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Increase in choline acetyltransferase activity in septum of rats after transient forebrain ischemia: A possible role of factors released in the hippocampus
Neuroscience Letters, 105 (1989) 321-325
321
Elsevier Scientific Publishers Ireland Ltd. 06409
Increase in choline acetyltransferase activity in septum of rats after transient forebrain ischemia: a possible role of factors released in the hippocampus Kinya Hisanaga 1,2,*, Kyuya Kogure 2, Hiroko Tsukui 3, N o b u y u k i Takei 1,**, Chika Nishio 3 and Hiroshi H a t a n a k a 1,3 1Department of Neuroscience, Mitsubishi Kasai Institute of Life Sciences, Tokyo (Japan), 2Department of Neurology, Tohoku University School of Medicine, Sendal (Japan) and 3Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Osaka (Japan) (Received 22 May 1989; Revised version received 29 June 1989; Accepted 3 July 1989)
Key words." Ischemia; Neurotrophic factor; Septal cholinergic neuron Choline acetyltransferase (CHAT) activity increased in rat septum 2 weeks after a transient forebrain ischemia. Extracts were prepared from hippocampus in which CA 1 pyramidal neurons had been selectively destroyed by the ischemic insult. ChAT activity in septal neuronal cultures treated with these extracts for 6 days was significantly higher than that in control cultures.
Transient ischemia of the rodent forebrain produces a well documented pattern of damage in the dorsal hippocampus. The pyramidal neurons in the CA1 subfield are particularly vulnerable whereas neurons in the CA3 subfield and dentate gyrus are relatively spared [9, 10, 19]. There is evidence of injury in CA1 neurons of hippocampus by 24 h which progresses to full neuronal necrosis within 72 h, a process referred to as delayed neuronal cell death [9, 19]. The survival of septal cholinergic neurons is believed to be supported by neurotrophic factors (NTFs) released in their target tissue, hippocampus [4, 15]. Nerve growth factor (NGF), the best characterized of the NTFs, is produced in normal hippocampal pyramidal neurons [1], and supports the survival of septal cholinergic neurons [5]. Therefore, it might be expected that the damage to hippocampal pyramidal neu-
*Present address: Department of Neurology, University of California, San Francisco, CA 94121, U.S.A. **Present address: Department of Neurochemistry, National Institute of Neuroscience, NCNP, Ogawahigashi-cho, 4-1-1, Kodaira, Tokyo 187, Japan. Correspondence: H. Hatanaka, Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita-shi, Osaka 565, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
322
rons following global ischemia would produce effects on the septal cholinergic neurons which are receiving NTFs from the hippocampus. However, little morphological damage to septum has been observed following global ischemia in the model we have used [19]. We examined whether choline acetyltransferase (CHAT) activity in septum is affected by the transient forebrain ischemia. Male Wistar rats (11-12 weeks of age) were subjected to 20 min of forebrain ischemia by the modified method of Pulsinelli and Brierley [18, 19]. Briefly, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and both vertebral arteries were electrocauterized at their site of exposure in the alar foramina. On the following day, the rats were anesthetized with N20/O2 (70:30) containing 2% halothane and the bilateral common carotid arteries were exposed. After completion of the surgical procedures, halothane was discontinued and the animals were subjected to forebrain ischemia for 20 min by temporary clipping of bilateral common carotid arteries. Bilateral loss of righting reflex obtained within 1 min after carotid clipping, was used as criteria for forebrain ischemia. Previous studies [16, 17] have shown that this transient forebrain ischemia produces selective pyramidal cell necrosis in the CA1 subfield, in agreement with studies of Pulsinelli [18]. Control animals were prepared and handled similarly except for clipping of the bilateral common carotid arteries. The animals were killed after the survival period of 14 days by means of decapitation, Several regions of these brains were dissected on ice, and stored at -80°C. These regions involved hippocampus, septum, striatum and cerebral cortex (frontparietal region). The extracts of various brain regions were processed for examining ChAT activity according to the method of Fonnum [4] with the modification of Hatanaka and Tsukui [6]. The ChAT activities in various brain regions of rats which were subjected to a tran-
Specific ChAT activities ( pmol/min/mg protein ) 0 250 5O0 750 I ~
Neocortex
] Sham-operation Operation
Hippocamp°
Medial Septum~ Stratum
~
j
~ ~
/
~ ]
j~ ~ P
Fig. I. Specific C h A T activities in 4 forebrain regions of rats subjected to a transient forebrain ischemia. Forebrain ischemia was carried out in male Wistar adult rats as described in text, After 2 weeks, 4 regions involving hippocampus, septum, striatum and cerebral cortex were dissected out. Specific C h A T activities were determined as described previously [6]. Open and closed columns, data from sham-operated rats and operated rats, respectively. Values were means _+_ S.D. (n = 5).
