Brain Research, 530 (1990) 257-260
257
Elsevier BRES 15940
Postischemic alterations of spontaneous activities in rat hippocampal CA1 neurons Katsutoshi Furukawa, Kenjirou Yamana and Kyuya Kogure Department of Neurology, Institute of Brain Disease, Tohoku University School of Medicine, Sendai (Japan) (Accepted 24 April 1990)
Key words: Delayed neuronal death; Hippocampus; Ischemia; Neuronal activity; Rat Changes of spontaneous impulse discharges in rat hippocampal neurons during and after transient forebrain ischemia were investigated electrophysiologically. Spontaneous impulse frequencies of CA1 neurons before ischemia were varied from 0.4 to 20.0 impulses/s and its average was 5.8 + 1.2 (means + S.E., n = 36). These spontaneous discharges were completely suppressed during forebrain ischemia exept for the transient hyperactivity observed just after the beginning of ischemia. Recovery of spontaneous discharges of CA1 neurons from suppression induced by 5 min ischemia started at 5 min, and neuronal activities were restored to pre-ischemic levels approximately 30 min after reperfusion. On the other hand, spontaneous impulse frequencies at all time points recorded after 20 min ischemia were less than 40% of the pre-ischemic levels. These continuous suppression of spontaneous activity after 20 min ischemia may suggest that neuronal function is impaired during and/or in the early stages of reperfusion, and functional disorders precede morphological degeneration. INTRODUCTION D e l a y e d n e u r o n a l d e a t h f o u n d in rats 14 and gerbils 7 has b e e n u s e d as a m o d e l of n e u r o n a l d e g e n e r a t i o n in the c e n t r a l n e r v o u s system.
Following transient ischemia,
d e g e n e r a t i o n o f h i p p o c a m p a l C A 1 n e u r o n s occurs after a d e l a y of a few days. S e v e r a l studies h a d c h a r a c t e r i z e d the p a t h o m e c h a n i s m s of d e l a y e d n e u r o n a l d e a t h , with respect to m o r p h o l o g i c a l s-~°, b i o c h e m i c a l 12"16, circulat013'17'19 c h a n g e s . T h e r e are, h o w e v e r , few e l e c t r o p h y siological r e p o r t s and little is k n o w n a b o u t functional c h a n g e s o f v u l n e r a b l e n e u r o n s d u r i n g and after a transient i s c h e m i a . In t h e p r e s e n t study, we m e a s u r e d the s p o n t a n e o u s activity o f rat h i p p o c a m p a l C A 1 n e u r o n s d u r i n g and after
bilateral common carotid arteries and the trachea were isolated through a ventral, midline cervical incision. Cotton thread was looped around each common carotid artery and it was passed through a polyethylene tube (10 cm in length, PE-160, Intramedic). Transient ischemia and reperfusion was induced by drawing and releasing the thread manually 5. The head of the rat was fixed on a stereotaxic instrument and the rat was artificially ventilated with anesthetic gas. The respirator was regulated so as to maintain physiological conditions (PaO2>100 mgHg, PaCO2 35-40 mmHg). The temperature was kept close to 37 °C by a heating mat. For recording spontaneous activity of CA1 neurons, a small bony opening was made 3.9 mm posterior and 2.1 mm lateral to the bregma using a dental drill. The indifferent electrode was a stainless screw placed on the frontal bone, and the recording electrode, a sharpened tungsten wire insulated with lacquer (tip diameter less than 1 /tm) inserted through the opening. The electrode was advanced carefully till stable recordings from CA1 neurons could be obtained (the depth was about 2.0-2.3 mm from the surface of the brain).
