Brain Research, 512 (1990) 7-14 Elsevier

7

BRES 15282

Septo-hippocampal deafferentation protects CA1 neurons against ischemic injury Alastair M. Buchan* and William A. Pulsinelli Cerebrovascular Disease Research Center, Raymond and Beverly Sackler Foundation, Inc., Department of Neurology and Neuroscience, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Accepted 8 August 1989) Key words: Ischemia; Hippocampus; Perforant pathway; Sch~iffercollateral; Fimbria/fornix pathway

Excessive synaptic excitation caused by transient cerebral ischemia has been proposed to explain the greater vulnerability of specific neuronal populations to ischemic injury. We tested this hypothesis in rats by cutting, alone or in combination, the afferent fibers that travel in the fimbria/fornix, the perforant, or the Sch/iffer collateral pathways and innervate the right CA1 hippocampus. Seven to twelve days later the animals were subjected to 30 min of reversible forebrain ischemia. Irreversible damage to the CA1 neurons was assessed with the light microscope after 70-120 h of cerebral reperfusion. The left, unlesioned hippocampus served as a control. Simultaneous cutting of the 3 major afferent pathways significantly reduced CA1 neuronal damage compared to the unlesioned side (P < 0.001) or to sham-lesioned controls (P < 0.001). Selective lesions of the fimbria/fornix but not the perforant or the Sch~iffercollateral pathways also protected against ischemic CA1 damage. These data indicate that afferent fiber input modulates hippocampal damage caused by ischemia, but they are inconsistent with the hypothesis that excitatory afferent fibers, travelling in either the perforant or the Schfiffer collateral pathways alone, play a major role. Neurotransmitters, other than those activating excitatory amino acid receptors or yet-to-be-identifiedsynaptic events, may be invoked to explain the spatial and temporal sensitivity of hippocampal CA1 neurons to ischemia. INTRODUCTION Severe, transient cerebral ischemia can irreversibly d a m a g e specific populations of highly vulnerable neurons. P y r a m i d a l cells in the CA1 zone of hippocampus are amongst the most sensitive of such populations 3. T h e i r injury is characterized by a unique delay of 24 h in the onset of histologically detectable damage 15'23. The mechanisms responsible for this sensitivity and the unique time course of the d a m a g e are unknown. A b n o r m a l i t i e s in the synaptic neurochemistry of excitatory and inhibitory neurotransmitters may be importantly involved in the molecular mechanisms of such injury 6'12'17'33. Ischemia causes excessive neurotransm i t t e r release 2, disturbed n e u r o t r a n s m i t t e r reuptake 27, and changes in postsynaptic r e c e p t o r properties 32 which m a y act alone or in concert to influence postsynaptic n e u r o n a l damage. A n experimental strategy to test the hypothesis that ischemia causes a lethal imbalance between excitation and inhibition is to interrupt the afferent fiber input to sensitive neurons, allow time for degeneration of nerve terminals, and then examine the effect of ischemia on the postsynaptic neurons. Lesions of the

perforant path 34 and the entorhinal cortex 13 in rats have been r e p o r t e d to protect CA1 h i p p o c a m p a l neurons against ischemia. I n t e r r u p t i o n of the mossy fiber/Sch~ffer collateral pathway by lesions of the d e n t a t e granule cells 11 or lesions of the C A 3 neurons 2°, were also r e p o r t e d to protect the rat CA1 zone against ischemic injury. Preliminary reports of Pulsinelli 21'22, however, failed to show protection of CA1 neurons when either perforant or Schfiffer collateral pathways were transected in rats subjected to transient forebrain ischemia. The latter study did reveal that transection of the afferent fibers travelling in the fimbria/fornix significantly attenuated damage to these cells. The p u r p o s e of the present study was to confirm which afferent pathways to the hippocampus, if any, were i m p o r t a n t l y involved in modifying ischemic damage. MATERIALS AND METHODS Hippocampal deafferentation Male Wistar rats (175-250 g) were used in two separate but otherwise comparable studies. In one study W.P. transected the hippocampal afferent pathways, and in the other, this was done by A.B. The two studies were separated by approximately 18 months.