323
sient forebrain ischemia are shown in Fig. 1. Specific ChAT activity in the septum was significantly higher in the ischemic experimental compared to that in sham-operated control rats (n = 5 in both groups), with little change in the activities in other regions. It is still unknown why the ChAT activity in septum increased after the ischemic insult. Although it can not be ruled out that the ischemic insult might have direct effects on the ChAT activity in septal cholinergic neurons, it is also possible that substances released from damaged hippocampus were involved. It has been suggested that the potential of the central nervous system to recover from injury may depend in part on NTFs that support neuronal survival, promote the sprouting of neurites, and guide the neurites to targets [2]. It has been shown that neurotrophic activity is present in the non-injured brain in rats [3, 13, 15, 20], and brain injury results in significant increases in neurotrophic activity in the tissue adjacent to the lesion I11-14, 20]. Injury-induced NTFs have been considered to act preferentially on neurons in regions connected by afferent or efferent projections [12], as well as on those in the area of damage [20]. Recently, Nakagawa and Ishihara [11] showed that extracts of hippocampus lesioned by direct colchicine injections promoted the survival of peripheral cholinergic neurons in culture. We examined whether extracts prepared from hippocampus lesioned by forebrain ischemia affect the ChAT activity in septal neuronal cultures. The hippocampal tissues were dissected 2 weeks after ischemic insult as described above, and then homogenized with a Teflon homogenizer in ice-cold serum-free culture medium. The supernatant was collected after centrifugation by using a Beckman TL-100 apparatus at 4°C for 15 min at 360,000 g steriled through 0.22 pm filters (Spin-X, Costar) and applied to cultures at every change of medium. Postnatal cholinergic neuronal cultures were prepared as described previously [7, 8]. After the culture for 6 days, the ChAT activity in cultured cells was examined. As shown in Fig. 2, extracts prepared from ischemia-subjected rats (n = 5) resulted in significant high levels in ChAT activities of septal cell cultures (1.4-fold), but not equal amounts of protein concentration of extracts from sham-operated rats (n = 5). These results sug-
1 no d0't'oo
NGFpg/ml) H~,.Xsham-ope
(160pg/ml) ~ : ~ ~ H1~60pg/ml) EXoperation~ ~
ChAT activities ( pmol/min/well ) 1.0 1.5 2.0 J
~
,
~
I ~''~
I I:~
:~ p
Fig. 2. Effects of hippocampal extracts from sham-operated rats and operated rats on ChAT activity in cultured postnatal rat septal cholinergic neurons. Culture method from 2-week-old rats was the same as described previously [7, 8]. Hippocampal extracts (HpEX) from sham-operated and operated rats were added at the concentration o f 160/~g protein/ml into culture medium. ChAT activity determination was as described previously [6]. Values were means 4- S.D. (n =4).