t r a n s i e n t ischemia. T h e m a i n p u r p o s e o f the p r e s e n t
Chronic experiment
study is to a d v a n c e o u r u n d e r s t a n d i n g s of the p a t h o m e -
Twenty-four hours after vertebral artery cauterization, the rats were anesthetized with 1% halothane. Both common carotid arteries were isolated through a neck incision. The blood flow of the common carotid arteries was occluded by aneurysm clips. While ischemia of 5 or 20 min durations were applied, the anesthetic gas was changed to room air. After a 3-72 h recovery period, the rat was reanesthetized with 1% halothane and fixed on a stereotaxic instrument. Spontaneous action potentials were recorded as described above.
c h a n i s m s o f d e l a y e d n e u r o n a l d e a t h by clarifying changes in t h e electrical activity o f v u l n e r a b l e n e u r o n s . MATERIALS AND METHODS
Animal preparation Male Wistar rats weighing 270-310 g were used in this study. The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and their bilateral vertebral arteries were occluded by electrocautery as described by Pulsine,i and Brierley 14.
Acute experiment Twenty-four hours after occluding vertebral arteries, the rats were anesthetized with 1% halothane in 30% 02/70% N20. Their
Morphology At the end of experiments, we applied DC current (30 gA, 20 s) through recording electrode so as to make a burn at the recording site. Animals were perfusion-fixed with FAM via the ascending aorta and the brain was removed and immersed in fixative for another 12 h. The brain was then embedded in paraffin. Five-gm paraffin sections were stained with Cresyl violet and the burn at the
Correspondence: K. Furukawa, Department of Neurology, Institute of Brain Disease, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai, Japan. 0006-8993/90/$03.50 (C) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
258 A
B
__i ............... ----~
. . . . . . .
[~._:Jll
Jr - r ~ l f
l1
I~
l~'l
r~rr~ r 7 F l l l l ~
Irlr vl iTIIIIITpI[rTTITIIIII rj i
k
C
D I
2 sec.
I
Fig. l. Typical recording form of discharges of a hippocampal CA1 neuron. A: before ischemia. B: just after the ischemic insult. An arrowhead indicates the beginning of ischemia. C: 20 min after the reperfusion from 5 min ischemia. D: 2 h after the reperfusion.
recording site together with the morphological changes of CA1 neurons were examined by a light microscope. RESULTS Spontaneous impulse frequencies of hippocampal CA1 neurons from intact, anesthetized rats varied from 0.4 to 20.0 impulses/s, with their average value being 5.8 + 1.2 (mean + S.E., n = 36). During our recordings, spontaneous impulse frequencies were little changed over 2 h. In the control group, an average spontaneous impulse frequency of 6.6 + 1.3 impulses/s changed to 7.1 _+ 1.4 (n = 6) during 2 h of continuous recording. The activity of these neurons increased transiently for 10-15 s after the beginning of ischemia, but spontaneous discharges disappeared within 40 s. When the duration of ischemia was 5 min, recovery of spontaneous activity was noted about 5 min after cerebral reperfusion. Following transient hyperactivity of 10-30 min duration, spontaneous impulse discharges returned to the pre-ischemic level. A typical recording from hippocampal CA1 neuron is shown in Fig. 1, and the time-course of changes in spontaneous impulse frequencies after 5 min ischemia is shown in Figs. 2 and 3A. Transient hyperactivities 15 min after cerebral reperfusion were observed in neurons in 13 of 14 single unit recordings. Spontaneous impulse frequency 2 h after 5 min ischemia was 3.99 + 0.35, n = 14. In the chronic experiments, neuronal activity recorded 6-72 h after 5 min ischemia was similar to the control
level, but slightly depressed at 3 h: 2.34 + 0.57, n = 5 (Fig. 4). The pyramidal cells of CA1 region at 3 days after 5 min ischemia showed almost no necrotic change exept for a few animals whose 10% of pyramidal cells in paramedian CA1 region revealed necrosis. Changes in the spontaneous impulse frequencies of CA1 neurons induced by 20 min ischemia were quite different from those induces by 5 min ischemia (Fig. 3B). During a continuous recording period of 2 h, recovery of spontaneous activity was observed in only 1 of 5 neurons subjected to 20 min ischemia. The other 4 neurons showed only several impulse discharges during the 2-h reperfusion period. Such depressed spontaneous impulse discharge was observed in the chronic experiments (Fig. 4). Because of the depressed activities of CA1 neurons,
A 10
& 20
I Fig. 2. Change of spontaneous activity of a CA1 neuron. Each column indicates the number of impulses per 10 s. During ischemic periods of 5 min (horizontal bar under the histogram), impulse discharges were completely suppressed except for transient hyperactivity found in the first 20 s. Numerals under the histogram show the time after reperfusion (min). A vertical scale indicates 50 impulses per 10 s.