* Present address: Clinical Neurological Sciences, University Hospital, London, Ont., Canada N6A 5A5. Correspondence: W.A. Pulsinelli, Cerebrovascular Disease Research Center, Raymond and Beverly Sackler Foundation, Inc., Department of Neurology and Neuroscience, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, U.S,A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

Rats in the initial study were anesthetized with pentobarbital (60 mg/kg i.p.) and those in the second study were anesthetized with an N2/O2/halothane mixture, (70%:28%:2%). The rats were placed in stereotaxic ear bars and the skull was exposed using sterile techniques. A cranial window was made over the right parietal cortex with a dental drill and a dissecting microscope. The dura was resected with care so as to avoid damage to the sagittal sinus. The cortex and subcortical white matter overlying the right dorsal hippocampus was aspirated with a 21 gauge Luer stub connected to a vacuum pump. In sham-lesioned animals the surgical procedure was stopped at this point, and hemostasis was achieved. In experimental animals, the principal afferent fibers that innervate the CA1 zone of hippocampus were cut individually or in combination as follows. Fimbria/fornix lesions. The fimbria/fornix pathway connecting the septum to the dorsal hippocampus was visualized with a dissecting microscope and transected by aspiration with a 23 gauge needle connected to a vacuum pump (Figs. 1, 2A).This procedure also transected the septal pole of the hippocampus, the ventral commissure, some alvear fibers and a portion of the cingulum bundle. The completeness of this aspiration lesion was verified by direct visualization with the dissecting microscope at the time of the lesioning. Schg~ffer collateral lesions. Under direct visualization of the dorsal hippocampus, which was achieved by aspiration of the overlying neocortex, a No. 15 scalpel blade was inserted vertically just medial to the border between the CA1 and CA2/3 fields, This surgical cut was extended from the septal pole of the hippocampus caudally to a point where the dorsal hippocampus descended into the depths of the brain; a length along the septo-temporal axis of approximately 2 mm (Figs. 1, 2B). This cut severed fibers of passage that travelled from the regio inferior to the regio superior of hippocampus. The anterior-posterior and lateral placement of this lesion was verified by direct visualization with the dissecting microscope at the time of the lesioning. The vertical depth of the lesion was verified in every animal by microscopic examination of the coronal sections of the hippocampus used for grading CAI neuron damage. Perforantpathway lesions. The perforant pathway travelling along the medial border of the hippocampus was visualized by aspiration of the posterior parietal cortex and the splenium of the corpus

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callosum. Using a 23 gauge needle connected to a vacuum pump the entorhinal projections to the angular bundle (pcrforant path) werc transected together with the overlying alvcar fibers and fibers travelling in the posterior half of the dorsal commissural pathway. The lesion was extended from the medial border of the CA1 zone out laterally to approach the junction with CA2/3, producing an almost complete transection of regio superior (Figs. 1, 2C). The completeness of this aspiration lesion was verified by direct visualization with the dissecting microscope at the time of lesioning. Tricut lesion. In separate groups of rats the right dorsal hippocampus was exposed and all 3 pathways were lesioned as described above. Following hemostasis and wound closure all animals were allowed to recover for 7-12 days. The animals were housed in individual cages, given food and water ad libitum and maintained on a 12 h light/12 h dark cycle. Antibiotics were not given.

I

Fig. 1. Diagram showing the dorsal view of a rat brain and the location of the unilateral hippocampal lesions: (1) anterior lesion transecting the fimbria/fornix (see Fig. 2A); (2) Schaffer collateral lesion (see Fig. 2B); (3) posterior transection cutting through the perforant path (see Fig. 2C). Abbreviations: ant. comm., anterior commissure; ang. bun., angular bundle; dorsal hipp. comm., dorsal hippocampal commissure; fire., fimbria; vent. hipp. comm., ventral hippocampal commissure; ENT.CX., entorhinal cortex.

Fig. 2. A: a coronal view of the fimbria/fornix aspiration lesion, -1.8 mm Bregma. B: a coronal view of the knife cut (dark bar marked by arrow) which severs the Sch~iffer collateral fibers, -3.8 mm Bregma. C: a coronal view of the perforant pathway aspiration lesion, -4.3 mm Bregma. Diagrams are modified with permission from the atlas of Paxinos and Watson. Ctx = neocortex; CPu = caudate putamen; Th = thalamus; CA1, CA2, CA3 are the respective pyramidal zones of dorsal hippocampus: fi = fimbria.