324 gest that factors released in the h i p p o c a m p u s lesioned by f o r e b r a i n ischemia m a y have effects on septal cholinergic n e u r o n s which h a v e lost their n o r m a l target cells. It is u n k n o w n w h e t h e r N G F is i n v o l v e d in the increase o f C h A T activities in septum following transient ischemia. A recent r e p o r t suggested that increase o f N G F receptors in rat basal f o r e b r a i n after e x o g e n o u s N G F t r e a t m e n t f o l l o w i n g septohipp o c a m p a l tract t ra n s e c ti o n m a y result in C h A T i n d u c t i o n in the basal f o r e b r a i n [21]. T o clarify the influence o f transient f o r e b r a i n ischemia o n the s e p t o h i p p o c a m p a l system, it w o u l d be o f interest to investigate the change o f N G F receptors in s e p t u m f oll o wi n g ischemic insults, as well as the c h a n g e o f N G F c o n t e n t in h i p p o c a m p u s . W e gratefully a c k n o w l e d g e the c o n t r i b u t i o n s o f Dr. K o i c h i r o N i s h i o k a an d Dr. T s u t o m u A r a k i o f T o h o k u University. W e are indebted to Dr. F r a n k R. Sharp o f the U n i v e r s i t y o f C a l i f o r n i a at San F r a n c i s c o for his kind advice an d suggestions in p r e p a r i n g the m an u s c r i p t . 1 Ayer-Lelievre, C., Olson, L., Ebendal, T., Seiger, A. and Persson, H., Expression of the fl-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1 2 Cotman, C.W. and Nieto-Sampedro, M., Cell biology of synaptic plasticity, Science, 225 (1984) 1287-1294. 3 Crutcher, K.A. and Collins, F., In vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982) 67~8. 4 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407~409. 5 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain, Dev. Brain Res., 9 (1983) 45-52. 6 Hatanaka, H. and Tsukui, H., Differential effects of nerve growth factor and glioma-conditioned medium on neurons cultured from various regions of fetal rat central nervous system, Dev. Brain Res., 30 (1986) 47 56. 7 Hatanaka, H., Tsukui, H. and Nihonmatsu, !., Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor, Neurosci. Lett., 79 (1987) 85 90. 8 Hatanaka, H., Tsukui, H. and Nihonmatsu, I., Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats, Dev. Brain Res., 39 (1988) 85 ~95. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57 69. 10 Kirino. T., Tamura, A. and Sano, K., Delayed neuronal death in the rat hippocampus following transient forebrain ischemia, Acta Neuropathol., 64 (1984) 139 147. 11 Nakagawa, Y. and Ishihara, T., Enhancement of neurotrophic activity in cholinergic cells by hippocampat extract prepared from colchicine-lesioned rats, Brain Res., 439 (1988) 11 18. 12 Needels, D.L., Nieto-Sampedro, M. and Cotman, C.W., Induction of a neurite-promoting factor in rat brain following injury or deafferentation, Neuroscience, 18 (1986) 517-526. 13 Nieto-Sampedro, M., Lewis, E.R., Cotman, C.W., Manthorpe, M., Skaper, S.D., Barbin, G., Longo, F.M. and Varon, S., Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site, Science, 217 (1982) 860-861. 14 Nieto-Sampedro, M., Manthorpe, M., Barbin, G., Varon, S. and Cotman, C.W., Injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity, J. Neurosci., 3 (1983) 2219-2229. 15 Ojika, K. and Appel, S.H., Neurotrophic effects of hippocampal extracts on medial septal nucleus in
325
vitro, Proc. Natl. Acad. Sci. USA., 81 (1984) 2567-2571. 16 Onodera, H., Sato, G. and Kogure, K., Lesions to Schaffer collaterals prevent ischemic death of CA1 pyramidal cells, Neurosci. Lett., 68 (1986) 169-174. 17 Onodera, H. and Kogure, K., Autoradiographic localization of opioid and spirodecanone receptors in the gerbil hippocampus as compared with the rat hippocampus, J. Cereb. Blood Flow Metab., 8 (1988) 568-574. 18 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 19 Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 20 Whittemore, S., Nieto-Sampedro, M., Needels, D.L. and Cotman, C.W., Neuronotrophic factors for mammalian brain neurons: injury induction in neonatal, adult and aged rat brain, Dev. Brain Res., 20 (1985) 169-178. 21 Yip, H. and Williams, L.R., Plasticity of NGF receptors in the rat basal forebrain following axotomy and exogenous NGF treatment, Soc. Neurosci. Abstr., 103 (1988) 9.