259
(B)
t
8
|s
6
| m
_z ~.4
~.4 E
•
g
2
/
i
i
I
I
0.5
1
1.5
2
Time
0.5
1
1.5
2
Time (hour)
(hour)
Fig. 3. The time-course of the change of the spontaneous impulse frequencies of CA1 neurons after transient ischemia (A: 5 min, B: 20 min). The points indicate the mean frequency in 14 rats for 5 min ischemia and in 5 rats for 20 min ischemia, respectively. Vertical lines represent S.E.
it was very difficult in the chronic experiments to make recordings from rats subjected to 20 min ischemia. In particular, single unit recordings could be made from only 6 of 13 rats examined 3 h after 20 min ischemia, and 5 of 9 rats 6 h after 20 rain ischemia. At 3 days after 20 min ischemia, more than 50% of the pyramidal cells of CA1 region showed necrotic change in all of the examined rats. DISCUSSION In the present study, the spontaneous activity of hippocampal CA1 neurons was completely suppressed during the course of ischemic periods, and the forms of recovery from ischemia-induced suppression differed according to the duration of the ischemic period. The spontaneous activity of CA1 neurons recorded approximately 30 min after 5 min ischemia was indistinguishable from that recorded in the control group. However,
10
Cont.
3
6
24
72
Time (hour)
Fig. 4. Spontaneous impulse frequencies at 3, 6, 24 and 72 h after reperfusion in the chronic experiments. The closed, open, and hatched columns represent control, 5 min ischemia, and 20 min ischemia, respectively, n = 36 in the control experiments and n = 5-8 for each point in the chronic experiments. Vertical lines represent S.E.
neuronal activity after 20 min ischemia was strongly suppressed at all points in time examined in the present study. Thus, the results obtained in the present study indicate that the function of CA1 neurons in the rat hippocampus is not affected by 5 min ischemia, but is disordered by 20 min ischemia. Dysfunction of CA1 neurons by transient ischemia seemed to occur during ischemia and/or of early stages of reperfusion. In the previous studies of anoxic model using brain slices, hippocampal neurons hyperpolarized 5-15 mV in amplitude and 4-12 min in duration after the beginning of anoxic insult 4'zl. The hyperpolarization was followed by slow depolarization which reached a plateau level of about 25 mV above prehypoxic resting potential within 20 min 4. Interestingly, this slow depolarization occurs without impulse discharges. Although we did not refer to the membrane potentials of CA1 neurons, dysfunction of CA1 neurons may be triggered by the slow depolarization. It has been well known that hippocampal CA1 neurons are selectively vulnerable to transient forebrain ischemia. In the reports which described about the time-course of morphological change of hippocampus in detail, degeneration of CA1 neurons was not observed 6 h after transient ischemia, however, it was seen at 24 h after ischemia 15. Electron-microscopic observations found a similar time-course of neuronal degeneration 1°. Our morphological examination revealed similar results compare to the previous reports 3'15. Namely, most pyramidal cells of the CA1 region showed severe damage at 3 days after 20 min ischemia and a small percentage of pyramidal cells showed necrotic changes at 3 days after 5 min ischemia. This delay of structural damage found in morphological experiments is in striking contrast to the immediate functional damage observed in the present study. In spite of the delayed occurrence of structural degeneration, some cellular functions of CA1 neurons
260 such as protein synthesis2"6"21 and protein kinase C activity 13 were changed during ischemia, and/or in the early stage of reperfusion. These changes of cellular functions may effect the excitabilities of CA1 neurons. In Mongolian gerbils, it has been reported that the hyperactivity of hippocampal CA1 neurons is consistently observed after 24 h of 5 min ischemia, and followed by the selective degeneration of CA1 pyramidal cells 2°. The hyperactivity reported in Mongolian gerbils was supposed to be related to 'excitotoxicity' induced by excitatory amino acids. In the rats, release of exitatory amino acids such as glutamate and aspartate increases during ischREFERENCES 1 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 intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. 2 Bodsch, W., Takahashi, K., Barbier, A., Ophoff, B.G. and Hossmann, K.A., Cerebral protein synthesis and ischemia. In K. Kogure, K.-A. Hossmann, B.K. Siesj6 and EA. Welsh (Eds.), Molecular Mechanisms of lschemic Brain, (Progress in Brain Research, Voi. 63), Elsevier, Amsterdam, 1985, pp. 197-210. 3 Buchan, A. and Pulsinelli, W.A., Neurotransmitters and neurotrophic factors in cerebral ischemia towards a molecular explanation for selective neuronal injury and a new rationale for stroke treatment. In H. Takeshita (Ed.), International Symposium of Brain Resuscitation, Ube/Yamaguchi, Japan, 1988, pp. 5-11. 4 Fujiwara, N., Higashi, H., Shimoji, K. and Yoshimura, M., Effects of hypoxia on rat hippocampal neurons in vitro, J. Physiol., 384 (1987) 131-151. 5 Itoh, T., Kawakami, M., Yamauchi, Y., Shimizu, S. and Nakamura, M., Effect of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats, Stroke, 17 (1986) 1284-1287. 6 Kiessling, M., Dienel, G.A., Jacewicz, M. and Pulsinelli, W.A., Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis, J. Cereb. Blood Flow Metab., 6 (1986) 642-649. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. 8 Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta Neuropathol., 62 (1984) 201-208. 9 Kirino, T. and Sano, K., Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus, Acta Neuropathol., 62 (1984) 209-218. 10 Kirino, T., Tamura, A. and Sano, K., Selective vulnerability of the hippocampus to ischemia-reversible and irreversible types of ischemic cell damage. In K. Kogure, K.-A. Hossmann, B.K.
emia, but it returned to the pre-ischemic level approximately 20-25 min after reperfusion ~. Thus, changes in release of exitatory amino acids may not play an important role in neuronal excitability after reperfusion. Another possibility exists, viz, that the suppression of neuronal activity may due to changes in the balance between excitatory and inhibitory input. In the rat hippocampus, GABAergic interneurons are reportedly more resistant to ischemia than CA1 pyramidal cells TM. Therefore, depression of spontaneous activity of CA1 neurons after transient ischemia may be due to the predominant innervation of inhibitory neurons. Siesj6 and F.A. Welsh (Eds.), Molecular Mechanisms of lschemic Brain, (Progress in Brain Research, Vol. 63), Elsevier, Amsterdam, 1985, pp. 39-58. 11 Lebrond, J. and Krnjevi~, K., Hypoxic changes in hippocampal neurons, J. Neurophysiol., 61 (1989) 1-14. 12 Onodera, H., Iijima, K. and Kogure, K., Mononucleotide metabolism in the rat brain after transient ischemia, J. Neurochem., 46 (1986) 1704-1710. 13 Onodera, H., Araki, T. and Kogure, K., Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3H]phorbol 12,13-dibutyrate, Brain Research, 481 (1989) 1-7. 14 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 15 Pulsinelli, W.A., Brierly, J.B. and Plum, E, Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 16 Pulsinelli, W.A. and Duffy, T.E., Regional energy balance in rat brain after transient forebrain ischemia, J. Neurochem., 40 (1983) 1500-1503. 17 Pulsinelli, W.A., Levy, D.E. and Duffy, T.E., Regional cerebral blood flow and glucose metabolism following transient forebrain ischemia, Ann. Neurol., 11 (1982) 499-509. 18 Schlander, M., Hoyer, S. and Frotscher, M., Glutamate decarboxylase-immunoreactive neurons in the aging rat hippocampus are more resistant to ischemia than CA1 pyramidal cells, Neurosci. Lett., 91 (1988) 241-246. 19 Suzuki, R., Yamaguchi, T., Kirino, T., Orzi, F. and Klatzo, I., The effects of 5-minute ischemia in Mongolian gerbils, I. Blood-brain barrier, cerebral blood flow, glucose utilization changes, Acta Neuropathol., 60 (1983) 207-216. 20 Suzuki, R., Yamaguchi, T., Choh-Luh Li and Klatzo, I., The effects of 5-minute ischemia in Mongolian gerbils, II. Changes of spontaneous neuronal activity in cerebral cortex and CA1 sector of hippocampus, Acta Neuropathol., 60 (1983) 217-222. 21 Thilmann, R., Xie, Y., Kleihues, P. and Kiessling, M., Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus, Acta Neuropathol., 71 (1986) 88-93.
257
Elsevier BRES 15940
Postischemic alterations of spontaneous activities in rat hippocampal CA1 neurons Katsutoshi Furukawa, Kenjirou Yamana and Kyuya Kogure Department of Neurology, Institute of Brain Disease, Tohoku University School of Medicine, Sendai (Japan) (Accepted 24 April 1990)
Key words: Delayed neuronal death; Hippocampus; Ischemia; Neuronal activity; Rat Changes of spontaneous impulse discharges in rat hippocampal neurons during and after transient forebrain ischemia were investigated electrophysiologically. Spontaneous impulse frequencies of CA1 neurons before ischemia were varied from 0.4 to 20.0 impulses/s and its average was 5.8 + 1.2 (means + S.E., n = 36). These spontaneous discharges were completely suppressed during forebrain ischemia exept for the transient hyperactivity observed just after the beginning of ischemia. Recovery of spontaneous discharges of CA1 neurons from suppression induced by 5 min ischemia started at 5 min, and neuronal activities were restored to pre-ischemic levels approximately 30 min after reperfusion. On the other hand, spontaneous impulse frequencies at all time points recorded after 20 min ischemia were less than 40% of the pre-ischemic levels. These continuous suppression of spontaneous activity after 20 min ischemia may suggest that neuronal function is impaired during and/or in the early stages of reperfusion, and functional disorders precede morphological degeneration. INTRODUCTION D e l a y e d n e u r o n a l d e a t h f o u n d in rats 14 and gerbils 7 has b e e n u s e d as a m o d e l of n e u r o n a l d e g e n e r a t i o n in the c e n t r a l n e r v o u s system.
Following transient ischemia,
d e g e n e r a t i o n o f h i p p o c a m p a l C A 1 n e u r o n s occurs after a d e l a y of a few days. S e v e r a l studies h a d c h a r a c t e r i z e d the p a t h o m e c h a n i s m s of d e l a y e d n e u r o n a l d e a t h , with respect to m o r p h o l o g i c a l s-~°, b i o c h e m i c a l 12"16, circulat013'17'19 c h a n g e s . T h e r e are, h o w e v e r , few e l e c t r o p h y siological r e p o r t s and little is k n o w n a b o u t functional c h a n g e s o f v u l n e r a b l e n e u r o n s d u r i n g and after a transient i s c h e m i a . In t h e p r e s e n t study, we m e a s u r e d the s p o n t a n e o u s activity o f rat h i p p o c a m p a l C A 1 n e u r o n s d u r i n g and after
bilateral common carotid arteries and the trachea were isolated through a ventral, midline cervical incision. Cotton thread was looped around each common carotid artery and it was passed through a polyethylene tube (10 cm in length, PE-160, Intramedic). Transient ischemia and reperfusion was induced by drawing and releasing the thread manually 5. The head of the rat was fixed on a stereotaxic instrument and the rat was artificially ventilated with anesthetic gas. The respirator was regulated so as to maintain physiological conditions (PaO2>100 mgHg, PaCO2 35-40 mmHg). The temperature was kept close to 37 °C by a heating mat. For recording spontaneous activity of CA1 neurons, a small bony opening was made 3.9 mm posterior and 2.1 mm lateral to the bregma using a dental drill. The indifferent electrode was a stainless screw placed on the frontal bone, and the recording electrode, a sharpened tungsten wire insulated with lacquer (tip diameter less than 1 /tm) inserted through the opening. The electrode was advanced carefully till stable recordings from CA1 neurons could be obtained (the depth was about 2.0-2.3 mm from the surface of the brain).
t r a n s i e n t ischemia. T h e m a i n p u r p o s e o f the p r e s e n t
Chronic experiment
study is to a d v a n c e o u r u n d e r s t a n d i n g s of the p a t h o m e -
Twenty-four hours after vertebral artery cauterization, the rats were anesthetized with 1% halothane. Both common carotid arteries were isolated through a neck incision. The blood flow of the common carotid arteries was occluded by aneurysm clips. While ischemia of 5 or 20 min durations were applied, the anesthetic gas was changed to room air. After a 3-72 h recovery period, the rat was reanesthetized with 1% halothane and fixed on a stereotaxic instrument. Spontaneous action potentials were recorded as described above.
c h a n i s m s o f d e l a y e d n e u r o n a l d e a t h by clarifying changes in t h e electrical activity o f v u l n e r a b l e n e u r o n s . MATERIALS AND METHODS
Animal preparation Male Wistar rats weighing 270-310 g were used in this study. The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and their bilateral vertebral arteries were occluded by electrocautery as described by Pulsine,i and Brierley 14.
Acute experiment Twenty-four hours after occluding vertebral arteries, the rats were anesthetized with 1% halothane in 30% 02/70% N20. Their
Morphology At the end of experiments, we applied DC current (30 gA, 20 s) through recording electrode so as to make a burn at the recording site. Animals were perfusion-fixed with FAM via the ascending aorta and the brain was removed and immersed in fixative for another 12 h. The brain was then embedded in paraffin. Five-gm paraffin sections were stained with Cresyl violet and the burn at the
Correspondence: K. Furukawa, Department of Neurology, Institute of Brain Disease, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aobaku, Sendai, Japan. 0006-8993/90/$03.50 (C) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
258 A
B
__i ............... ----~
. . . . . . .
[~._:Jll
Jr - r ~ l f
l1
I~
l~'l
r~rr~ r 7 F l l l l ~
Irlr vl iTIIIIITpI[rTTITIIIII rj i
k
C
D I
2 sec.
I
Fig. l. Typical recording form of discharges of a hippocampal CA1 neuron. A: before ischemia. B: just after the ischemic insult. An arrowhead indicates the beginning of ischemia. C: 20 min after the reperfusion from 5 min ischemia. D: 2 h after the reperfusion.
recording site together with the morphological changes of CA1 neurons were examined by a light microscope. RESULTS Spontaneous impulse frequencies of hippocampal CA1 neurons from intact, anesthetized rats varied from 0.4 to 20.0 impulses/s, with their average value being 5.8 + 1.2 (mean + S.E., n = 36). During our recordings, spontaneous impulse frequencies were little changed over 2 h. In the control group, an average spontaneous impulse frequency of 6.6 + 1.3 impulses/s changed to 7.1 _+ 1.4 (n = 6) during 2 h of continuous recording. The activity of these neurons increased transiently for 10-15 s after the beginning of ischemia, but spontaneous discharges disappeared within 40 s. When the duration of ischemia was 5 min, recovery of spontaneous activity was noted about 5 min after cerebral reperfusion. Following transient hyperactivity of 10-30 min duration, spontaneous impulse discharges returned to the pre-ischemic level. A typical recording from hippocampal CA1 neuron is shown in Fig. 1, and the time-course of changes in spontaneous impulse frequencies after 5 min ischemia is shown in Figs. 2 and 3A. Transient hyperactivities 15 min after cerebral reperfusion were observed in neurons in 13 of 14 single unit recordings. Spontaneous impulse frequency 2 h after 5 min ischemia was 3.99 + 0.35, n = 14. In the chronic experiments, neuronal activity recorded 6-72 h after 5 min ischemia was similar to the control
level, but slightly depressed at 3 h: 2.34 + 0.57, n = 5 (Fig. 4). The pyramidal cells of CA1 region at 3 days after 5 min ischemia showed almost no necrotic change exept for a few animals whose 10% of pyramidal cells in paramedian CA1 region revealed necrosis. Changes in the spontaneous impulse frequencies of CA1 neurons induced by 20 min ischemia were quite different from those induces by 5 min ischemia (Fig. 3B). During a continuous recording period of 2 h, recovery of spontaneous activity was observed in only 1 of 5 neurons subjected to 20 min ischemia. The other 4 neurons showed only several impulse discharges during the 2-h reperfusion period. Such depressed spontaneous impulse discharge was observed in the chronic experiments (Fig. 4). Because of the depressed activities of CA1 neurons,
A 10
& 20
I Fig. 2. Change of spontaneous activity of a CA1 neuron. Each column indicates the number of impulses per 10 s. During ischemic periods of 5 min (horizontal bar under the histogram), impulse discharges were completely suppressed except for transient hyperactivity found in the first 20 s. Numerals under the histogram show the time after reperfusion (min). A vertical scale indicates 50 impulses per 10 s.
259
(B)
t
8
|s
6
| m
_z ~.4
~.4 E
•
g
2
/
i
i
I
I
0.5
1
1.5
2
Time
0.5
1
1.5
2
Time (hour)
(hour)
Fig. 3. The time-course of the change of the spontaneous impulse frequencies of CA1 neurons after transient ischemia (A: 5 min, B: 20 min). The points indicate the mean frequency in 14 rats for 5 min ischemia and in 5 rats for 20 min ischemia, respectively. Vertical lines represent S.E.
it was very difficult in the chronic experiments to make recordings from rats subjected to 20 min ischemia. In particular, single unit recordings could be made from only 6 of 13 rats examined 3 h after 20 min ischemia, and 5 of 9 rats 6 h after 20 rain ischemia. At 3 days after 20 min ischemia, more than 50% of the pyramidal cells of CA1 region showed necrotic change in all of the examined rats. DISCUSSION In the present study, the spontaneous activity of hippocampal CA1 neurons was completely suppressed during the course of ischemic periods, and the forms of recovery from ischemia-induced suppression differed according to the duration of the ischemic period. The spontaneous activity of CA1 neurons recorded approximately 30 min after 5 min ischemia was indistinguishable from that recorded in the control group. However,
10
Cont.
3
6
24
72
Time (hour)
Fig. 4. Spontaneous impulse frequencies at 3, 6, 24 and 72 h after reperfusion in the chronic experiments. The closed, open, and hatched columns represent control, 5 min ischemia, and 20 min ischemia, respectively, n = 36 in the control experiments and n = 5-8 for each point in the chronic experiments. Vertical lines represent S.E.
neuronal activity after 20 min ischemia was strongly suppressed at all points in time examined in the present study. Thus, the results obtained in the present study indicate that the function of CA1 neurons in the rat hippocampus is not affected by 5 min ischemia, but is disordered by 20 min ischemia. Dysfunction of CA1 neurons by transient ischemia seemed to occur during ischemia and/or of early stages of reperfusion. In the previous studies of anoxic model using brain slices, hippocampal neurons hyperpolarized 5-15 mV in amplitude and 4-12 min in duration after the beginning of anoxic insult 4'zl. The hyperpolarization was followed by slow depolarization which reached a plateau level of about 25 mV above prehypoxic resting potential within 20 min 4. Interestingly, this slow depolarization occurs without impulse discharges. Although we did not refer to the membrane potentials of CA1 neurons, dysfunction of CA1 neurons may be triggered by the slow depolarization. It has been well known that hippocampal CA1 neurons are selectively vulnerable to transient forebrain ischemia. In the reports which described about the time-course of morphological change of hippocampus in detail, degeneration of CA1 neurons was not observed 6 h after transient ischemia, however, it was seen at 24 h after ischemia 15. Electron-microscopic observations found a similar time-course of neuronal degeneration 1°. Our morphological examination revealed similar results compare to the previous reports 3'15. Namely, most pyramidal cells of the CA1 region showed severe damage at 3 days after 20 min ischemia and a small percentage of pyramidal cells showed necrotic changes at 3 days after 5 min ischemia. This delay of structural damage found in morphological experiments is in striking contrast to the immediate functional damage observed in the present study. In spite of the delayed occurrence of structural degeneration, some cellular functions of CA1 neurons
260 such as protein synthesis2"6"21 and protein kinase C activity 13 were changed during ischemia, and/or in the early stage of reperfusion. These changes of cellular functions may effect the excitabilities of CA1 neurons. In Mongolian gerbils, it has been reported that the hyperactivity of hippocampal CA1 neurons is consistently observed after 24 h of 5 min ischemia, and followed by the selective degeneration of CA1 pyramidal cells 2°. The hyperactivity reported in Mongolian gerbils was supposed to be related to 'excitotoxicity' induced by excitatory amino acids. In the rats, release of exitatory amino acids such as glutamate and aspartate increases during ischREFERENCES 1 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 intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. 2 Bodsch, W., Takahashi, K., Barbier, A., Ophoff, B.G. and Hossmann, K.A., Cerebral protein synthesis and ischemia. In K. Kogure, K.-A. Hossmann, B.K. Siesj6 and EA. Welsh (Eds.), Molecular Mechanisms of lschemic Brain, (Progress in Brain Research, Voi. 63), Elsevier, Amsterdam, 1985, pp. 197-210. 3 Buchan, A. and Pulsinelli, W.A., Neurotransmitters and neurotrophic factors in cerebral ischemia towards a molecular explanation for selective neuronal injury and a new rationale for stroke treatment. In H. Takeshita (Ed.), International Symposium of Brain Resuscitation, Ube/Yamaguchi, Japan, 1988, pp. 5-11. 4 Fujiwara, N., Higashi, H., Shimoji, K. and Yoshimura, M., Effects of hypoxia on rat hippocampal neurons in vitro, J. Physiol., 384 (1987) 131-151. 5 Itoh, T., Kawakami, M., Yamauchi, Y., Shimizu, S. and Nakamura, M., Effect of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats, Stroke, 17 (1986) 1284-1287. 6 Kiessling, M., Dienel, G.A., Jacewicz, M. and Pulsinelli, W.A., Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis, J. Cereb. Blood Flow Metab., 6 (1986) 642-649. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. 8 Kirino, T. and Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta Neuropathol., 62 (1984) 201-208. 9 Kirino, T. and Sano, K., Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus, Acta Neuropathol., 62 (1984) 209-218. 10 Kirino, T., Tamura, A. and Sano, K., Selective vulnerability of the hippocampus to ischemia-reversible and irreversible types of ischemic cell damage. In K. Kogure, K.-A. Hossmann, B.K.
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