t o hy-
of synaptic
transmission was observed indicating enhancement evoked neurotransmitter take)
or
an
increased
release (or decreased post-synaptic
of
re-up-
responsiveness.
Since we did not detect significantly increased glutamate
concentrations
in t h e
post-hypoxic
period
we
favor the latter explanation. This work was supported by the Medical Research Council (Canada) and Queen's University Faculty of Medicine Trust Fund. D.H.P. is a Roy J. Carver Clinician Scientist, and J.W.G. is a Medical Research Council of Canada Scholar. The authors would like to recognize Mr. James Reynolds for his expertise with the EAA analysis. l Andersen, P., Bliss, T.V.P. and Skrede, K.K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208221. 2 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebra] microdialysis, Z Neurochem, 43 (1984) 1369-1374. 3 Clark, G.D. and Rothman, S.M., Blockade of excitatory amino acid receptors protects anoxic hippocampal slices, Neuroscience, 21 (1987) 665-671. 4 Hagberg, H., Andersson, P., Kjellmer, 1., Thiringer, K. and Thordstein, M., Extracellular overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia, Neurosci. Lett., 78 (1987) 311-317.
5 Pellegrini-Giampietro, D.E., Cherici, G., Alesiani, M., Carla, V. and Moroni, F., Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage, J. Neurosci., 10 (1990) 1035-1041. 6 Penning, D.H., Patrick, J., Jimmo, S. and Brien, J.F., Release of glutamate and gamma-aminobutyric acid in the ovine fetal hippocampus: ontogeny and effect of hypoxia, J. Dee. PhysioL, 16 (1991) 301-307. 7 Reynolds, J.N., Racz, W.J., Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices, Can. J. Physiol. Pharmacol., 65 (1987) 791-798. 8 Roberts, E.L. and Sick, T.J., Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices, Brain Res., 570 (1992) 225-230. 9 Rothman, S., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J. Neurosci., 4 (1984) 1884-1891. 10 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 11 Schiff, S.J. and Somjen, G.G., Hyperexcitability following moderate hypoxia in hippocampal tissue slices, Brain Res., 337 (1985) 337-340. 12 Schiff, S.J. and Somjen, G.G., The effect of graded hypoxia on the hippocampal slice: an in vitro model of the ischemic penumbra, Stroke, 18 (1987) 30-37. 13 Silverstein, F.S., Chen, R. and Johnston, M.V., The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain, Neurosci. Lett., 71 (1986) 13-18. 14 Symon, L., The relationship between CBF, evoked potentials and the clinical features in cerebral ischaemia, Acta Neurol. Scand., 62 (1980) 175-190.
301
© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 25753
Effect of hypoxia on glutamate effiux and synaptic transmission in the guinea pig hippocampus * Donald H. Penning a Joanne W. Goh b, Hossam E1-Beheiry c and James F. Brien
b
" Department of Anesthesia, University of Iowa, Iowa City, I 0 52242 (USA) and Department of b Pharmacology and Toxicology and CAnaesthesia, Queen's University, Kingston, Ont. (Canada)
(Accepted 27 April 1993)
Key words: Hippocampus; Hypoxia; Synaptic transmission; Glutamate
Simultaneous assessment of synaptic activity and glutamate efflux in guinea pig hippocampal brain slices was made before, during and after a 10-min period of hypoxia. Spontaneous glutamate efflux was assessed by determining glutamate concentration in the superfusion medium at discrete times using high performance liquid chromatography (HPLC). Synaptic activity was assessed using extracellular recording of the evoked population spike in the CA l region following stimulation of the Schaffer collateral pathway. Hypoxia decreased (P < 0.05) the amplitude of the population spike by 3 min and abolished it by 5 min. This was accompanied by an increase (P < 0.05) in glutamate concentration in the superfusate at 10 min. Following re oxygenation, the population spike returned to baseline amplitude by 5 min and was greater (P < 0.05) than baseline at 10 and 20 min of recovery. Glutamate concentration returned to baseline levels by 1 min of recovery. This experimental preparation can be used to explore the temporal relationship between glutamate efflux and synaptic activity during hypoxia. The results of this study indicate that, in the hippocampal CA1 region, post-synaptic elements are more sensitive than their presynaptic counterparts to hypoxia.