321
Elsevier Scientific Publishers Ireland Ltd. 06409
Increase in choline acetyltransferase activity in septum of rats after transient forebrain ischemia: a possible role of factors released in the hippocampus Kinya Hisanaga 1,2,*, Kyuya Kogure 2, Hiroko Tsukui 3, N o b u y u k i Takei 1,**, Chika Nishio 3 and Hiroshi H a t a n a k a 1,3 1Department of Neuroscience, Mitsubishi Kasai Institute of Life Sciences, Tokyo (Japan), 2Department of Neurology, Tohoku University School of Medicine, Sendal (Japan) and 3Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Osaka (Japan) (Received 22 May 1989; Revised version received 29 June 1989; Accepted 3 July 1989)
Key words." Ischemia; Neurotrophic factor; Septal cholinergic neuron Choline acetyltransferase (CHAT) activity increased in rat septum 2 weeks after a transient forebrain ischemia. Extracts were prepared from hippocampus in which CA 1 pyramidal neurons had been selectively destroyed by the ischemic insult. ChAT activity in septal neuronal cultures treated with these extracts for 6 days was significantly higher than that in control cultures.
Transient ischemia of the rodent forebrain produces a well documented pattern of damage in the dorsal hippocampus. The pyramidal neurons in the CA1 subfield are particularly vulnerable whereas neurons in the CA3 subfield and dentate gyrus are relatively spared [9, 10, 19]. There is evidence of injury in CA1 neurons of hippocampus by 24 h which progresses to full neuronal necrosis within 72 h, a process referred to as delayed neuronal cell death [9, 19]. The survival of septal cholinergic neurons is believed to be supported by neurotrophic factors (NTFs) released in their target tissue, hippocampus [4, 15]. Nerve growth factor (NGF), the best characterized of the NTFs, is produced in normal hippocampal pyramidal neurons [1], and supports the survival of septal cholinergic neurons [5]. Therefore, it might be expected that the damage to hippocampal pyramidal neu-
*Present address: Department of Neurology, University of California, San Francisco, CA 94121, U.S.A. **Present address: Department of Neurochemistry, National Institute of Neuroscience, NCNP, Ogawahigashi-cho, 4-1-1, Kodaira, Tokyo 187, Japan. Correspondence: H. Hatanaka, Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita-shi, Osaka 565, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
322
rons following global ischemia would produce effects on the septal cholinergic neurons which are receiving NTFs from the hippocampus. However, little morphological damage to septum has been observed following global ischemia in the model we have used [19]. We examined whether choline acetyltransferase (CHAT) activity in septum is affected by the transient forebrain ischemia. Male Wistar rats (11-12 weeks of age) were subjected to 20 min of forebrain ischemia by the modified method of Pulsinelli and Brierley [18, 19]. Briefly, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and both vertebral arteries were electrocauterized at their site of exposure in the alar foramina. On the following day, the rats were anesthetized with N20/O2 (70:30) containing 2% halothane and the bilateral common carotid arteries were exposed. After completion of the surgical procedures, halothane was discontinued and the animals were subjected to forebrain ischemia for 20 min by temporary clipping of bilateral common carotid arteries. Bilateral loss of righting reflex obtained within 1 min after carotid clipping, was used as criteria for forebrain ischemia. Previous studies [16, 17] have shown that this transient forebrain ischemia produces selective pyramidal cell necrosis in the CA1 subfield, in agreement with studies of Pulsinelli [18]. Control animals were prepared and handled similarly except for clipping of the bilateral common carotid arteries. The animals were killed after the survival period of 14 days by means of decapitation, Several regions of these brains were dissected on ice, and stored at -80°C. These regions involved hippocampus, septum, striatum and cerebral cortex (frontparietal region). The extracts of various brain regions were processed for examining ChAT activity according to the method of Fonnum [4] with the modification of Hatanaka and Tsukui [6]. The ChAT activities in various brain regions of rats which were subjected to a tran-
Specific ChAT activities ( pmol/min/mg protein ) 0 250 5O0 750 I ~
Neocortex
] Sham-operation Operation
Hippocamp°
Medial Septum~ Stratum
~
j
~ ~
/
~ ]
j~ ~ P
Fig. I. Specific C h A T activities in 4 forebrain regions of rats subjected to a transient forebrain ischemia. Forebrain ischemia was carried out in male Wistar adult rats as described in text, After 2 weeks, 4 regions involving hippocampus, septum, striatum and cerebral cortex were dissected out. Specific C h A T activities were determined as described previously [6]. Open and closed columns, data from sham-operated rats and operated rats, respectively. Values were means _+_ S.D. (n = 5).