9 Forebrain ischemia Sham-lesioned and hippocampal afferent-lesioned animals were anesthetized as described above with halothane and prepared for 4-vessel occlusion (4-VO) ischemia by the modified surgical method 242s. The rats were allowed to recover from surgery overnight, during which time they were fasted, but allowed free access to water. On the day of the experiment the ventral neck incision was locally anesthetized with lidocaine and both carotid arteries were exposed and occluded in the awake rat; the vertebral arteries had been previously cauterized. Only those animals that lost the righting reflex while maintaining unassisted respiration for 30 min were included in the study. After 30 min the carotid artery clasps were released and the restoration of carotid artery blood flow was verified by direct visualization of the vessels. The ventral neck wound was closed with a suture. Body temperature was maintained at 37 + 0.5 °C with a rectal thermistor connected to a heating lamp until the rats had recovered thermal homeostasis (approximately 2 h after unclasping). Animals that developed seizures after cerebral reperfusion were excluded from the study.

TABLE I

CA1 hippocampal damage - Study 1 Values differ slightly from the preliminary report zl because of repeated blinded grading of the injury.

Lesion

Sham controls Lesioned animals Tricut Fimbria/fornix Sch/iffer collateral Perforant path

N

Mean grade CA1 neuronal damage (+_S.E.) Intact left hippocampus

Deafferented right hippocampus

10

1.7 + 0.3

2.0 + 0.3

8 11 8 12

3.0 3.0 2.3 + 0.3 2.5 + 0.3

1.3 + 1.5 + 2.7 + 2.1 +

0.2* 0.2* 0.1 0.3

* P < 0.001 compared to left hippocampus (intact) of same group.

Histopathological analysis The rats were reanesthetized at 70-120 h after cerebral recirculation, and the brains were either frozen in freon cooled over dry ice or fixed by transcardial perfusion with FAM (40% formaldehyde/ glacial acetic acid/methanol, 1:1:8) after brief saline perfusion. The perfused-fixed brains were left in situ overnight at 4 °C before removal. Coronal sections, 20/tm thick from frozen tissue, or 7/~m thick from paraffin-embedded tissue, were cut at 3 different levels of the dorsal hippocampus. These coronal levels were chosen to span the complete longitudinal axis of the dorsal CA1 hippocampus. These sections were stained with hematoxylin and eosin and examined with the light microscope by an observer blinded to the experimental conditions. In the initial study where W.P. made the lesions, A.B. graded the histological injury and the order was reversed for the second study. Ischemic injury to neurons in the CA1 zone was graded on a scale of 0-3 with 0: normal hippocampus; 1: 50% of neurons damaged. Irreversible ischemic damage was accepted in any neuron showing a shrunken cell body with eosinophilic cytoplasm and a dark pyknotic nucleus, homogenizing cell change or naked nuclei 3'24. In animals subjected to the tricut lesion much of the lateral portion of the CA1 hippocampus was surgically damaged; accordingly, in these animals only the medial one-half of the CA1 zone was graded for ischemic injury.

to h i p p o c a m p u s o r i n f e c t i o n p r e c l u d e d e v a l u a t i o n . In the s e c o n d study, 67 animals w e r e s u b j e c t e d to h i p p o c a m p a l deafferentation;

4 were

lost d u r i n g

surgery,

9 were

i n c o m p l e t e l y u n r e s p o n s i v e d u r i n g i s c h e m i a , 11 d i e d with r e s p i r a t o r y arrest o r seizures and 1 d e v e l o p e d an abscess in the l e s i o n e d h i p p o c a m p u s . This m o r b i d i t y and m o r tality was d i s t r i b u t e d r e l a t i v e l y e q u a l l y a m o n g the e x p e r i m e n t a l g r o u p s in the t w o e x p e r i m e n t s . T h e results of t h e s e studies a r e p r e s e n t e d in Tables I and II. In the s h a m - l e s i o n e d c o n t r o l a n i m a l s s u b j e c t e d to t r a n s i e n t f o r e b r a i n i s c h e m i a , the s e v e r i t y of h i s t o p a t h o logical d a m a g e to n e u r o n s in t h e left and right C A 1 p y r a m i d a l z o n e s was similar. In c o n t r a s t , d a m a g e in the deafferented hippocampus

of a n i m a l s with e i t h e r the

tricut lesion o r with selective t r a n s e c t i o n o f the f i m b r i a / fornix p a t h w a y was significantly less c o m p a r e d to the c o n t r a l a t e r a l intact h i p p o c a m p u s .