T h e r e l e a s e o f excitatory a m i n o acids ( E A A ) , such as g l u t a m a t e , in the b r a i n is i m p l i c a t e d as a causative a g e n t in n e u r o n a l cell d e a t h following h y p o x i a / i s c h e m i a 9'1°. R e l e a s e o f g l u t a m a t e in h i p p o c a m p a l neurons d u r i n g h y p o x i a / i s c h e m i a has b e e n d e m o n s t r a t e d in vivo 2'4 a n d in vitro 5. G l u t a m a t e 1° o r o n e o f its r e c e p t o r a n a l o g s 13 i n j e c t e d into t h e b r a i n is neurotoxic, a n d this effect can b e a t t e n u a t e d by E A A r e c e p t o r a n t a g o n i s t s 3. H y p o x i a / i s c h e m i a r a p i d l y i n t e r f e r e s with synaptic transmission. In t h e h u m a n , a d e c r e a s e in c e r e b r a l cortical b l o o d flow to less t h a n 1 6 - 2 0 m l / 1 0 0 g b r a i n / m i n is a s s o c i a t e d with a loss o f cortical e v o k e d p o t e n t i a l s 14. S h o r t p e r i o d s o f h y p o x i a can reversibly abolish t h e p o p u l a t i o n spike r e c o r d e d in t h e C A l region following s t i m u l a t i o n o f t h e S c h a f f e r c o l l a t e r a l p a t h w a y in h i p p o c a m p a l slices, a n d t h e r e is an enh a n c e d a m p l i t u d e o f t h e p o p u l a t i o n spike d u r i n g recovery f r o m hypoxia 11'12. T h e p r e s e n t study was u n d e r t a k e n in g u i n e a pig
h i p p o c a m p a l slices to d e t e r m i n e the effect of an in vitro e p i s o d e o f m o d e r a t e hypoxia on t h e e x t r a c e l l u l a r p o p u l a t i o n spike o f C A 1 p y r a m i d a l n e u r o n s , following s t i m u l a t i o n o f t h e s t r a t u m r a d i a t u m , a n d on t h e efflux o f g l u t a m a t e a n d to d e t e r m i n e t h e t e m p o r a l r e l a t i o n ship b e t w e e n t h e s e two variables. T r a n s v e r s e l y s e c t i o n e d h i p p o c a m p a l slices (500 tzm thick) w e r e o b t a i n e d f r o m a d u l t m a l e D u n k i n - H a r t l e y g u i n e a pigs. P r i o r to sacrifice, t h e a n i m a l s w e r e p l a c e d on an ice p a c k for 30 m i n while b e i n g a n a e s t h e t i z e d with 2 % h a l o t h a n e . B o d y t e m p e r a t u r e was not m e a sured. T h e b r a i n was t h e n r a p i d l y r e m o v e d a n d b o t h h i p p o c a m p i w e r e d i s s e c t e d o u t for slice p r e p a r a t i o n . Slices w e r e cut using a M c l l w a i n tissue c h o p p e r a n d p l a c e d in ice-cold artificial c e r e b r o s p i n a l fluid ( A C S F ) c o n t a i n i n g (mM): NaC1 120, KC1 3.1, N a H 2 P O 4 1.3, N a H C O 3 26, CaC12 2, MgC12 1.5, a n d d e x t r o s e 10; p H 7.4 a n d g a s s e d with 95% 0 2, 5 % C O 2. T h e p r o c e d u r e f r o m sacrifice of t h e a n i m a l to p l a c e m e n t of slices in the A C S F s u p e r f u s i o n m e d i u m r e q u i r e d less t h a n 2
Correspondence: D.H. Penning, The University of Iowa, Department of Anesthesia, 200 Hawkins Drive SE618, Iowa City, IO 52242-1079, USA.
Fax: (1) (319) 356-2940. * Presented in part at the Annual Meeting of the Canadian Anaesthetists' Society, Toronto, June 1992.
302 min. 12-15 slices were sandwiched between two nylon nets stretched over Plexiglas rings of diameter 1" o.d., 7 / 8 " i.d. and 7 / 8 " o.d., 3 / 4 " i.d.. The tissue was then allowed to equilibrate at room temperature for 2 h before experiments were initiated. At the end of the equilibration period, the sandwiched slices were placed in a Plexiglas tissue chamber recessed into an aluminum block. Slices were completely submerged in the ACSF. A series of resistors located within the block allowed for variable adjustment of the bath temperature using an external controller. ACSF solution was gassed with 95% 0 2, 5% CO 2 and was delivered to the chamber at a flow rate of 2.5 ml/min. To ensure efficient superfusion of the hippocampal slices, the inlet for the ACSF medium was located below the slices, and superfusate was directed through a channel before being removed by vacuum suction. An additional gas line for dispensing 95% 0 2 / 5 % CO 2 was placed directly over the submerged slices to ensure maximal oxygenation of the tissue, and the temperature of the superfusion medium was maintained at 35 _+ 0.2°C. The condition of hypoxia was produced by using 95% N 2, 5% CO 2 as the gas blown over the slices and bubbled through the ACSF solution. In a separate series of experiments (n = 5),the pH and gas tensions (_+ S.D.) of the tissue bath ACSF were measured during control and hypoxic conditions. The pH, pO 2 and pCO 2 values for control were 7.28_+ 0.02, 630_+ 17, and 47.1 _+ 1.6, respectively, and for hypoxic conditions were 7.25 _+ 0.01, 64 _+ 2, and 49.9 _+ 0.1, respectively. CA~ neuronal population spikes were recorded in the pyramidal cell body layer using extracellular recording electrodes (borosilicate glass, resistance 2-3 MO, filled with 4 M NaCI) pulled on a F l a m i n g / B r o w n microelectrode puller (Sutter Instruments). These near-maximal synaptic responses were evoked by stimulation of the stratum radiatum utilizing negative pulses