323
sient forebrain ischemia are shown in Fig. 1. Specific ChAT activity in the septum was significantly higher in the ischemic experimental compared to that in sham-operated control rats (n = 5 in both groups), with little change in the activities in other regions. It is still unknown why the ChAT activity in septum increased after the ischemic insult. Although it can not be ruled out that the ischemic insult might have direct effects on the ChAT activity in septal cholinergic neurons, it is also possible that substances released from damaged hippocampus were involved. It has been suggested that the potential of the central nervous system to recover from injury may depend in part on NTFs that support neuronal survival, promote the sprouting of neurites, and guide the neurites to targets [2]. It has been shown that neurotrophic activity is present in the non-injured brain in rats [3, 13, 15, 20], and brain injury results in significant increases in neurotrophic activity in the tissue adjacent to the lesion I11-14, 20]. Injury-induced NTFs have been considered to act preferentially on neurons in regions connected by afferent or efferent projections [12], as well as on those in the area of damage [20]. Recently, Nakagawa and Ishihara [11] showed that extracts of hippocampus lesioned by direct colchicine injections promoted the survival of peripheral cholinergic neurons in culture. We examined whether extracts prepared from hippocampus lesioned by forebrain ischemia affect the ChAT activity in septal neuronal cultures. The hippocampal tissues were dissected 2 weeks after ischemic insult as described above, and then homogenized with a Teflon homogenizer in ice-cold serum-free culture medium. The supernatant was collected after centrifugation by using a Beckman TL-100 apparatus at 4°C for 15 min at 360,000 g steriled through 0.22 pm filters (Spin-X, Costar) and applied to cultures at every change of medium. Postnatal cholinergic neuronal cultures were prepared as described previously [7, 8]. After the culture for 6 days, the ChAT activity in cultured cells was examined. As shown in Fig. 2, extracts prepared from ischemia-subjected rats (n = 5) resulted in significant high levels in ChAT activities of septal cell cultures (1.4-fold), but not equal amounts of protein concentration of extracts from sham-operated rats (n = 5). These results sug-
1 no d0't'oo
NGFpg/ml) H~,.Xsham-ope
(160pg/ml) ~ : ~ ~ H1~60pg/ml) EXoperation~ ~
ChAT activities ( pmol/min/well ) 1.0 1.5 2.0 J
~
,
~
I ~''~
I I:~
:~ p
Fig. 2. Effects of hippocampal extracts from sham-operated rats and operated rats on ChAT activity in cultured postnatal rat septal cholinergic neurons. Culture method from 2-week-old rats was the same as described previously [7, 8]. Hippocampal extracts (HpEX) from sham-operated and operated rats were added at the concentration o f 160/~g protein/ml into culture medium. ChAT activity determination was as described previously [6]. Values were means 4- S.D. (n =4).
324 gest that factors released in the h i p p o c a m p u s lesioned by f o r e b r a i n ischemia m a y have effects on septal cholinergic n e u r o n s which h a v e lost their n o r m a l target cells. It is u n k n o w n w h e t h e r N G F is i n v o l v e d in the increase o f C h A T activities in septum following transient ischemia. A recent r e p o r t suggested that increase o f N G F receptors in rat basal f o r e b r a i n after e x o g e n o u s N G F t r e a t m e n t f o l l o w i n g septohipp o c a m p a l tract t ra n s e c ti o n m a y result in C h A T i n d u c t i o n in the basal f o r e b r a i n [21]. T o clarify the influence o f transient f o r e b r a i n ischemia o n the s e p t o h i p p o c a m p a l system, it w o u l d be o f interest to investigate the change o f N G F receptors in s e p t u m f oll o wi n g ischemic insults, as well as the c h a n g e o f N G F c o n t e n t in h i p p o c a m p u s . W e gratefully a c k n o w l e d g e the c o n t r i b u t i o n s o f Dr. K o i c h i r o N i s h i o k a an d Dr. T s u t o m u A r a k i o f T o h o k u University. W e are indebted to Dr. F r a n k R. Sharp o f the U n i v e r s i t y o f C a l i f o r n i a at San F r a n c i s c o for his kind advice an d suggestions in p r e p a r i n g the m an u s c r i p t . 1 Ayer-Lelievre, C., Olson, L., Ebendal, T., Seiger, A. and Persson, H., Expression of the fl-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1 2 Cotman, C.W. and Nieto-Sampedro, M., Cell biology of synaptic plasticity, Science, 225 (1984) 1287-1294. 3 Crutcher, K.A. and Collins, F., In vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity?, Science, 217 (1982) 67~8. 4 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407~409. 5 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.. and Thoenen, H., NGF-mediated increase of choline acetyltransferase (CHAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain, Dev. Brain Res., 9 (1983) 45-52. 6 Hatanaka, H. and Tsukui, H., Differential effects of nerve growth factor and glioma-conditioned medium on neurons cultured from various regions of fetal rat central nervous system, Dev. Brain Res., 30 (1986) 47 56. 7 Hatanaka, H., Tsukui, H. and Nihonmatsu, !., Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor, Neurosci. Lett., 79 (1987) 85 90. 8 Hatanaka, H., Tsukui, H. and Nihonmatsu, I., Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats, Dev. Brain Res., 39 (1988) 85 ~95. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57 69. 10 Kirino. T., Tamura, A. and Sano, K., Delayed neuronal death in the rat hippocampus following transient forebrain ischemia, Acta Neuropathol., 64 (1984) 139 147. 11 Nakagawa, Y. and Ishihara, T., Enhancement of neurotrophic activity in cholinergic cells by hippocampat extract prepared from colchicine-lesioned rats, Brain Res., 439 (1988) 11 18. 12 Needels, D.L., Nieto-Sampedro, M. and Cotman, C.W., Induction of a neurite-promoting factor in rat brain following injury or deafferentation, Neuroscience, 18 (1986) 517-526. 13 Nieto-Sampedro, M., Lewis, E.R., Cotman, C.W., Manthorpe, M., Skaper, S.D., Barbin, G., Longo, F.M. and Varon, S., Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site, Science, 217 (1982) 860-861. 14 Nieto-Sampedro, M., Manthorpe, M., Barbin, G., Varon, S. and Cotman, C.W., Injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity, J. Neurosci., 3 (1983) 2219-2229. 15 Ojika, K. and Appel, S.H., Neurotrophic effects of hippocampal extracts on medial septal nucleus in
325
vitro, Proc. Natl. Acad. Sci. USA., 81 (1984) 2567-2571. 16 Onodera, H., Sato, G. and Kogure, K., Lesions to Schaffer collaterals prevent ischemic death of CA1 pyramidal cells, Neurosci. Lett., 68 (1986) 169-174. 17 Onodera, H. and Kogure, K., Autoradiographic localization of opioid and spirodecanone receptors in the gerbil hippocampus as compared with the rat hippocampus, J. Cereb. Blood Flow Metab., 8 (1988) 568-574. 18 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 19 Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 20 Whittemore, S., Nieto-Sampedro, M., Needels, D.L. and Cotman, C.W., Neuronotrophic factors for mammalian brain neurons: injury induction in neonatal, adult and aged rat brain, Dev. Brain Res., 20 (1985) 169-178. 21 Yip, H. and Williams, L.R., Plasticity of NGF receptors in the rat basal forebrain following axotomy and exogenous NGF treatment, Soc. Neurosci. Abstr., 103 (1988) 9.