In n e i t h e r study did

t r a n s e c t i o n o f the Sch~ffer c o l l a t e r a l o r the p e r f o r a n t

Statistical analysis

p a t h w a y s significantly a l t e r t h e g r a d e o f injury to C A 1

Differences in the grade of neuronal damage between the intact (left) hippocampus and the deafferented (right) hippocampus of the same animal were statistically analyzed with the Mann-Whitney U-test. The Mann-Whitney U-test and the Bonferroni correction for multiple comparisons were used to compare ischemic damage in animals with lesions of the right hippocampus to the right hippocampus of sham-lesioned animals.

h i p p o c a m p u s . A small s e g m e n t of the m o s t lateral C A 1 z o n e ipsilateral to lesions o f t h e p e r f o r a n t p a t h w a y was

TABLE II

CA1 hippocampal damage - Study 2 RESULTS

Lesion

N

In t h e initial study, 49 of 151 animals w e r e available for histological analyses a f t e r 30 min o f transient f o r e b r a i n i s c h e m i a and at least 3 days of p o s t i s c h e m i c survival. T h e

Intact left hippocampus

Deafferented right hippocampus

3.0

3.0

2.8 + 0.1 3.0 2.9 + 0.1 2.9 + 0.1

0.5 + 1.8 + 2.9 + 2.7 +

that d i e d s e c o n d a r y to r e s p i r a t o r y arrest o r n e u r o g e n i c

Sham controls Lesioned animals Tricut Fimbria/fornix Sch/iffer collateral Perforant path

p u l m o n a r y e d e m a d u r i n g the i s c h e m i c insult, o r to p o s t i s c h e m i c seizures, a n d 9 in which t r a u m a t i c d a m a g e

*P < 0.001 compared to left hippocampus (intact) of same group. **P < 0.001 compared to right hippocampus of sham controls.

102 a n i m a l s n o t available for study i n c l u d e d : 6 d e a t h s r e l a t e d to surgical p r o c e d u r e s , 8 animals that w e r e i n a d e q u a t e l y u n r e s p o n s i v e during i s c h e m i a , 79 animals

5

Mean grade CA1 neuronal damage (+ S.E.)*

12 12 4 9

0.3*'** 0.3* 0.1 0.1

11

Fig. 3. C. (A) A low power (40x) photomicrograph of the left (intact) and right (fimbria/fornix lesioned) hippocampus from a rat subjected to 30 min of forebrain ischemia and 3 days post ischemic survival. The arrows indicate the CA1 pyramidal cell zone at the precise area shown at higher magnification in (B) and (C). (B) and (C) are high power (350x) views of the intact and lesioned sides, respectively. Note the loss of CA1 neurons in the intact (B) hippocampus and preservation of neurons in the deafferented (C) region.

spared in some animals causing a lower grade of damage in the right hippocampus of this group. This response, however, was inconsistent between animals and did not achieve statistical significance. Animals with the fimbria/fornix transection showed the greatest protection in the medial one-half of the dorsal CA1 zone compared with the more lateral one-half of this zone. A zone of almost complete protection was seen in the medial one half of the dorsal CA1 zone in 7 of 11 animals in the initial study, and in 8 of 12 animals in the second study (Fig. 3). Gliosis was present in the deafferented dorsal hippocampus of all animals, and was especially prominent in those animals with fimbria/fornix lesions. This gliosis was most common in the stratum radiatum but also occurred in a few animals in the stratum oriens. No consistent relationship, however, existed between the degree and/or location of gliosis and the protection of CA1 neurons against ischemic injury. Gliosis was also noted in the ipsilateral thalamus of all animals whether subjected either to hippocampal deafferentation or to sham lesions.

DISCUSSION Results from this study demonstrate that the exquisite sensitivity of hippocampal CA1 neurons to ischemia can be attenuated by transecting specific afferent fiber pathways travelling to hippocampus. In two separate, but otherwise comparable studies (differing only in date and surgeon), cutting the major afferent pathways to the CA1 neurons afforded almost complete protection to the medial one-half of the CA1 zone against 30 min of severe forebrain ischemia. Furthermore, it was discovered that selective cutting of fibers travelling in the vicinity of the fimbria/fornix pathway accounted for the greatest proportion of this protection. Lesions of the perforant pathway, and the Sch/iffer collateral pathway failed to significantly influence outcome. These results contradict previously published studies that reported protection of CA1 pyramidal neurons against ischemia by lesions involving the perforant 13'34 and association fiber afferents n,2°. Differences in experimental design and lesion technique must be considered