CONTROL
ti 0
q
5 MIN HYPOXIA
l -~t
delivered through concentric bipolar electrodes (SNEX-100, Rhodes Electronics) connected to a Grass S-88 stimulator and photoelectric constant current stimulus isolation unit (PSIU6). Stimulation frequency was once every 10 s. Signals were detected through an Axoclamp-2A amplifier and displayed on an oscilloscope. Responses were monitored for at least 15 min prior to experimental manipulation to ensure stability and for at least 20 min after the hypoxic episode. Following stabilization of the hippocampal CA~ electrophysiological signal, ACSF sampling for the determination of glutamate concentration as an index of spontaneous glutamate effiux and population spike recordings were made before, during and after a 10-min period of hypoxia. Sampling of the superfusate (0.6-ml samples taken over a 30-s period) and collection of electrophysiological signals occurred simultaneously at 1, 3, 5, and 10 min during hypoxia and 1, 3, 5, 10, and 20 min post-hypoxia. Quantitation of glutamate in the superfusate samples collected at discrete experimental times was conducted by an established procedure involving reversephase h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC) with fluorescence detection of the thio-substituted isoindole derivative formed by precolumn derivatization with o - p h t h a l a l d e h y d e and /3mercaptoethanol 6'7. The lower limit of quantitative sensitivity and the within-day coefficient of variation of the method were determined previously and were 9 pmol, and < 10%, respectively. Five consecutive traces of the evoked population spikes were averaged at various times using pClamp software (Axon Instruments Inc.) on an IBM-AT clone microcomputer. Data were also stored in its entirety on VHS videotape following digitization by a VR-10A Digital Data Recorder (Instrutech Corp.). Data analysis consisted of an average of the downward and up-
10 MIN HYPOXIA
t
10MIN RECOVERY
4 1
Fig. 1. Effect of hypoxia on the CA 1 population spike. Responses were evoked by stimulation of the stratum radiatum input and recorded in the CA t pyramidal cell body layer. Records shown are an average of 5 consecutive sweeps each. Stimulation rate was once every 10 s and duration of hypoxia (95% N 2, 5% C O ~ ) w a s 10 min.
303 ward components of the population spike, and normalization of experimental values to pre-treatment controis. Data for glutamate concentration in the superfusate were expressed as percent of control value. Statistical analysis involved repeated-measures ANOVA followed by Newman-Keul's post-hoc test for a statistically significant (P < 0.05) F statistic. Values reported are mean + S.E.M. (n = 8). The effect of hypoxia on the extracellular recording from the CA 1 region of a hippocampal slice is shown in Fig. 1. There was a complete loss of the population spike after 5 min of hypoxia, which was sustained for the remainder of the 10-min period of hypoxia. There was no change in the presynaptic volley. By 10 min of recovery, the presynaptic volley remained unchanged, but the population spike amplitude was increased. While the magnitude of the change varied between the eight experimental animals, the pattern of the change was always the same. The results for all eight animals are presented in Table I. The population spike amplitude was decreased significantly (P < 0.05) by 3 min of hypoxia, and was essentially abolished at 5 min and remained so at 10 min of hypoxia. During the reoxygenation phase, the population spike recovered to its control amplitude by 5 min and was significantly increased (P < 0.05) over control amplitude at 10 and 20 min. Glutamate concentration in the superfusate was significantly increased ( P < 0.05) at 10 min of hypoxia. During re-oxygenation, glutamate concentration rapidly returned to control value. The loss of the population spike that occurs with hypoxia precedes the appearance of glutamate in the artificial CSF. Our data indicate that the rise in glutamate efflux occurred after the loss in synaptic transmission since glutamate efflux did not change between 3 and 5 min, at which time the population spike had nearly disappeared. The observation that the population spike, the size of which is a reflection of the number of post-synaptic neurons recruited to fire action potentials 1, was markedly reduced prior to any changes in glutamate efflux suggests that the CA l
neurons are more sensitive to hypoxic insult than presynaptic terminals. The rise in glutamate efflux at 10 min of hypoxia may be mediated by depolarization of presynaptic elements through increased extracellular K ÷ accumulation. It has been reported that exposure of hippocampal slices to anoxia produces a gradual rise in extracellular K ÷ concentrations which is maximal at approximately 10 min 8. We do not feel that the delay in appearance of increased glutamate concentrations in the ACSF following the loss of synaptic activity represents the transit-time from the extracellular space to the ACSF since there is almost immediate return to control concentrations of glutamate during the onset of the re-oxygenation phase. Another finding of this study is the increased population spike amplitude during recovery from hypoxia. Recovery of synaptic transmission was seen in each slice following 10 min of hypoxia. The population spike amplitude was back to control values by 5 min and was significantly increased at 10-20 min of recovery. The stimulus characteristics and electrode placement were not altered during the experiment. Evidence for this is the stable baseline between collection time-points during the control period and the constant amplitude of the presynaptic volley. It remains to be determined whether the increase in amplitude of the population spike seen in the present investigation is due to an enhancement of evoked neurotransmitter release (or decreased re-uptake) or an increased post-synaptic responsiveness. Other investigators have measured glutamate release during conditions of hypoxia/ischemia for both in vitro 5 and in vivo experiments 2, and have demonstrated the electrophysiological effects of hypoxia in brain slices 11'12. We believe this is the first report combining these two experimental indices of synaptic activity. Our experimental preparation allows the opportunity to explore the temporal relationship between glutamate release and synaptic activity during hypoxia. The results of our studies indicate that, in the hippocampal CA 1 region, post-synaptic elements are more
TABLE I
Mean population spike amplitude and glutamate efflux from hippocampal slices during and following hypoxia Values are mean ( + S.E.M., n = 8)
Hypoxia (min) % Control Population spike ( + S.E.M.) Glutamate * * ( + S.E.M.)
1 101 (6) 140 (29)
3 28 * (18) 130 (15)
Recovery (min) 5 4 * (4) 98 (8)
10 0 * 149 * (22)
1 8* (5) 106 (14)
3 49 * (21) 103 (9)
5 99 (13) 95 (10)
10 132 * (7) 115 (15)
20 143 * (13) 127 (8)
* Significantly different from pre-hypoxia control ( P <0.05); ** the control value for the glutamate concentration in the superfusate was 24.4 + 6.0 p m o l / m l .
304 sensitive than
their
presynaptic
counterparts
poxia. F u r t h e r , a p o s t - h y p o x i c e n h a n c e m e n t
t o hy-
of synaptic
transmission was observed indicating enhancement evoked neurotransmitter take)
or
an
increased
release (or decreased post-synaptic
of
re-up-
responsiveness.
Since we did not detect significantly increased glutamate
concentrations
in t h e
post-hypoxic
period
we
favor the latter explanation. This work was supported by the Medical Research Council (Canada) and Queen's University Faculty of Medicine Trust Fund. D.H.P. is a Roy J. Carver Clinician Scientist, and J.W.G. is a Medical Research Council of Canada Scholar. The authors would like to recognize Mr. James Reynolds for his expertise with the EAA analysis. l Andersen, P., Bliss, T.V.P. and Skrede, K.K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208221. 2 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebra] microdialysis, Z Neurochem, 43 (1984) 1369-1374. 3 Clark, G.D. and Rothman, S.M., Blockade of excitatory amino acid receptors protects anoxic hippocampal slices, Neuroscience, 21 (1987) 665-671. 4 Hagberg, H., Andersson, P., Kjellmer, 1., Thiringer, K. and Thordstein, M., Extracellular overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia, Neurosci. Lett., 78 (1987) 311-317.
5 Pellegrini-Giampietro, D.E., Cherici, G., Alesiani, M., Carla, V. and Moroni, F., Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage, J. Neurosci., 10 (1990) 1035-1041. 6 Penning, D.H., Patrick, J., Jimmo, S. and Brien, J.F., Release of glutamate and gamma-aminobutyric acid in the ovine fetal hippocampus: ontogeny and effect of hypoxia, J. Dee. PhysioL, 16 (1991) 301-307. 7 Reynolds, J.N., Racz, W.J., Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices, Can. J. Physiol. Pharmacol., 65 (1987) 791-798. 8 Roberts, E.L. and Sick, T.J., Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices, Brain Res., 570 (1992) 225-230. 9 Rothman, S., Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death, J. Neurosci., 4 (1984) 1884-1891. 10 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 11 Schiff, S.J. and Somjen, G.G., Hyperexcitability following moderate hypoxia in hippocampal tissue slices, Brain Res., 337 (1985) 337-340. 12 Schiff, S.J. and Somjen, G.G., The effect of graded hypoxia on the hippocampal slice: an in vitro model of the ischemic penumbra, Stroke, 18 (1987) 30-37. 13 Silverstein, F.S., Chen, R. and Johnston, M.V., The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain, Neurosci. Lett., 71 (1986) 13-18. 14 Symon, L., The relationship between CBF, evoked potentials and the clinical features in cerebral ischaemia, Acta Neurol. Scand., 62 (1980) 175-190.