Hippocampal neurotrophic factor: Characterization and response to denervation

BrainResearch, 363 (1986)299-306 Elsevier

299

BRE 11380

Hippocampal Neurotrophic Factor: Characterization and Response to Denervation ANNE M. HEACOCK, AMY R. SCHONFELD and ROBERT KATZMAN Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY10461 (U.S.A.)

(Accepted May 21st, 1985) Key words: cholinergic - - gliosis - - chick ciliary ganglion - - methotrexate - - neurotrophic factor

The rat hippocampus contains an acidic macromolecular neurotrophic factor (NTF) which supports the survival of cultured chick ciliary ganglion cells. Hippocampal NTF increases approximately 2-fold from birth to adulthood with no further change with aging. Disruption of extrinsic inputs to the hippocampus from the entorhinal cortex, locus coeruleus or contralateral hippocampus, but not from the septum, results in an increase in the concentration of NTF in the hippocampus. Destruction of intrinsic hippocampal neurons by kainic acid treatment is accompanied by a large increase in hippocampal NTF, a result consistent with a glial origin for the factor. This conclusion is supported by the finding that a lesion-induced rise in NTF can be suppressed by administration of methotrexate, an inhibitor of gliosis. INTRODUCTION Many types of cultured neuronal cells exhibit a requirement for trophic substances which affect their ability to survive or grow neurites 36,37. In vivo, neuronal death often results following injury or during development, when peripheral neurons are experimentally deprived of contact with their target organ. These observations have led to the hypothesis that a diffusible neurotrophic factor (NTF), present in target tissues, may promote neuronal survival and that a deficiency in such substances may contribute to the selective neuronal loss in such pathological conditions as Alzheimer's disease 2,39. In addition to the well-characterized nerve growth factor, other NTFs, active on sympathetic, parasympathetic and sensory neurons, have been found in various sources, including conditioned culture medium and extracts of heart, skeletal muscle and other tissues7,8. Speculation about a role for NTFs in CNS development, plasticity and response to injury has increased with the discovery of trophic activity in mammalian brain 6,13,21,25,26 and recent reports of a requirement of cultured CNS neurons for such NTFsS,Zl,zs, 28.29. A physiological function for NTF is supported by the observations that stimulation of endogenous NTF

production by lesioning adult rat brain27 or administration of injured brain extract, containing NTF, to neonatal rats 28 maximizes the survival of transplanted embryonic neurons. In addition, we recently found that prelesioning of the hippocampus increases both hippocampal NTF and sprouting of cholinergic axons into intrahippocampal iris implants 33. In this report, we further characterize hippocampal NTF, examine its changes with development, response to denervation and present evidence consistent with a glial origin for NTF. The hippocampus is especially well-suited to studies of the role of NTFs in neuronal plasticity since its major inputs have been well-defined anatomically and are accessible to experimental manipulation. In addition, the hippocampus is known to undergo reactive synaptogenesis following selective lesions 12. Extrinsic afferents to the hippocampal formation originate from the entorhinal cortex, septum, locus coeruleus and the contralateral hippocampus. In the present study, in order to examine the role each of these inputs might have in regulating hippocampal NTF content, each pathway was lesioned and the effect of the corresponding hippocampal extracts on the survival of cultured chick embryo ciliary ganglion cells was determined. Disruption of any of the above in-

Correspondence: A.M. Heacock. Present address: University of Michigan, Neuroscience Laboratory, 1103 E. Huron, Ann Arbor, MI 48109, U.S.A.

300 puts, with the exception of that from the septum, resulted in an increase in hippocampal NTF. The largest increase in NTF was produced by kainic acid treatment. This result, taken together with the finding that a lesion-induced rise in NTF could be suppressed by methotrexate, an inhibitor of gliosis, suggests a role for glia in NTF production. Portions of this work have been presented in abstract form17,is. MATERIALSAND METHODS

Tissue culture Culture of chick ciliary ganglion cells was carried out essentially as described by Varon et al. 38. Ganglia were removed from 8-day chick embryos (Spafas, Storrs, CT), dissociated in 0.08% trypsin, rinsed, diluted to 20,000 neurons/ml with modified Eagles Basal medium containing 10% fetal calf serum (Gibco), then added (5000 neurons/well) to 16 mm polyornithine-coated multiwell plates (Falcon). Each well contained 0.25 ml of control medium or medium supplemented with trophic factor. In some experiments, culture wells were pre-coated with chick heart conditioned medium (HCM)I,n in order to facilitate neurite outgrowth. Culture plates were incubated in 5% CO2, 95% air at 37 °C for 24 h, then the medium was aspirated and the cells were fixed overnight in 2% glutaraldehyde. Cell survival was determined by counting the number of phase-bright cells in each of three 1 x 8 mm radial strips per well, in duplicate wells for each supplement. The mean cell count per strip (minus the background survival in unsupplemented wells) was then multiplied by 25 to give the total number of neurons per well. In the absence of trophic supplement, less than 5% of the neurons survived, while maximal survivals of up to 50% were obtained with chick eye NTF 23 (gift of M. Manthorpe) or saturating amounts of hippocampal extract. One trophic unit (TU) is defined as the amount required for one-half maximal survival; data are expressed as TU/mg protein. As an internal control for each experiment, wells containing excess chick eye NTF were always included.

Hippocampal extract preparation Normal Sprague-Dawley or Fischer 344 rats or those subjected to the surgical procedures described below were killed by decapitation, brains were re-

moved and right and left hippocampi were dissected on ice, then frozen separately at -70 °C. For assay of trophic content, tissue was thawed and each hippocampus was homogenized in 0.6 ml 0.01 M sodium phosphate buffer, pH 7.4, then centrifuged at 100,000 g for 30 min. The pellet was discarded and aliquots of the supernatant were reserved for protein determination22 or diluted with tissue culture medium for assay of trophic content. The hippocampal extract, prior to dilution, could be stored at -70 °C for as long as 4 weeks without loss of activity. In some experiments, aliquots of the homogenate, before centrifugation, were reserved, at -20 °C, for choline acetyltransferase (CAT) determination 15.

Surgery Stereotaxic surgery 31 on female Sprague-Dawley rats (240-260 g) was carried out using a combination of ketamine and xylazine for anesthesia. For unilateral lesions of the fornix-fimbria, a fine glass rod was inserted at the midline, 7.5 mm anterior to the interaural line and 5.0 mm deep from the cortical surface. The glass rod was then moved 4.5 mm laterally, transecting the right fornix-fimbria pathway. Partial ablation of the right entorhinal cortex was accomplished using a radiofrequency lesion generator (65 °C for 20-30 s) with the probe inserted at two sites: 1.0 and 2.0 mm anterior to the interaural line, 4.5 and 4.8 mm lateral and 5.1 and 6.1 mm ventral, respectively. Some of these animals also received a 10/A intraventricular injection (frontal horn of the lateral ventricle) of either Hank's balanced salt solution (BSS) or 100 ktg methotrexate (MTX, Sigma), freshly dissolved in Hank's BSS. This amount of MTX, given at the time of entorhinal cortex lesion, is reported to suppress the hippocampal glial reaction examined at 5 weeks following the lesion 3. For lesions of the locus coeruleus, the radiofrequency probe was inserted 1.2 mm posterior, 1.3 mm lateral and 7.2 mm ventral and the pulse applied at 65 °C for 10 s. With the latter animals, the superior cervical ganglion (SCG) was also removed ipsilaterally. Interhippocampal connections were interrupted by making midline lesions with a knife throughout the extent of the dorsal and ventral hippocampal commissures. For all of the above lesions, correct positioning was verified histologically. Survival times (1-8 weeks) are as indicated in Results. Right and

301 left hippocampi were r e m o v e d and assayed separately for trophic activity. F o r lesions of the f o r n i x fimbria or entorhinal cortex, the left h i p p o c a m p u s served as a control. F o r commissurotomy or locus coeruleus lesions, right and left hippocampi were assayed separately, then the values averaged, since no right/left difference was a p p a r e n t following these lesions. In all of the experiments r e p o r t e d here, each set of control and lesioned tissues was assayed at the same time on the same p r e p a r a t i o n of ciliary ganglion cells in o r d e r to minimize possible effects of interassay variability. In another group of animals, kainic acid (8 mg/kg) was administered intraperitoneally and after 7 days, the right hippocampus was r e m o v e d for N T F determination, while the left h i p p o c a m p u s was e x a m i n e d histologically for p y r a m i d a l cell disruption in areas C A 3 and CA43°. RESULTS

Neurotrophic activity in normal adult rat hippocampus A d d i t i o n of h i p p o c a m p a l extract enhanced the survival of chick e m b r y o ciliary ganglion cells in a conc e n t r a t i o n - d e p e n d e n t m a n n e r (Fig. 1), with saturation occurring at a p p r o x i m a t e l y 80 fig protein/ml. Consequently, in subsequent assays, concentrations

of h i p p o c a m p a l extract of 10-40/~g/ml were used. While there was no detectable n e u r i t e - p r o m o t i n g activity in h i p p o c a m p a l extract, even at the highest concentrations examined, if the culture dishes were first p r e t r e a t e d with a n e u r i t e - p r o m o t i n g factor from H C M , extensive neuritic outgrowth then occurred. No neurotoxic activity was detectable when varying amounts of h i p p o c a m p a l extract were a d d e d to culture wells containing chick eye N T F , nor did the presence of the latter further enhance the neuronal survival p r o d u c e d by saturating amounts of h i p p o c a m pal extract. Trophic activity was not confined to the hippocampus, but was also present in frontal cortex and cerebellum, although at slightly lower concentrations (60% and 70%, respectively, of h i p p o c a m p a l values). H i p p o c a m p a l N T F appears to be a protein since it was sensitive to trypsin t r e a t m e n t and 93% of the activity was lost after heating for 5 min at 60 °C. N T F activity was retained by an A m i c o n PM10 filter, indicating a molecular weight greater than 10,000 daltons. It is an acidic protein since it b o u n d to a D E A E Biogel A column (in 0.01 M sodium p h o s p h a t e buffer, p H 7.4) and could be eluted with 0.1 M NaC1, resulting in an a p p r o x i m a t e l y 3-fold purification.

Changes in hippocampal NTF with development If the main function of h i p p o c a m p a l N T F was in

20~1

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0 40 0')

0 ee" 0

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5O0 0 'Io

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HIPPOCAMPAL PROTEIN~g/ml)

Fig. 1. Dose-response of ciliary ganglion cell survival to hippocampal NTF. Hippocampal extract (0.25 ml), diluted with culture medium to the concentrations indicated, was added to culture wells containing 0.25 ml of cell suspension (20,000 neurons/ml) and neuronal survival was determined after 24 h. Maximum survival attained was 37%, while in unsupplemented wells, survival was only 3.3% of the plated neurons. The values shown were obtained after subtraction of this background survival. Duplicate determinations differed by less than 12%.

20

or¢, I-

O

3

8

14

21

28

~f,

4'~ ADULT 1 YR 2 'YRS

DAYS POSTNATAL

Fig. 2. Developmental changes in hippocampal trophic activity. Hippocampal extract was prepared from Fischer rats of the indicated ages. The data shown are from one of 3 similar experiments in which 2-3 pooled hippocampi were assayed in duplicate for survival-promoting activity for chick ciliary ganglion cells. Duplicate determinations differed by less than 15%.

302 synapse formation and selective neuronal survival

stantial reduction in hippocampal C A T activity, had

during d e v e l o p m e n t , its concentration in developing

no concomitant effects on hippocampal N T F at any

hippocampus would be expected to be higher than in

of the time points e x a m i n e d (Table I). In contrast to

the adult, with perhaps a further decline on aging.

this lack of effect of f o r n i x - f i m b r i a transection, dis-

The data in Fig. 2 show that this does not appear to be

ruption of other extrinsic afferents was found to in-

the case. H i p p o c a m p a l N T F content was relatively

crease hippocampal N T F (Table II). Thus, lesions of

low at birth, increasing gradually, by approximately

the entorhinal cortex (EC) resulted in over a 2-fold

2-fold to the adult levels, with no further change in

e n h a n c e m e n t after 3 weeks which persisted for up to

aged rats. This result suggests that in addition to pos-

8 weeks (data not shown). Though the effect at one

sible functions during d e v e l o p m e n t , N T F may also be

week was not statistically significant, small early

involved in maintenance of neuronal survival and

changes might likely be obscured when the whole

synaptic connections in the mature hippocampus.

hippocampus is examined rather than the presumed

R e s p o n s e to lesion

lowing commissurotomy,

affected region, the dentate gyrus. A t one week folhippocampal

NTF

was

Interruption of the cholinergic input to the hippo-

minimally elevated (40%, Table II). Disruption of

campus from the septum by unilateral partial transec-

the noradrenergic input by lesions of the locus coeru-

tion of the f o r n i x - f i m b r i a , while resulting in a sub-

leus and unilateral r em o v al of the superior cervical

TABLE I Effect of unilateralfornix-fimbria transection on hippocampal N T F content

For each animal, NTF content and CAT activity (mean ± S.E.M.) in the denervated hippocampus were compared with that in the control hippocampus. Values for normal unoperated animals (n=9) were: NTF = 42.7 ± 2.9; CAT = 26.4 ± 1.1. Time post-lesion

n

1 2 4 8

10 3 4 3

week weeks weeks weeks

N T F ( TU/mg)

CA T (nmol/mg/h)

Denervated

Control

Denervated

Control

42.7 ± 42.9 ± 39.3 + 56.4 +

37.7 ± 44.2 ± 42.1 + 51.2 ±

10.2 ± 9.9 ± 9.9 + 15.4 +

29.5 ± 31.6 ± 26.7 ± 28.8 +

3.5 5.3 2.8 10.6

5.7 7.5 4.9 6.7

1.1 1.0 1.0 1.8

2.7 2.9 1.8 2.3

TABLE II Effect of various lesions on hippocampal N T F content

Survival times following the lesions were one week, except where indicated. Data show mean + S.E.M. (range). Type of lesion

n

TU/mg protein Lesioned

Entorhinal cortex 1 week 3 weeks

3 3

Commissurotomy

7

Locus coeruleus + SCG

6

Kainic acid

3

43.3 ± 8.8 (31.8 - 59.9) 76.7 ± 1.7 (73.3 - 78.8) 66.6 _+5.2 (48.9 - 86.4) 51.6 _+2.7 (44.5 - 62.4) 177.6 ± 30.3 (124 - 229)

* Left hippocampus, contralateral to lesion served as control. Normal hippocampi from unoperated animals served as control. *** t-test, lesioned vs control. **

n

p***

Control

34.8 + 2.1" (31.9 - 38.9) 35.5 ± 3.6* (29.2 - 35.5) 47.5 ± 3.3** (38.8 - 58.3) 31.8 ± 2.5** (26.1 - 38.3) 43.1 _+6.5** (31.9 - 59.9)

3

n.S.

3

< 0.001

6

< 0.05

5

< 0.001

4

< 0.001

303 ganglion gave rise to a 63% increase in NTF. The fact that there was no right/left difference in trophic activity in these animals suggests that SCG removal did not substantially contribute to this increase. In addition to these studies of removal of extrinsic input, the role of hippocampal interneurons in N T F production was also examined. Hippocampal pyramidal cells in area CA3 and CA4 are particularly sensitive to systemic kainic acid administration 30. However, these cells do not appear to be a major source of NTF, since, rather than a decrease, very large increases in NTF (over 4-fold) were found at one week after kainic acid injection. This result is compatible with a glial origin for NTF, since kainate is known to cause a pronounced glial reaction.

Suppression of gliosis with methotrexate In addition to kainic acid treatment, at least two other lesions found to cause increases in hippocampal NTF are also known to produce a glial response in the hippocampus - - commissurotomy 4 and entorhinal

2.0

m

(16) r/)

cortex lesion 32, thus further suggesting a glial role in NTF production. A recent report that methotrexate suppresses the hippocampal glial response to entorhinal cortex lesion 3 prompted use of this drug to examine the relationship between gliosis and NTF content (Fig. 3). Rats given single intraventricular injections of MTX (100 pg) at the time of entorhinal cortex (EC) lesion showed, at 5 weeks, only a 1.17-fold enhancement of hippocampal NTF. This can be compared to the 1.8-fold increase in rats receiving E C lesion alone. The effect of M T X was confined to the lesioned side, since the left hippocampal trophic content (when compared to saline-injected animals) was not altered following MTX injection, indicating that MTX does not have a general toxic effect on the NTF assay. This conclusion is further supported by the observation that delay of the methotrexate treatment until two weeks after the E C lesion, at which time the glial reaction had passed its peak, resulted in no suppression of NTF content (data not shown). While systemically administered M T X has been reported to result in immunosuppression 9, such effects do not apparently play a role here since intraventricular M T X caused no change in spleen or thymus wet weights. DISCUSSION

e,,,.

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n

oo)

T (5)

-~

.O

e-

e~ O

eE

Control

EC lesion

EC les~on

+ MTX Fig. 3. Methotrexate suppresses the EC lesion-induced rise in hippocampal NTF. Rats were injected intraventricularly with MTX (100pg) or vehicle at the time of right entorhinal cortex lesion. After 5 weeks, NTF content (TU/mg protein) in right and left hippocampus was determined. Data are expressed as mean right/left ratio + S.E.M. Control values of 48.9 TU/mg in unoperated animals did not differ from those of left hippocampus in operated animals. *, different from control, P < 0.01; **, different from EC lesion, P < 0.005.

The rat hippocampal neurotrophic factor described here is an acidic protein of molecular weight greater than 10,000 daltons which promotes survival of chick ciliary ganglion cells and is present at low concentrations for the first few weeks after birth, then increases to adult levels. These properties distinguish it both from the basic pig brain factor of Thoenen 6, active on sensory neurons, and from the hippocampal NTF recently reported by Ojika and Appe129 to stimulate neurite outgrowth and cholinergic activity in rat embryo septal explants. The molecular weight of this latter factor was less than 5000 daltons and the activity peaked at 2 - 3 weeks postnatal. The NTF reported by Nieto-Sampedro et al. to be present in brain wound fluid 26 and in brain tissue (including hippocampus) of developing 24 and adult rats27 appears to be more similar to the one described in the present report. The brain wound fluid NTF is active on cultured chick dorsal root ganglion, superior cervical ganglion and spinal cord, in addition to ciliary ganglion, and was recently reported to enhance

304 survival of rat striatal neurons 2s. In addition, there was a brief report 13 of a hippocampal factor which stimulates the rate of neurite extension in chick ciliary and sympathetic ganglion cells but which has not been further characterized. From the data available thus far, there appears to be little indication either of the localization of NTF to a particular brain region, although it may be enriched in hippocampus, or of specificity as to neurotransmitter phenotype, although many of the aforementioned responding cultured cells are cholinergic. Such questions must await further purification of the active component(s). If the idea that target-derived trophic substances are important for maintenance of neuronal survival and function is applied to CNS trophic factors, then it might be expected that alterations in trophic content would occur following disruption of intracerebral connections. In the peripheral nervous system, partial denervation of cardiac 10, skeletaP 9,2° or iris 14 muscle enhances their production of survival- or neurite-promoting trophic substances. A recent report presents evidence for a similar phenomenon in the CNS 16. Survival of neonatal rat SCG neurons implanted adjacent to the hippocampus in adult rats is greatly enhanced by unilateral fornix-fimbria transection but not by lesions of the perforant path 16. The result was interpreted to reflect the release from the hippocampus of atrophic factor active on noradrenergic neurons whose production was regulated by cholinergic input from the septum. This is in contrast to the situation reported here for the hippocampal 'cholinergic' NTF which was not altered by fornixfimbria transection but was increased following disruption of pathways from the entorhinal cortex, locus coeruleus/SCG or contralateral hippocampus. These results, taken together, could mean that a reciprocal relationship exists such that the 'cholinergic' NTF is controlled by non-cholinergic (e.g. noradrenergic) afferents. Such a suggestion must, however, be tempered by consideration of the dissimilarities in experimental paradigm between the studies of Gage et al.16 and those reported here. A rise in hippocampal trophic activity (for chick ciliary ganglion cells) following entorhinal cortex lesion (15 days) was also reported by Nieto-Sampedro et al. 27. This effect appears to be rather long-lasting (at least 8 weeks) but is not clearly present at one week postlesion. While it seems reasonable to conclude that the enhancement

in hippocampal NTF following EC lesion is due to the resultant deafferentation, diffusion of trophic factor from the site of the lesion, as observed by Nieto-Sampedro et al.27 in the cortex, cannot be ruled out. However, if such were the case, the lesions of the fornixfimbria should also have given rise to enhanced hippocampal NTF. The fact that entorhinal cortex lesion also results in reactive synaptogenesis in the hippocampus brings up the question of how well the timecourse of the response of hippocampal NTF correlates with that for the sprouting phenomenon. Although the increase in terminal density seems to be an early event, plateauing by 12 days, synapse density is reported to continue to increase for up to 7 months 35. Thus while the data is not at present consistent with a role for NTF in the initial sprouting response, it may be important for synapse formation and maintenance. Another point that should be addressed is that while electrolytic lesions of the locus coeruleus, in combination with SCG removal, does result in an increase in hippocampal NTF, fornixfimbria transection, which disrupts the dorsal pathway from the locus coeruleus, does not give such an increase. It may be that the more extensive disruption of noradrenergic afferents in the former procedure is required for alterations in NTF content to occur. The largest increase in hippocampal NTF (4-fold) was produced by systemic administration of kainic acid. This result agrees with that reported by NietoSampedro et al. for intraventricularly injected kainate 27 and, because of the gliosis elicited by this treatment, suggests a glial origin for hippocampal NTF. Such a source is also consistent with the time-course of developmental changes in NTF and with the observation of a glial response following lesions to the entorhinal cortex 3~ and the commissural associational pathway 4, as well as after direct injury to the brain9, all of which result in an increase in NTF. This possible glial origin is further supported by the finding that methotrexate, which was recently reported to suppress trauma-induced glial proliferation 9 and to partially block the hippocampal glial response to entorhinal cortex lesions 3, also suppressed the lesion-induced rise in hippocampal NTF. In summary, the present study has shown that rat hippocampus contains a macromolecular survivalpromoting factor active on peripheral cholinergic

305 n e u r o n s , which m a y h a v e a glial origin, and w h o s e

e n d o g e n o u s N T F , a c a p a b i l i t y which m i g h t be of

c o n c e n t r a t i o n s e e m s to be r e g u l a t e d by n o n - c h o l i n e r -

s o m e t h e r a p e u t i c use in c e r t a i n p a t h o l o g i c a l condi-

gic afferents to the h i p p o c a m p u s . T h e possible prac-

tions c h a r a c t e r i z e d by n e u r o n a l loss.

tical applications of such trophic substances w e r e recently u n d e r l i n e d by t h e o b s e r v a t i o n s that h i p p o c a m pal w o u n d fluid e n h a n c e d

survival of e m b r y o n i c

ACKNOWLEDGEMENTS

striatal implants 28 and that purified chick eye N T F inj e c t e d into the s e p t u m e n h a n c e d cholinergic sprout-

This study was s u p p o r t e d by the N a t i o n a l I n s t i t u t e

ing into i n t r a h i p p o c a m p a l iris i m p l a n t s 34. M o r e de-

of A g i n g (1 R03 A G 0 3 9 4 1 - 0 1 ) ( A . R . S . ) , t h e W o o d -

tailed study of the cellular origin and r e g u l a t i o n of

Kalb F o u n d a t i o n and a P o t a m k i n - L e r n e r F e l l o w s h i p

trophic activity c o u l d l e a d to a m e a n s of increasing

(A.M.H.).

REFERENCES

15 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 16 Gage, F.H., Bj6rklund, A. and Stenevi, U., Denervation releases a neuronal survival factor in adult rat hippocampus, Nature (London), 308 (1984) 637=639. 17 Heacock, A.M., Schonfeld, A.R. and Katzman, R., Cholinergic hippocampal neurotrophic factor: developmental changes and response to injury, Soc. Neurosci. Abstr., 9 (1983) 840. 18 Heacock, A.M., Schonfeld, A.R. and Katzman, R., Relation of hippocampal trophic activity to cholinergic nerve sprouting, Soc. Neurosci. Abstr., 10 (1984) 1052. 19 Henderson, C.E., Huchet, M. and Changeux, J.-P., Denervation increases a neurite-promoting activity in extracts of skeletal muscle, Nature (London), 302 (1982) 609-611. 20 Hill, M.A. and Bennett, M.R., Cholinergic growth factor from skeletal muscle elevated following denervation, Neurosci. Lett., 35 (1983) 31-35. 21 Kligman, D., Isolation of a protein from bovine brain which promotes neurite extension from chick embryo cerebral cortex neurons in defined medium, Brain Research, 205 (1982) 93-100. 22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R,J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Manthorpe, M., Skaper, S., Adler, R., Landa, K. and Varon, S., Cholinergic neuronotrophic factors: Iractionation of an extract from selected chick embryonic eye tissues, J. Neurochem., 34 (1980) 69-75. 24 Manthorpe, M., Nieto-Sampedro, M., Skaper, S.D., Lewis, E.R., Barbin, G., Longo, F.M., Cotman, C.W. and Varon, S., Neuronotrophic activity in brain wounds of the developing rat. Correlation with implant survival in the wound cavity, Brain Research, 267 (1983) 47-56. 25 Muller, H., Beckh, S. and Seifert, W., Neurotrophic factor for central neurons, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1248-1252. 26 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. 27 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

1 Adler, R. and Varon, S., Cholinergic neuronotrophic factors: V. Segregation of survival and neurite-promoting activities in heart conditioned media, Brain Research, 188 (1980) 437-448. 2 Appel, S.H., A unifying hypothesis for the cause of amyotrophic lateral sclerosis, Parkinsonism and Alzheimer disease, Ann. Neurol., 10 (1981) 499-505. 3 Avendano, C., Suppression of reactive glial proliferation in the denervated dentate gyrus of the rat: effects on the pattern of acetylcholinesterase staining in the molecular layer, Brain Research, 265 (1983) 160-162. 4 Avendano, C. and Cowan, W.M., A study of glial cell proliferation in the molecular layer of the dentate gyrus of the rat following interruption of the ventral hippocampal commissure, Anat. Embrvol., 157 (1979) 347-366. 5 Barbin, G., Selak, I., Manthorpe, M. and Varon, S., Use of central neuronal cultures for the detection of neuronotrophic agents, Neuroscience, 12 (1984) 33-43. 6 Barde, Y.-A., Edgar, D. and Thoenen, H., Purification of a new neurotrophic factor from mammalian brain, EMBOJ., 1 (1982) 549-553. 7 Barde, Y.-A., Edgar, D. and Thoenen, H., New neurotrophic factors, Ann. Rev. Physiol., 45 (1983) 601-612. 8 Berg, D.K., New neuronal growth factors, Ann. Rev. Neurosci., 7 (1984) 149-170. 9 Billingsley, M.L., Hall, N. and Mandel, H.G., Trauma-induced glial proliferation of the immune system, Immunopharmacology, 5 (1982) 95-101. 10 Boon, Y.H., Hendry, I.A., Hill, C.E. and Watters, D.J., Effects of cardiac denervation on levels of neuronal survival factors for cultured autonomic neurones, Dev. Brain Res., 12 (1984) 154-157. 11 Collins, F., Induction of neurite outgrowth by a conditioned-medium factor bound to the culture substratum, Proc. Natl. Acad. Sci. U.S.A.. 75 (1978) 5210-5213. 12 Cotman, C.W. and Nadler, J.V., Reactive synaptogenesis in the hippocampus. In C.W. Cotman (Ed.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 227-271. 13 Crutcher, K.A. and Collins, F., In vitro evidence for two distinct hippocampal growth factors: basis of neuronal plasticity, Science, 217 (1982) 67-68. 14 Ebendal, T., Olson, L., Seiger, A. and Hedlund, K.O., Nerve growth factors in the rat iris, Nature (London), 286 (1980) 25-28.

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28

29

30

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implants in the wound cavity, J. Neurosci., 3 (1983) 2219-2229. Nieto-Sampedro, M., Whittemore, S.R., Needels, D.L., Larson, J. and Cotman, C.W., The survival of brain transplants is enhanced by extracts from injured brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6250-6254. Ojika, K. and Appel, S.H., Neurotrophic effects of hippocampal extracts on medial septal nucleus in vitro, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)2567-2571. