- Email: [email protected]
Changes in content of neuroactive amino acids and acetylcholine in the rat hippocampus following transient forebrain ischemia
Neurochem. Int. Vol. 21, No. 2, pp. 243-249, 1992
0197-0186/92$5.00+0.00 Copyright© 1992PergamonPress Ltd
Printedin Great Britain.All rights reserved
CHANGES IN CONTENT OF NEUROACTIVE AMINO ACIDS A N D ACETYLCHOLINE IN THE RAT HIPPOCAMPUS FOLLOWING TRANSIENT FOREBRAIN ISCHEMIA MASASHI KATSURA, TOSHIAKI IINO a n d KINYA KURIYAMA* Department of Pharmacology, Kyoto Prefectural of University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan (Received 22 September 1991 ; accepted 27 December 1991)
Abstract--Changes in content of selected neuroactive amino acids [glutamicacid, aspartic acid, glycine,7aminobutyric acid (GABA) and taurine] and acetylcholine (ACh) in the rat hippocampus following transient forebrain ischemia were investigated using male Wistar rats. Rats were allowed to survive for 1 or 5 days following 10 or 20 min of 4-vessel occlusion, and killed by a focused microwave irradiation. A significantreduction in all neuroactive amino acids examined except GABA was noted in the hippocampus on the fifth day. One day after the 4-vessel occlusion for I0 min, no significant effect on the content of neuroactive amino acids in all brain areas was observed. ~,-Aminobutyricacid content in the hippocampus was only significantly reduced on the fifth day after the occlusion for 20 min. Similarly, a significant decrease in ACh content in the hippocampus was observed on the fifth day after the occlusion for 20 min. Considering the data that a significantloss of neuronal cells in the hippocampus (delayed neuronal death) was detected only 5 days after the 4-vessel occlusion, it can be said that the alterations in the hippocampus of neuroactive amino acids such as glutamic acid, aspartic acid, glycineand taurine are more sensitivethan those in GABA and ACh against cerebral ischemia. A possible correlation of these changes of neuroactive amino acids in the occurrence of delayed neuronal death in the hippocampus is also suggested.
It is well known that transient cerebral ischemia causes variable brain damage in proportion to its degree and duration. Pulsinelli et al. (1982) have reported that neurons in certain brain regions such as hippocampus, striatum, neocortex and thalamus are selectively vulnerable to ischemia. Hippocampal pyramidal cells in the CA1 layer are among the most vulnerable neurons to transient ischemia (Pulsinelli et al., 1982; Kirino, 1982). The death of these cells occurs gradually over a period of 2 days and this phenomenon has been termed "delayed neuronal death" (Kirino, 1982; Kirino et al., 1984; Johansen et al., 1987; Munekata and Hossmann, 1987). Although detailed mechanisms underlying these ischemia-induced neuronal degenerations or the death of hippocampal CA 1 neurons have not been clarified, it has been suggested that the neurotoxic action of synaptically released excitatory amino acids in areas vulnerable to ischemia plays an important role in the pathogenesis of ischemic neuronal death (Benveniste et al., 1984; Rothman and Olney, 1986; Onodera et
*Author to whom correspondence should be addressed.
al., 1986). Many studies have been conducted to clar-
ify the morphological changes seen after ischemia (Kirino, 1982; Johansen et al., 1983; Kirino et al., 1984; Schmidt-Kastner and Hossman, 1988) and the alterations in specific neuronal enzymatic signals such as glutamic acid decarboxylase (GAD, Francis and Pulsinelli, 1982 ; Schlander et al., 1988 ; Johansen et al., 1989), choline acetyltransferase (CAT, Francis and Pulsinelli, 1982) and cysteine-sulfinic acid decarboxylase (CSAD, Chan-Palay et al., 1982). In spite of the fact that the majority of hippocampal pyramidal cells is presumed to be glutamatergic and/or aspartatergic (Storm-Mathisen and Opsahl, 1978; Zaczek et al., 1979), few data on possible alterations in the activities of these enzymes related to the metabolism of excitatory amino acids. Recently, microdialysis technique (Ungerstedt, 1984) has been introduced and neuronal vulnerabilities following transient forebrain ischemia, deafferentation or kainic acid-induced cerebral lesion has been examined (Butcher and Hameberger, 1987 ; Matsumoto et al., 1991). In this study, the content of these neuroactive amino acids in various cerebral regions including hippocampus following transient 243
244
MASASHI KATSURA et al.
cerebral ischemia was measured. The effects o f transient cerebral ischemia on A C h c o n t e n t a n d morphological features in h i p p o c a m p a l n e u r o n s were also examined.
EXPERIMENTAL PROCEDURES
Animals Male Wistar rats weighing 250-300 g were purchased from Japan SLC, Inc. (Hamamatsu, Japan). They were used for the experiments after breeding for a week with laboratory chow (MF, Oriental Yeast Co., Ltd., Chiba, Japan) and tap water ad libitum. Application of cerebral ischemia Rats were subjected to forebrain ischemia by the 4-vessel occlusion procedure described by Pulsinelli and Brierlay (1979). Briefly, the rats were anesthetized with a mixture of 70% N20, 0.5% halothane and 30% O: and the vertebral arteries were electrocauterized. On the following day, the rats were reanesthetized and the carotid arteries were ligated by clips to produce the 4-vessel occlusion, and the rats exhibiting isoelectronic electroencephalogram were subjected to experiments. After 10 or 20 min occlusion, the clips were removed and carotid blood flow was reperfused. Histological examination Brain was perfused with 300 mt of 10% formalin saline via the left ventricle of the heart. The skull was then opened 1 h after the perfusion and the brain was removed and refixed for several days in 20% formalin. After dehydration and paraffin embedding, 4/~m of coronal section was stained with hematoxylin-eosin. Extraction of neuroactive amino acids and acetylcholine Rats were killed by focused microwave irradiation on the head (5 kW, 1.1 s). One or 5 days after the 4-vessel occlusion, the dorsal hippocampus, dorsolateral striatum and frontal cortex (lateral portion) were dissected out according to the method of Glowinski and Iversen (1%6). The tissue samples were subjected to extract neuroactive amino acids with 50 mM perchloric acid and diluted with 0.1 N HCI. Acetylcholine was extracted from the tissue samples according to the method of Potter et al. (1983) with a minor modification. The tissue samples were homogenized with 2 ml of 1 N formic acid:acetone (15:85) mixture containing I nM ethylhomocholine as an internal standard, and centrifuged at 1,700 g for 20 rain at 4°C. The supematant obtained was washed with diethyl ether and finally lyophilized at -20°C. After resuspending the lyophilized sample in distilled water, it was mixed with a solution containing KI-solution and 1 mM tetraethylammonium chloride before centrifuging at 10,000 g for 3 min at room temperature. In order to remove excessive iodine, the pellet was dissolved in acetonitrile and anion exchange resin (200-400 mesh, AG lX8 chloride form, BIO-RAD) was added. The iodine-free supernatant was then dried under a N2 stream and resuspended in distilled water for ACh determination.
Determinations o['neuroactive amino acid~ and acetylcholine To determine the content of cerebral amino acids, the extract from cerebral tissue homogenate was analyzed by a high performance liquid chromatography (HPLC) with fluorescent detection following post-labeling with o-phthalaldehyde by the method of Ida and Kuriyama (1963). For the measurement of cerebral ACh content, the extract was analyzed by a HPLC equipped with a precolumn (ACODS), a separate column (Eicompak AC-Gel), an immobilized column (AC-Enzympak, Eicom, Kyoto, Japan) and an electrochemical detector (VMD-101A, Yanaco, Kyoto, Japan) operating at a flow rate of 1 ml/min. The mobile phase consisted of 100 mM phosphate buffer (pH 8.0), 0.8 mM sodium 1-decansulfonate and 0.6 mM tetramethylammonium chloride. The detector potential was maintained at 450 mV against an Ag/AgC1 reference electrode (Potter et al., 1983 ; Fujimori and Yamamoto, 1987). Reagents Sodium 1-decansulfonate, tetramethylammonium chloride and tetraethylammonium chloride were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan) and Kanto Chemical Co., Ltd. (Tokyo, Japan), respectively. Other reagents used in this study were commercially available and of analytical grade. Statistics Each value was expressed as the mean + SEM, and statistical significance was determined by Student's or Aspin Welch's t-test. RESULTS Histological observations As s h o w n in Fig. 1, n e u r o n s in the h i p p o c a m p a l C A I region were morphologically intact o n the 1st day after 10 m i n o f 4-vessel occlusion a n d similar to those in s h a m - o p e r a t e d groups (data not shown). On the fifth day after 10 m i n of occlusion, n e u r o n a l damage in the h i p p o c a m p a l region became apparent. In the s h a m - o p e r a t e d group, average n e u r o n a l density in the C A I region was 121 _+ 7 cells/mm ( N = 5), while that in the g r o u p applied 10 m i n o f i s c h e m i a was 10 + 3 cells/mm (N = 5). In addition, the average n e u r o n a l density in the h i p p o c a m p a t CA1 region in sham-operated g r o u p was n o t different f r o m t h a t in n o n - t r e a t e d group (data n o t shown). The n e u r o n a l damages in the h i p p o c a m p a l C A I region o n the fifth day following 20 m i n of the occlusion was similar to t h a t in 10 min-occluded group, a l t h o u g h the extent o f n e u r o n a l d a m a g e s was more obvious t h a n t h a t in 10 m i n - o c c l u d e d g r o u p (data not shown). Content o f neuroactive amino acids One day after 10 a n d 20 m i n o f occlusion, the content of neuroactive a m i n o acids in the h i p p o c a m p u s showed n o significant changes [Figs 2(A) a n d 3(A)].
Amino acids and ACh contents and cerebral ischemia
245
Fig. 1. Histological changes in the CA1 region of rat hippocampus on the fifth day following 4-vessel occlusion. (A), (C): Sham-operated. The hippocampal CAI neurons are intact. (B), (D): 5 days after ischemic insults. Almost all hippocampal CA1 neurons are degenerated. Hematoxylin-eosin stain. Bar = 100 gm. DG : dentate gyrus.
On the other hand, all neuroactive amino acids measured, except GABA, significantly decreased in the hippocampus on the fifth day following 10 and 20 min of ischemia [taurine: 60% and 61%; aspartic acid: 70% and 87%; glutamic acid: 56% and 68%; glycine: 49% and 44%; G A B A : 66% and 73% . Figs 2(B) and 3(B)]. In contrast, G A B A content in the hippocampus was only significantly reduced in the 20 min occluded group. In other brain areas studied, the content of neuroactive amino acids showed no significant alteration following 10 and 20 min of occlusion (data not shown). Acetylcholine content
On the first day following 10 and 20 min of ischemia, the content of ACh showed no significant change in any brain areas. On the other hand, ACh content in the hippocampus on the fifth day following 20 min of occlusion decreased by 25% (P < 0.01) in comparison with that on the fifth day following 10 min of ischemia (Fig. 4).
DISCUSSION Neurons in the hippocampus are vulnerable to cerebral ischemia in man as well as experimental animals (Brierley, 1984). It has been shown that the CAI pyramidal cells and the CA3c/hilar somatostatinergic neurons are especially vulnerable to ischemia. CA1 pyramidal cells exhibit the "delayed neuronal death" phenomenon (Kirino, 1982; Kirino et al., 1984; Johansen et al., 1987; Munekata and Hossman, 1987). Namely, the ischemic loss of CA1 neurons delays 4 days following the loss of hilar somatostatinergic neurons during the first 2 days after the ischemia. This vulnerability of CA 1 pyramidal cells is believed to be due to the excitotoxicities of glutamate and aspartate mediated by N-methyl-D-aspartate (NMDA) type receptor (Rothman, 1984; SchmidtKastner and Hossmann, 1988). These ischemia-induced neuronal damages have been evaluated with regard to biochemical and immunohistochemical markers such as G A D for GABAergic neuron (Francis and Pulsinelli, 1982 ; Schlander et al.,
246
MASASHI KATSURA et al. Amino acids content 15
10
5
:
i
(/~mol/g we~ weight.)
O
Aspartic acid
0
5
10
i
i
T
**
t'nl GABA
I
i
~~
acid
I
15 [
~
I
coo
~ Ischemio
I
I
I
Fig. 2. Changes in neuroactive amino acid contents in hippocampus on the first and fifth day alter 10 min of cerebral ischemia. Rats were killed by microwave irradiation on the head on the first (A) and fifth (B) day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral amino acid contents by H P L C with fluorescent detection after post-labeling with o-phthalaldehyde. *P < 0.05 and **P < 0:01, compared with each control value. Each column represents the mean_+ SEM (N = 5 8).
Amino acids content (/~mol/g wet weight] 15 1
(A)
10 r
5 I
1 doy
~ /
-~N~
5 i
10 i
x~'~ **
L-
Taurine
s doy~
15
(B)
/
acid
,
oo.J °-,. ooo **F
-~
Glutomic acid
Glycine
l
I
GABA I
I
I
~ I
Ischemio
l: Control
: I
I
Fig. 3. Changes in neuroactive amino acid contents in the hippocampus on the first and fifth day after 20 min of cerebral ischemia. Rats were killed by microwave irradiation on the head on the first (A) and fifth (B) day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral amino acid content by H P L C with fluorescent detection after post-labeling with o-phthalaldehyde. *P < 0.05 and **P < 0.0l, compared with each control value. Each column represents the mean + SEM (N = 4 8).
Amino acids and ACh contents and cerebral ischemia
50
O
E 25 E
'l/
I
J: Control : Ischemio
O U e-
10 m i n
20 min
Fig. 4. Changes in ACh content in the hippocampus on the fifth day after 10 and 20 min of cerebral ischemia. Rats were sacrificed by microwave irradiation on the head on the fifth day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral ACh contents by HPLC with electrochemical detection. **P < 0.01, compared with each control value. Each column represents the mean_+ SEM (N = 3-5). 1988; Johansen et al., 1989), CAT for cholinergic neuron (Francis and Pulsinelli, 1982) and CSAD for CSAD-positive (taurine) neuron (Chan-Palay et al., 1982), and these neurons in the hippocampus are relatively resistant to ischemic insult. Although the majority of pyramidal cells in the hippocampal CAI region has been considered to be glutamatergic and/or aspartatergic (Storm-Mathisen and Opsahl, 1978; Zaczek et al., 1979), the vulnerability of these neurons in the hippocampus has not been clearly evaluated. Recently, Matsumoto et al. (1991) have reported that the measurement of a high potassium-evoked release of neuroactive amino acids in vivo is useful for evaluating the vulnerability of neurons in rat hippocampal pyramidal cells after transient forebrain ischemia. We could not find, however, any reports on the changes in the content of neuroactive amino acids after cerebral ischemia. In this study, therefore, we have attempted to evaluate the vulnerability of neurons possessing neuroactive amino acids following transient ischemia by measuring their contents in the hippocampus and other brain regions. In addition, possible correlation of these changes with ACh content and morphological features in the hippocampus has also been attempted. Histologically, pyramidal cells in the hippocampal CA1 region demonstrated a marked necrosis on the fifth day after the occlusion (Fig. 1). This result is in line with findings by others suggestive of a hierarchy of neurons in the susceptibility to ischemic injury (selective neuronal vulnerability) as reported pre-
247
viously (Johansen et al., 1983; Kirino et al., 1984; Matsumoto et al., 1991). All neuroactive amino acids in the hippocampus showed a significant decrease on the fifth day after 20 min of ischemia (Fig. 3). These decreases were similar to those after 10 min ofischemia (Fig. 2). On the other hand, the content of neuroactive amino acids in other brain areas had no significant alterations on the fifth day following 10 min of ischemia. Furthermore, ACh content in the hippocampus was significantly reduced only on the fifth day after 20 min of ischemia (Fig. 4). These results suggest that both the contents of neuroactive amino acids and ACh in the hippocampus may be reduced following transient forebrain ischemia, but neurons containing of neuroactive amino acids may be more sensitive against ischemiainduced deterioration than that of ACh. The pyramidal cells in the hippocampal CA1 region have glutamatergic and aspartatergic neuronal inputs from the CA3 pyramidal cells (Schaffer collateralcommissural, Collingridge et al., 1983). On the other hand, these neurons in the hippocampal CA 1 region send outputs to the lateral septum (Storm-Mathisen and Opsahl, 1978 ; Yu et al., 1989) and the collaterals to hippocampal interneurons (Somogyi et al., 1983). GABAergic interneurons in the hippocampus mediate recurrent inhibition or feed-forward inhibition to the pyramidal neurons (Alger and Nicoll, 1982). On the other hand, taurine is one of the most abundant amino acids in the brain and considered to act as a neuromodulator which regulates calcium transport (Kuriyama et al., 1978). Some reports also have suggested that taurine coexists with G A B A in the same neuron (Chan-Palay et al., 1982), although the function of taurine in the brain is still obscure. Many studies conducting to clarify the effects of cerebral ischemia on hippocampal GAD, CAT and CSAD activities have indicated no significant changes in these enzyme activities in the hippocampus to ischemia insult (Francis and Pulsinelli, 1982; Schlander et al., 1988 ; Johansen et al., 1989 ; Chan-Palay et al., 1982), suggesting that the observed decrease in the contents of both neuroactive amino acids and ACh in the hippocampus after transient forebrain ischemia may be due to the reduction of intact neurons in this brain region rather than the changes in activities of these enzymes involved in biosyntheses of neuroactive amino acids and ACh. Previously reported evidences have suggested that the abnormal release of glutamate during ischemia induces an excessive calcium influx and subsequent non-specific activation of phospholipases which cause hydrolytic breakdown of membranous phospholipids
248
MASASHI KATSURA et al.
(Westerberg et al., 1987 ; Nedergard, 1988). Similarly, other neuroactive amino acids have been reported to be abnormally released during and after ischemia when monitored by brain dialysis (Benveniste et al., 1984 ; Matsumoto et al., 1991). It has been postulated that the activation of protein kinase C (PKC), which is highly concentrated in rat brain (Kikkawa et al., 1982), and translocation of the enzyme may play an important role in the postischemic modulation of synaptic efficacy and in the damage of C A 1 pyramidal cells (Onodera et al., 1989). Moreover, possible involvement of a similar mechanism in the release of neurotransmitters and synaptic plasticity have also been suggested (Nishizuka, 1986; Malenka et al., 1986). Considering these data, it may be possible that the increased release of neuroactive amino acids during transient forebrain ischemia and/or the neuronal damage through the activation of P K C may induce the secondary decreases in the contents of neuroactive amino acids and A C h through the death of CA1 pyramidal cells in the hippocampus. The detailed mechanisms underlying the decreases in the contents of neuroactive amino acids and ACh following transient cerebral ischemia remain to be elucidated in future studies.
REFERENCES
Alger B. E. and Nicoll R. A. (1982) Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studies in vitro. J. Physiol. 328, !05-123. Benveniste H., Drejer J., Schousboe A. and Diemer N. H. (1984) Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by microdialysis. J. Neurochem. 43, 1369-1376. Brierley J. B. (1984) Cerebral hypoxia, in Greenfield," Neuropathology (Adams J. H., Corsellis J. A. N. and Duchen L. W., eds), pp. 43-85. Edward Arnold Press, London. Butcher S. P. and Hamberger A. (1987) In vivo studies on extracellular, and veratrine-releasable, pools of endogenous amino acids in the rat striatum: effect of corticostriatal deafferentation or kainic acid lesion. J. Neurochem. 48, 713-721. Chan-Palay V., Lin C. T., Palay S., Yamamoto M. and Wu J. Y. (1982) Taurine in the mammalian cerebellum : demonstration by autoradiography with [3H]taurine and immunocytochemistry with antibodies against the taurinesynthesizing enzyme, cysteine sulfinic acid decarboxylase. Proc. natn. Acad. Sci. 79, 2695-2699. Collingridge G. L., Kehl S. J. and McLennan H. (1983) Excitatory amino acids in synaptic transmission in Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334, 33-46. Francis A. and PulsineUi W. A. (1982) The response of GABAergic and cholinergic neurons to transient cerebral ischemia. Brain Res. 243, 271-278. Fujimori K. and Yamamoto K. (1987) Determination of
acetylcholine and choline in perchlorate extracts of brain tissue using liquid chromatography-electrochemistry with an immobilized-enzyme reactor. J. Chromatoyr. 414, 167 173. Glowinski J. and Iversen L. L. (1966). Regional studies of catecholamines in rat brain I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13, 655--669. Ida S. and Kuriyama K. (1983) Simultaneous determination of CSA and CA in rat brain by high performance liquid chromatography. Analyt. Biochem. 130, 95 101. Johansen F. F., Jorgensen M. B. and Diemer N. H. (1983) Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat. Acta Neuropathol. 61, 135-140. Johansen F. F., Zimmer J. and Diemer N. H. (1987) Early loss of somatostatin neurons in dendate hitus after cerebral ischemia in the rat precedes CA I pyramidal cell loss. Acta Neuropathol. 73, 110-114. Johansen F. F., Lin C. T., Schousboe A. and Wu J. Y. (1989) Immunocytochemical investigation of L-glutamic acid decarboxylase in the rat hippocampal formation: the influence of transient cerebral ischemia. J. comp. Neurol. 281, 40-53. Kikkawa E., Takai Y., Minakuchi R., Inohara S. and Nishizuka Y. (1982) Calcium-activated phospholipiddependent protein kinase from rat brain. Subcellular distribution, purification and properties. J. biol. Chem. 257, 13341-13348. Kirino T. (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57~9. Kirino T., Tamura A. and Sano K. (1984) Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol. 64, 139-147. Kuriyama K., Muramatsu M., Nakagawa K. and Kakita K. (1978) Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissue. In : Taurine and Neuroloyicat Disorders (Barbeau A. and Huxtable R. J., eds), pp. 201-221. Raven Press, New York. Malenka R. C., Madison D. V. and Nicoll R. A. (1986) Potentiation of synaptic transmission in the hippocampus by phorbol esters. Nature 321, 175-177. Matsumoto K., Ueda S., Hashimoto T. and Kuriyama K. (1991) Evaluation of isehemic neuronal injury after transient forebrain ischemia in the rat hippocampus using in vivo mierodialysis. Brain Res. 543, 236-242. Munekata K. and Hossmann K. A. (1987) Effect of 5-minute ischemia on regional pH and energy state of the gerbil brain: relation to selective vulnerability of the hippocampus. Stroke 18, 412~417. Nedergaad M. (1988) Mechanisms of brain damage in focal cerebral ischemia. Acta Neurol. Scand. 77, 81-101. Nishizuka Y. (1986) Studies and perspectives of protein kinase C. Science 233, 305-312. Onodera H., Sato G. and Kogure K. (1986) Lesions to Schaffer collaterals prevent ischemic death of CAI pyramidal cells. Neurosci. Letl. 68, 169-174. Onodera H., Araki T. and Kogure K. (1989) Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate. Brain Res~ 481, 1 7. Potter P. E., Meek J. L. and NeffN. H. (1983) Acety!choline and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41,188 194.
Amino acids and ACh contents and cerebral ischemia Pulsinelli W. A. and Brierley J. B. (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267-272. Pulsinelli W. A., Brierley J. B. and Plum F. (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11,491-498. Rothman S. M. (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J. Neurosci. 4, 1884-1891. Rothman S. M. and Olney J. W. (1986) Glutamate and pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19, 105-111. Schlander M., Hoyer S. and Frotscher M. (1988) Glutamate decarboxylase-immunoactive neurons in the aging rat hippocampus and more resistant to ischemia than CA1 pyramidal cells. Neurosci. Left. 91, 241-246. Schmidt-Kastner R. and Hossmann K. A. (1988) Distribution of ischemic neuronal damage in the dorsal hippocampus of rat. Acta Neuropathol. 76, 411-421. Somogyi P., Nunzi M. G. and Smith A. D. (1983) A new type of specific interneuron in the monkey hippocampus
249
forming synapses exclusively with the axion initial segments of pyramidal cells. Brain Res. 259, 137-142. Storm-Mathisen J. and Opsahl M. W. (1978) Aspartate and/or glutamate may be transmitter in hippocampal efferents to septum and hypothalamus. Neurosci. Lett. 9, 65-70. Ungerstedt U. (1984) Measurement of neurotransmitters release by intracranial dialysis. In : Measurement of Neurotransmitter Release in Vivo ; Methods in the Neurascience, Vol. 6 (Marsaden C. A., eds), pp. 81-106. John Wiley,
Chichester. Westerberg E., Deshpande J. K. and Wieloch T. (1987) Regional differences in arachidonic acid relesae in rat hippocampus CA1 and CA2 regions during cerebral ischemia. J. cereb. Blood Flow Metab. 7, 189-192. Yu A. C. H., Gregory G. A. and Chan P. H. (1989) Hypoxiainduced dysfunctions and injury of astrocytes in primary cell cultures. J. cereb. Blood Flow Metab. 9, 20-28. Zaczek R., Hedreen J. C. and Coyle J. T. (1979) Evidence for a hippocampal-septal glutamatergic pathway in the rat. Exp. Neurol. 65, 14:~156.
0197-0186/92$5.00+0.00 Copyright© 1992PergamonPress Ltd
Printedin Great Britain.All rights reserved
CHANGES IN CONTENT OF NEUROACTIVE AMINO ACIDS A N D ACETYLCHOLINE IN THE RAT HIPPOCAMPUS FOLLOWING TRANSIENT FOREBRAIN ISCHEMIA MASASHI KATSURA, TOSHIAKI IINO a n d KINYA KURIYAMA* Department of Pharmacology, Kyoto Prefectural of University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan (Received 22 September 1991 ; accepted 27 December 1991)
Abstract--Changes in content of selected neuroactive amino acids [glutamicacid, aspartic acid, glycine,7aminobutyric acid (GABA) and taurine] and acetylcholine (ACh) in the rat hippocampus following transient forebrain ischemia were investigated using male Wistar rats. Rats were allowed to survive for 1 or 5 days following 10 or 20 min of 4-vessel occlusion, and killed by a focused microwave irradiation. A significantreduction in all neuroactive amino acids examined except GABA was noted in the hippocampus on the fifth day. One day after the 4-vessel occlusion for I0 min, no significant effect on the content of neuroactive amino acids in all brain areas was observed. ~,-Aminobutyricacid content in the hippocampus was only significantly reduced on the fifth day after the occlusion for 20 min. Similarly, a significant decrease in ACh content in the hippocampus was observed on the fifth day after the occlusion for 20 min. Considering the data that a significantloss of neuronal cells in the hippocampus (delayed neuronal death) was detected only 5 days after the 4-vessel occlusion, it can be said that the alterations in the hippocampus of neuroactive amino acids such as glutamic acid, aspartic acid, glycineand taurine are more sensitivethan those in GABA and ACh against cerebral ischemia. A possible correlation of these changes of neuroactive amino acids in the occurrence of delayed neuronal death in the hippocampus is also suggested.
It is well known that transient cerebral ischemia causes variable brain damage in proportion to its degree and duration. Pulsinelli et al. (1982) have reported that neurons in certain brain regions such as hippocampus, striatum, neocortex and thalamus are selectively vulnerable to ischemia. Hippocampal pyramidal cells in the CA1 layer are among the most vulnerable neurons to transient ischemia (Pulsinelli et al., 1982; Kirino, 1982). The death of these cells occurs gradually over a period of 2 days and this phenomenon has been termed "delayed neuronal death" (Kirino, 1982; Kirino et al., 1984; Johansen et al., 1987; Munekata and Hossmann, 1987). Although detailed mechanisms underlying these ischemia-induced neuronal degenerations or the death of hippocampal CA 1 neurons have not been clarified, it has been suggested that the neurotoxic action of synaptically released excitatory amino acids in areas vulnerable to ischemia plays an important role in the pathogenesis of ischemic neuronal death (Benveniste et al., 1984; Rothman and Olney, 1986; Onodera et
*Author to whom correspondence should be addressed.
al., 1986). Many studies have been conducted to clar-
ify the morphological changes seen after ischemia (Kirino, 1982; Johansen et al., 1983; Kirino et al., 1984; Schmidt-Kastner and Hossman, 1988) and the alterations in specific neuronal enzymatic signals such as glutamic acid decarboxylase (GAD, Francis and Pulsinelli, 1982 ; Schlander et al., 1988 ; Johansen et al., 1989), choline acetyltransferase (CAT, Francis and Pulsinelli, 1982) and cysteine-sulfinic acid decarboxylase (CSAD, Chan-Palay et al., 1982). In spite of the fact that the majority of hippocampal pyramidal cells is presumed to be glutamatergic and/or aspartatergic (Storm-Mathisen and Opsahl, 1978; Zaczek et al., 1979), few data on possible alterations in the activities of these enzymes related to the metabolism of excitatory amino acids. Recently, microdialysis technique (Ungerstedt, 1984) has been introduced and neuronal vulnerabilities following transient forebrain ischemia, deafferentation or kainic acid-induced cerebral lesion has been examined (Butcher and Hameberger, 1987 ; Matsumoto et al., 1991). In this study, the content of these neuroactive amino acids in various cerebral regions including hippocampus following transient 243
244
MASASHI KATSURA et al.
cerebral ischemia was measured. The effects o f transient cerebral ischemia on A C h c o n t e n t a n d morphological features in h i p p o c a m p a l n e u r o n s were also examined.
EXPERIMENTAL PROCEDURES
Animals Male Wistar rats weighing 250-300 g were purchased from Japan SLC, Inc. (Hamamatsu, Japan). They were used for the experiments after breeding for a week with laboratory chow (MF, Oriental Yeast Co., Ltd., Chiba, Japan) and tap water ad libitum. Application of cerebral ischemia Rats were subjected to forebrain ischemia by the 4-vessel occlusion procedure described by Pulsinelli and Brierlay (1979). Briefly, the rats were anesthetized with a mixture of 70% N20, 0.5% halothane and 30% O: and the vertebral arteries were electrocauterized. On the following day, the rats were reanesthetized and the carotid arteries were ligated by clips to produce the 4-vessel occlusion, and the rats exhibiting isoelectronic electroencephalogram were subjected to experiments. After 10 or 20 min occlusion, the clips were removed and carotid blood flow was reperfused. Histological examination Brain was perfused with 300 mt of 10% formalin saline via the left ventricle of the heart. The skull was then opened 1 h after the perfusion and the brain was removed and refixed for several days in 20% formalin. After dehydration and paraffin embedding, 4/~m of coronal section was stained with hematoxylin-eosin. Extraction of neuroactive amino acids and acetylcholine Rats were killed by focused microwave irradiation on the head (5 kW, 1.1 s). One or 5 days after the 4-vessel occlusion, the dorsal hippocampus, dorsolateral striatum and frontal cortex (lateral portion) were dissected out according to the method of Glowinski and Iversen (1%6). The tissue samples were subjected to extract neuroactive amino acids with 50 mM perchloric acid and diluted with 0.1 N HCI. Acetylcholine was extracted from the tissue samples according to the method of Potter et al. (1983) with a minor modification. The tissue samples were homogenized with 2 ml of 1 N formic acid:acetone (15:85) mixture containing I nM ethylhomocholine as an internal standard, and centrifuged at 1,700 g for 20 rain at 4°C. The supematant obtained was washed with diethyl ether and finally lyophilized at -20°C. After resuspending the lyophilized sample in distilled water, it was mixed with a solution containing KI-solution and 1 mM tetraethylammonium chloride before centrifuging at 10,000 g for 3 min at room temperature. In order to remove excessive iodine, the pellet was dissolved in acetonitrile and anion exchange resin (200-400 mesh, AG lX8 chloride form, BIO-RAD) was added. The iodine-free supernatant was then dried under a N2 stream and resuspended in distilled water for ACh determination.
Determinations o['neuroactive amino acid~ and acetylcholine To determine the content of cerebral amino acids, the extract from cerebral tissue homogenate was analyzed by a high performance liquid chromatography (HPLC) with fluorescent detection following post-labeling with o-phthalaldehyde by the method of Ida and Kuriyama (1963). For the measurement of cerebral ACh content, the extract was analyzed by a HPLC equipped with a precolumn (ACODS), a separate column (Eicompak AC-Gel), an immobilized column (AC-Enzympak, Eicom, Kyoto, Japan) and an electrochemical detector (VMD-101A, Yanaco, Kyoto, Japan) operating at a flow rate of 1 ml/min. The mobile phase consisted of 100 mM phosphate buffer (pH 8.0), 0.8 mM sodium 1-decansulfonate and 0.6 mM tetramethylammonium chloride. The detector potential was maintained at 450 mV against an Ag/AgC1 reference electrode (Potter et al., 1983 ; Fujimori and Yamamoto, 1987). Reagents Sodium 1-decansulfonate, tetramethylammonium chloride and tetraethylammonium chloride were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan) and Kanto Chemical Co., Ltd. (Tokyo, Japan), respectively. Other reagents used in this study were commercially available and of analytical grade. Statistics Each value was expressed as the mean + SEM, and statistical significance was determined by Student's or Aspin Welch's t-test. RESULTS Histological observations As s h o w n in Fig. 1, n e u r o n s in the h i p p o c a m p a l C A I region were morphologically intact o n the 1st day after 10 m i n o f 4-vessel occlusion a n d similar to those in s h a m - o p e r a t e d groups (data not shown). On the fifth day after 10 m i n of occlusion, n e u r o n a l damage in the h i p p o c a m p a l region became apparent. In the s h a m - o p e r a t e d group, average n e u r o n a l density in the C A I region was 121 _+ 7 cells/mm ( N = 5), while that in the g r o u p applied 10 m i n o f i s c h e m i a was 10 + 3 cells/mm (N = 5). In addition, the average n e u r o n a l density in the h i p p o c a m p a t CA1 region in sham-operated g r o u p was n o t different f r o m t h a t in n o n - t r e a t e d group (data n o t shown). The n e u r o n a l damages in the h i p p o c a m p a l C A I region o n the fifth day following 20 m i n of the occlusion was similar to t h a t in 10 min-occluded group, a l t h o u g h the extent o f n e u r o n a l d a m a g e s was more obvious t h a n t h a t in 10 m i n - o c c l u d e d g r o u p (data not shown). Content o f neuroactive amino acids One day after 10 a n d 20 m i n o f occlusion, the content of neuroactive a m i n o acids in the h i p p o c a m p u s showed n o significant changes [Figs 2(A) a n d 3(A)].
Amino acids and ACh contents and cerebral ischemia
245
Fig. 1. Histological changes in the CA1 region of rat hippocampus on the fifth day following 4-vessel occlusion. (A), (C): Sham-operated. The hippocampal CAI neurons are intact. (B), (D): 5 days after ischemic insults. Almost all hippocampal CA1 neurons are degenerated. Hematoxylin-eosin stain. Bar = 100 gm. DG : dentate gyrus.
On the other hand, all neuroactive amino acids measured, except GABA, significantly decreased in the hippocampus on the fifth day following 10 and 20 min of ischemia [taurine: 60% and 61%; aspartic acid: 70% and 87%; glutamic acid: 56% and 68%; glycine: 49% and 44%; G A B A : 66% and 73% . Figs 2(B) and 3(B)]. In contrast, G A B A content in the hippocampus was only significantly reduced in the 20 min occluded group. In other brain areas studied, the content of neuroactive amino acids showed no significant alteration following 10 and 20 min of occlusion (data not shown). Acetylcholine content
On the first day following 10 and 20 min of ischemia, the content of ACh showed no significant change in any brain areas. On the other hand, ACh content in the hippocampus on the fifth day following 20 min of occlusion decreased by 25% (P < 0.01) in comparison with that on the fifth day following 10 min of ischemia (Fig. 4).
DISCUSSION Neurons in the hippocampus are vulnerable to cerebral ischemia in man as well as experimental animals (Brierley, 1984). It has been shown that the CAI pyramidal cells and the CA3c/hilar somatostatinergic neurons are especially vulnerable to ischemia. CA1 pyramidal cells exhibit the "delayed neuronal death" phenomenon (Kirino, 1982; Kirino et al., 1984; Johansen et al., 1987; Munekata and Hossman, 1987). Namely, the ischemic loss of CA1 neurons delays 4 days following the loss of hilar somatostatinergic neurons during the first 2 days after the ischemia. This vulnerability of CA 1 pyramidal cells is believed to be due to the excitotoxicities of glutamate and aspartate mediated by N-methyl-D-aspartate (NMDA) type receptor (Rothman, 1984; SchmidtKastner and Hossmann, 1988). These ischemia-induced neuronal damages have been evaluated with regard to biochemical and immunohistochemical markers such as G A D for GABAergic neuron (Francis and Pulsinelli, 1982 ; Schlander et al.,
246
MASASHI KATSURA et al. Amino acids content 15
10
5
:
i
(/~mol/g we~ weight.)
O
Aspartic acid
0
5
10
i
i
T
**
t'nl GABA
I
i
~~
acid
I
15 [
~
I
coo
~ Ischemio
I
I
I
Fig. 2. Changes in neuroactive amino acid contents in hippocampus on the first and fifth day alter 10 min of cerebral ischemia. Rats were killed by microwave irradiation on the head on the first (A) and fifth (B) day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral amino acid contents by H P L C with fluorescent detection after post-labeling with o-phthalaldehyde. *P < 0.05 and **P < 0:01, compared with each control value. Each column represents the mean_+ SEM (N = 5 8).
Amino acids content (/~mol/g wet weight] 15 1
(A)
10 r
5 I
1 doy
~ /
-~N~
5 i
10 i
x~'~ **
L-
Taurine
s doy~
15
(B)
/
acid
,
oo.J °-,. ooo **F
-~
Glutomic acid
Glycine
l
I
GABA I
I
I
~ I
Ischemio
l: Control
: I
I
Fig. 3. Changes in neuroactive amino acid contents in the hippocampus on the first and fifth day after 20 min of cerebral ischemia. Rats were killed by microwave irradiation on the head on the first (A) and fifth (B) day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral amino acid content by H P L C with fluorescent detection after post-labeling with o-phthalaldehyde. *P < 0.05 and **P < 0.0l, compared with each control value. Each column represents the mean + SEM (N = 4 8).
Amino acids and ACh contents and cerebral ischemia
50
O
E 25 E
'l/
I
J: Control : Ischemio
O U e-
10 m i n
20 min
Fig. 4. Changes in ACh content in the hippocampus on the fifth day after 10 and 20 min of cerebral ischemia. Rats were sacrificed by microwave irradiation on the head on the fifth day after the occlusion. The extract from each cerebral tissue homogenate was analyzed to examine cerebral ACh contents by HPLC with electrochemical detection. **P < 0.01, compared with each control value. Each column represents the mean_+ SEM (N = 3-5). 1988; Johansen et al., 1989), CAT for cholinergic neuron (Francis and Pulsinelli, 1982) and CSAD for CSAD-positive (taurine) neuron (Chan-Palay et al., 1982), and these neurons in the hippocampus are relatively resistant to ischemic insult. Although the majority of pyramidal cells in the hippocampal CAI region has been considered to be glutamatergic and/or aspartatergic (Storm-Mathisen and Opsahl, 1978; Zaczek et al., 1979), the vulnerability of these neurons in the hippocampus has not been clearly evaluated. Recently, Matsumoto et al. (1991) have reported that the measurement of a high potassium-evoked release of neuroactive amino acids in vivo is useful for evaluating the vulnerability of neurons in rat hippocampal pyramidal cells after transient forebrain ischemia. We could not find, however, any reports on the changes in the content of neuroactive amino acids after cerebral ischemia. In this study, therefore, we have attempted to evaluate the vulnerability of neurons possessing neuroactive amino acids following transient ischemia by measuring their contents in the hippocampus and other brain regions. In addition, possible correlation of these changes with ACh content and morphological features in the hippocampus has also been attempted. Histologically, pyramidal cells in the hippocampal CA1 region demonstrated a marked necrosis on the fifth day after the occlusion (Fig. 1). This result is in line with findings by others suggestive of a hierarchy of neurons in the susceptibility to ischemic injury (selective neuronal vulnerability) as reported pre-
247
viously (Johansen et al., 1983; Kirino et al., 1984; Matsumoto et al., 1991). All neuroactive amino acids in the hippocampus showed a significant decrease on the fifth day after 20 min of ischemia (Fig. 3). These decreases were similar to those after 10 min ofischemia (Fig. 2). On the other hand, the content of neuroactive amino acids in other brain areas had no significant alterations on the fifth day following 10 min of ischemia. Furthermore, ACh content in the hippocampus was significantly reduced only on the fifth day after 20 min of ischemia (Fig. 4). These results suggest that both the contents of neuroactive amino acids and ACh in the hippocampus may be reduced following transient forebrain ischemia, but neurons containing of neuroactive amino acids may be more sensitive against ischemiainduced deterioration than that of ACh. The pyramidal cells in the hippocampal CA1 region have glutamatergic and aspartatergic neuronal inputs from the CA3 pyramidal cells (Schaffer collateralcommissural, Collingridge et al., 1983). On the other hand, these neurons in the hippocampal CA 1 region send outputs to the lateral septum (Storm-Mathisen and Opsahl, 1978 ; Yu et al., 1989) and the collaterals to hippocampal interneurons (Somogyi et al., 1983). GABAergic interneurons in the hippocampus mediate recurrent inhibition or feed-forward inhibition to the pyramidal neurons (Alger and Nicoll, 1982). On the other hand, taurine is one of the most abundant amino acids in the brain and considered to act as a neuromodulator which regulates calcium transport (Kuriyama et al., 1978). Some reports also have suggested that taurine coexists with G A B A in the same neuron (Chan-Palay et al., 1982), although the function of taurine in the brain is still obscure. Many studies conducting to clarify the effects of cerebral ischemia on hippocampal GAD, CAT and CSAD activities have indicated no significant changes in these enzyme activities in the hippocampus to ischemia insult (Francis and Pulsinelli, 1982; Schlander et al., 1988 ; Johansen et al., 1989 ; Chan-Palay et al., 1982), suggesting that the observed decrease in the contents of both neuroactive amino acids and ACh in the hippocampus after transient forebrain ischemia may be due to the reduction of intact neurons in this brain region rather than the changes in activities of these enzymes involved in biosyntheses of neuroactive amino acids and ACh. Previously reported evidences have suggested that the abnormal release of glutamate during ischemia induces an excessive calcium influx and subsequent non-specific activation of phospholipases which cause hydrolytic breakdown of membranous phospholipids
248
MASASHI KATSURA et al.
(Westerberg et al., 1987 ; Nedergard, 1988). Similarly, other neuroactive amino acids have been reported to be abnormally released during and after ischemia when monitored by brain dialysis (Benveniste et al., 1984 ; Matsumoto et al., 1991). It has been postulated that the activation of protein kinase C (PKC), which is highly concentrated in rat brain (Kikkawa et al., 1982), and translocation of the enzyme may play an important role in the postischemic modulation of synaptic efficacy and in the damage of C A 1 pyramidal cells (Onodera et al., 1989). Moreover, possible involvement of a similar mechanism in the release of neurotransmitters and synaptic plasticity have also been suggested (Nishizuka, 1986; Malenka et al., 1986). Considering these data, it may be possible that the increased release of neuroactive amino acids during transient forebrain ischemia and/or the neuronal damage through the activation of P K C may induce the secondary decreases in the contents of neuroactive amino acids and A C h through the death of CA1 pyramidal cells in the hippocampus. The detailed mechanisms underlying the decreases in the contents of neuroactive amino acids and ACh following transient cerebral ischemia remain to be elucidated in future studies.
REFERENCES
Alger B. E. and Nicoll R. A. (1982) Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studies in vitro. J. Physiol. 328, !05-123. Benveniste H., Drejer J., Schousboe A. and Diemer N. H. (1984) Elevation of the extracellular concentrations of glutamate and aspartate in the rat hippocampus during transient cerebral ischemia monitored by microdialysis. J. Neurochem. 43, 1369-1376. Brierley J. B. (1984) Cerebral hypoxia, in Greenfield," Neuropathology (Adams J. H., Corsellis J. A. N. and Duchen L. W., eds), pp. 43-85. Edward Arnold Press, London. Butcher S. P. and Hamberger A. (1987) In vivo studies on extracellular, and veratrine-releasable, pools of endogenous amino acids in the rat striatum: effect of corticostriatal deafferentation or kainic acid lesion. J. Neurochem. 48, 713-721. Chan-Palay V., Lin C. T., Palay S., Yamamoto M. and Wu J. Y. (1982) Taurine in the mammalian cerebellum : demonstration by autoradiography with [3H]taurine and immunocytochemistry with antibodies against the taurinesynthesizing enzyme, cysteine sulfinic acid decarboxylase. Proc. natn. Acad. Sci. 79, 2695-2699. Collingridge G. L., Kehl S. J. and McLennan H. (1983) Excitatory amino acids in synaptic transmission in Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334, 33-46. Francis A. and PulsineUi W. A. (1982) The response of GABAergic and cholinergic neurons to transient cerebral ischemia. Brain Res. 243, 271-278. Fujimori K. and Yamamoto K. (1987) Determination of
acetylcholine and choline in perchlorate extracts of brain tissue using liquid chromatography-electrochemistry with an immobilized-enzyme reactor. J. Chromatoyr. 414, 167 173. Glowinski J. and Iversen L. L. (1966). Regional studies of catecholamines in rat brain I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13, 655--669. Ida S. and Kuriyama K. (1983) Simultaneous determination of CSA and CA in rat brain by high performance liquid chromatography. Analyt. Biochem. 130, 95 101. Johansen F. F., Jorgensen M. B. and Diemer N. H. (1983) Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat. Acta Neuropathol. 61, 135-140. Johansen F. F., Zimmer J. and Diemer N. H. (1987) Early loss of somatostatin neurons in dendate hitus after cerebral ischemia in the rat precedes CA I pyramidal cell loss. Acta Neuropathol. 73, 110-114. Johansen F. F., Lin C. T., Schousboe A. and Wu J. Y. (1989) Immunocytochemical investigation of L-glutamic acid decarboxylase in the rat hippocampal formation: the influence of transient cerebral ischemia. J. comp. Neurol. 281, 40-53. Kikkawa E., Takai Y., Minakuchi R., Inohara S. and Nishizuka Y. (1982) Calcium-activated phospholipiddependent protein kinase from rat brain. Subcellular distribution, purification and properties. J. biol. Chem. 257, 13341-13348. Kirino T. (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57~9. Kirino T., Tamura A. and Sano K. (1984) Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol. 64, 139-147. Kuriyama K., Muramatsu M., Nakagawa K. and Kakita K. (1978) Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissue. In : Taurine and Neuroloyicat Disorders (Barbeau A. and Huxtable R. J., eds), pp. 201-221. Raven Press, New York. Malenka R. C., Madison D. V. and Nicoll R. A. (1986) Potentiation of synaptic transmission in the hippocampus by phorbol esters. Nature 321, 175-177. Matsumoto K., Ueda S., Hashimoto T. and Kuriyama K. (1991) Evaluation of isehemic neuronal injury after transient forebrain ischemia in the rat hippocampus using in vivo mierodialysis. Brain Res. 543, 236-242. Munekata K. and Hossmann K. A. (1987) Effect of 5-minute ischemia on regional pH and energy state of the gerbil brain: relation to selective vulnerability of the hippocampus. Stroke 18, 412~417. Nedergaad M. (1988) Mechanisms of brain damage in focal cerebral ischemia. Acta Neurol. Scand. 77, 81-101. Nishizuka Y. (1986) Studies and perspectives of protein kinase C. Science 233, 305-312. Onodera H., Sato G. and Kogure K. (1986) Lesions to Schaffer collaterals prevent ischemic death of CAI pyramidal cells. Neurosci. Letl. 68, 169-174. Onodera H., Araki T. and Kogure K. (1989) Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate. Brain Res~ 481, 1 7. Potter P. E., Meek J. L. and NeffN. H. (1983) Acety!choline and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41,188 194.
Amino acids and ACh contents and cerebral ischemia Pulsinelli W. A. and Brierley J. B. (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267-272. Pulsinelli W. A., Brierley J. B. and Plum F. (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11,491-498. Rothman S. M. (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J. Neurosci. 4, 1884-1891. Rothman S. M. and Olney J. W. (1986) Glutamate and pathophysiology of hypoxic-ischemic brain damage. Ann. Neurol. 19, 105-111. Schlander M., Hoyer S. and Frotscher M. (1988) Glutamate decarboxylase-immunoactive neurons in the aging rat hippocampus and more resistant to ischemia than CA1 pyramidal cells. Neurosci. Left. 91, 241-246. Schmidt-Kastner R. and Hossmann K. A. (1988) Distribution of ischemic neuronal damage in the dorsal hippocampus of rat. Acta Neuropathol. 76, 411-421. Somogyi P., Nunzi M. G. and Smith A. D. (1983) A new type of specific interneuron in the monkey hippocampus
249
forming synapses exclusively with the axion initial segments of pyramidal cells. Brain Res. 259, 137-142. Storm-Mathisen J. and Opsahl M. W. (1978) Aspartate and/or glutamate may be transmitter in hippocampal efferents to septum and hypothalamus. Neurosci. Lett. 9, 65-70. Ungerstedt U. (1984) Measurement of neurotransmitters release by intracranial dialysis. In : Measurement of Neurotransmitter Release in Vivo ; Methods in the Neurascience, Vol. 6 (Marsaden C. A., eds), pp. 81-106. John Wiley,
Chichester. Westerberg E., Deshpande J. K. and Wieloch T. (1987) Regional differences in arachidonic acid relesae in rat hippocampus CA1 and CA2 regions during cerebral ischemia. J. cereb. Blood Flow Metab. 7, 189-192. Yu A. C. H., Gregory G. A. and Chan P. H. (1989) Hypoxiainduced dysfunctions and injury of astrocytes in primary cell cultures. J. cereb. Blood Flow Metab. 9, 20-28. Zaczek R., Hedreen J. C. and Coyle J. T. (1979) Evidence for a hippocampal-septal glutamatergic pathway in the rat. Exp. Neurol. 65, 14:~156.