12 as a partial explanation for these conflicting data. Anatomical descriptions of the perforant and Schfiffer collateral fiber pathways lead us to believe that our deafferentation techniques have successfully severed the majority of these fibers which innervate the CA1 neurons of dorsal hippocampus. The lateral and medial perforant pathway fibers join and ascend along the medial margin of the hippocampus as a distinct fiber bundle (angular bundle) to a level as far rostrally as the dorsal hippocampal commissure. At this point, fibers that have yet to penetrate the hippocampus descend and travel longitudinally within the hippocampus for a variable distance to reach either the CA1 pyramidal neurons or the dentate granule layer l~. Aspiration of the angular bundle and the regio superior at the level of the dorsal hippocampal commissure, as was accomplished in our study (Figs. 1, 2C), would sever the majority of ipsilateral and contralateral perforant fibers innervating the CA1 zone of the ipsilateral dorsal hippocampus. Electrophysiological recordings I and radiotracer-autoradiographic studies 3° of Sch~ffer collateral fibers in the rat are consistent with a lamellar CA3/CA1 unit that is oriented essentially perpendicular to the longitudinal axis of the hippocampus. However, these same autoradiographic studies 3° also indicate considerable longitudinal divergence of the Schfiffer collateral fibers with terminals innervating CA1 neurons over rostral-caudal distances of several millimeters. These latter Schfiffer collateral fibers probably reach the appropriate longitudinal lamellae by traversing the stratum radiatum of the regio inferior in conjunction with the longitudinal association bundle 9"3°'31. The latter fiber bundle traverses long distances within the stratum radiatum of the regio inferior. If the above topographic orientation of the Schfiffer collateral fibers is correct, then our knife cut (Figs. 1, 2B), which extends for approximately 2 mm along the longitudinal axis of the dorsal hippocampus and separates CA1 from CA2/CA3 neurons, would sever the majority of Schfiffer collateral fibers innervating the dorsal C A I hippocampus. The variability of neuronal injury associated with all animal models of transient global ischemia requires care and attention to statistical design. Accordingly, we have gone on to replicate the results of our study in 3 separate experiments using large numbers of animals; two of the studies form the basis for this report and the results of a third 4 are in preparation. With the exception of a study by Lasner et al. 16 (see below), we are unaware of other published efforts to replicate the claims that perforant or Schfiffer collateral pathway lesions protect CA1 neurons against ischemia. Wieloch et al. 34 reported significantly fewer injured neurons in the dorsal hippocampus of 4 rats whose

perforant pathways were cut prior to cerebral ischemia, but failed to find protection with lesions of the fimbria/ fornix. In our study, using larger numbers of animals, we noted some preservation of neurons in the lateral CA I zone (CAlb) of rats with perforant-path lesions, but such protection was inconsistent between animals, and was not significantly different from non-lesioned controls. Our observations agree with those of Lasner et al. ~ who reported that in gerbils entorhinal lesions made prior to ischemia only partially preserved neurons in the most lateral segment of the CA1 zone. A concern not addressed by Wieloch et al. 34 is that transection of the perforant pathway results in transection of CA1 axons travelling to subiculum and therefore in retrograde degeneration and loss of some CA1 pyramidal cells 3-4 days after the lesion 14. Accordingly, in perforant-pathway lesioned animals, fewer CA1 neurons are exposed to the subsequent ischemic insult. If the absolute number of ischemia-injured neurons is then used as an endpoint measure 34, it is possible to inadvertently detect fewer injured cells in the deafferented versus the intact hippocampus. A similar loss of neurons in the fimbria/fornix lesioned hippocampus is not a problem, since Kameyama et al. ~4 found no decrease in the numbers of CA1 pyramidal neurons in the hippocampi of rats with such lesions. Two additional studies that report protection of the rat CA1 hippocampus against ischemia are difficult to evaluate objectively because of design questions. In the studies of JOrgensen et al. ~x in which the entorhinal cortex was ablated and the study of Johansen et a l . l t in which the dentate gyrus was degranulated unilaterally by injections of colchicine, the numbers of normal pyramidal neurons were counted in consecutive 300 ~m lengths of the CA1 zone as an endpoint measure. The CAI zone at the anterior-posterior level of dorsal hippocampus examined in the latter studies measures approximately 3 mm in lateral width and contains approximately 500-700 pyramidal neurons 34, i.e. 50-70 neurons per 300 ~m length. Curiously, although both studies were performed in the same species and by the same investigators, one study 1~ reported approximately 50 pyramidal neurons but the other ~ only 18 pyramidal neurons per 300 ktm length in the normal hippocampus. An explanation for this discrepancy was not presented. More importantly, since neither study examined more than 4 consecutive 300 ~m lengths of the CA1 zone, less than one-third of the available CA1 neurons were counted. Accordingly, the arbitrary selection of 300 ~m lengths within the whole CA1 field of hippocampus could lead to selection bias and erroneous results. The potential weaknesses of this counting procedure are further emphasized by the observations of Lasher et al. ~ who reported protection of

13 only 30% of C A l b neurons, i.e. but no protection of C A l a neurons in gerbils subjected to entorhinal lesions and global ischemia. M o r e o v e r , the conclusions of Johansen et al. 11 that colchicine-induced destruction of the d e n t a t e gyrus p r o t e c t e d CA1 neurons against ischemia must be t e m p e r e d by the fact that colchicine is known to acutely alter the CA1 p y r a m i d a l cells and to block axonal t r a n s p o r t in all cell types. Such nonselective effects may have influenced fiber pathways o t h e r than those travelling via the trisynaptic circuit. In a n o t h e r study, O n o d e r a et al. 2° p r o d u c e d lesions of the C A 3 neurons with kainic acid injections, and rep o r t e d that in 9 of 13 lesioned rats there was m a r k e d a t t e n u a t i o n of d a m a g e to CA1 neurons following an ischemic insult. Unfortunately, no quantitative analysis of h i p p o c a m p a l injury was presented in this study. A weakness of all of these studies, including ours, is the absence of n e u r o t r a n s m i t t e r m e a s u r e m e n t s to validate the specificity and completeness of a particular afferent fiber lesion. H o w e v e r , based on reports of n e u r o t r a n s m i t t e r depletion after pathway lesions similar to ours 5'7'18, and o t h e r experiments which define pathw a y - n e u r o t r a n s m i t t e r relationships ~9'2s'29, our data weigh against a singular role for excitatory amino a c i d - m e d i a t e d injury to CA1 h i p p o c a m p a l neurons. We

REFERENCES 1 Andersen, P., Bliss, T. and Skrede, K., Lamellar organization of hippocampal excitatory pathways, Exp. Brain Res., 13 (1971) 222-238. 2 Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N., Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. 3 Brierley, J., Cerebral hypoxia. In W. Blackwood and J. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold Publishers, 1976, pp. 43-85. 4 Buchan, A. and Pulsinelli, W., Fimbria/fornix lesions protect hippocampal neurons from ischemic damage, Stroke, 19 (1988) 146. 5 Fonnum, F. and Walaas, I., The affect of intrahippocampal kainic acid injections and surgical lesions on neurotransmitter in hippocampus and septum, J. Neurochem., 31 (1978) 1173-1181. 6 Francis, A. and Pulsinelli, W., Response of GABAergic and cholinergic neurons to transient forebrain ischemia, Brain Research, 243 (1982) 271-278. 7 Gage, E, Bj6rklund, A. and Stenevi, U., Re-innervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Research, 268 (1983) 27-37. 8 Gage, E, Bjorklund, A. and Stenevi, U., Denervation releases a neuronal survival factor in adult rat hippocampus, Nature (Lond.), 308 (1984) 637-639. 9 Hjorth-Simonsen, A., Some intrinsic connections of the hippocampus in the rat: an experimental analysis, J. Comp. Neurol., 147 (1973) 145-162. 10 Hjorth-Simonsen, A. and Jeune, B., Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation, J. Comp. Neurol., 144 (1972) 215-232.

found that, when severed, glutamatergic pathways originating in the entorhinal cortex, or in the Sch/iffer collateral system, failed to alter injury of CA1 neurons. In the absence of n e u r o t r a n s m i t t e r m e a s u r e m e n t s , however, we cannot be certain that all fibers of the latter pathways were cut or that fibers i n t e r r u p t e d by the fimbria/fornix lesions did not involve o t h e r excitatory amino acid pathways. Accordingly, the hypothesis that excitatory amino acids are involved in ischemic injury to neurons remains viable. Nevertheless, given the anatomical selectivity of the fimbria/fornix lesions, and the fact that afferent fibers travelling in this p a t h w a y contain, for example, acetylcholine and several m o n o a m i n e s , it is possible that neurotransmitters o t h e r than the excitatory amino acids are involved in ischemic cell death. In addition, o t h e r effects brought about by transection of the fimbria/fornix, which include an uncoupling of septohippocampal theta driving 26, proliferation of glial cells in the dorsal h i p p o c a m p u s , and perhaps the synthesis and release of specific n e u r o h u m o r a l or n e u r o t r o p h i c factors s, must all be considered as possible factors in the protective nature of this lesion. Acknowledgements. This study was supported in part by a grant from U.S. Public Health Service (NS-03346). A.B. was a Centennial Fellow of the Canadian M.R.C.

11 Johansen, F., J0rgensen, M. and Diemer, N., Ischemic CA1 pyramidal cell loss is prevented by preischemic colchicine destruction of dentate gyrus granule cells, Brain Research, 377 (1986) 344-347. 12 JOrgensen, M. and Diemer, N., Selective neuron loss after cerebral ischemia in the rat: possible role of transmitter glutamate, Acta Neurol. Scand., 66 (1982) 536-546. 13 JOrgensen, M., Johansen, E and Diemer, N., Removal of the entorhinal cortex protects hippocampal CA1 neurons from ischemic damage, Acta Neuropathol., 73 (1987) 189-194. 14 Kameyama, M., Wasterlain, C., Ackerman, R., Finch, D., Lear, J. and Kuhl, D., Neuronal response of the hippocampal formation to injury: blood flow, glucose metabolism and protein synthesis, Exp. Neurol., 79 (1983) 329-346. 15 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 237 (1982) 57-69. 16 Lasner, T., Crain, B. and Nadler, J., Entorhinal cortical lesions partially prevent hippocampal cell death after transient forebrain ischemia in the gerbil, Soc. Neurosci. Abstr., 13 (1987) 1498. 17 Meldrum, B., Metabolic effects of prolonged epileptic seizures and the causation of epileptic brain damage. In E Rose (Ed.), Metabolic Disorders of the Nervous System, Pitman, London, 1981, 175-187. 18 Nadler, J. and Smith, E., Perforant path lesion depletes glutamate content of facia dentata synaptosomes, Neurosci. Leu., 25 (1981) 275-280. 19 Nadler, J., White, F., Vaca, K., Perry, B. and Cotman, C., Biochemical correlates of transmission mediated by glutamate and aspartate, J. Neurochem., 31 (1978) 147-155. 20 Onodera, H., Sato, G. and Kogure, K., Lesions of the Sch~iffer collaterals prevent ischemic death of CA1 pyramidal cells, Neurosci. Lett., 68 (1986) 169-174. 21 Pulsinelli, W., Hippocampal deafferentation lessens ischemic injury to CA1 pyramidal neurons, J. Cereb. Blood Flow Metabol., Vol. 5: Supp. 3 (1985) 309-310.

14 22 Pulsinelli, W., Selective neuronal vulnerability: morphological and molecular characteristics, Prog. Brain Res., 63 (1985) 29-38. 23 Pulsinelli, W., Brierley, J. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 24 Pulsinelli, W. and Buchan, A., The 4-vessel occlusion rat model: method for complete occlusion of the vertebral arteries and control of the collateral circulation, Stroke, 19 (1988) 913-914. 25 Pulsinelli, W. and Duffy, T., Regional energy balance in rat brain after transient forebrain ischemia, J. Neurochem.. 40 (1983) 1500-1503. 26 Rawlins, J., Feldon, J. and Gray, J., Septo-hippocampal connections and the hippocampal theta rhythm, Exp. Brain Res., 37 (1979) 49-63. 27 Silverstein, E , Buchanan, K. and Johnston, M., Hypoxiaischemia causes severe but reversible depression of striatal synaptosomal 3H-glutamate uptake, Ann. Neurol., 18 (1985) 122. 28 Storm-Mathisen, J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fiber systems.

29

30

3t 32

33 34

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Septo-hippocampal deafferentation protects CA1 neurons against ischemic injury.

Excessive synaptic excitation caused by transient cerebral ischemia has been proposed to explain the greater vulnerability of specific neuronal popula...
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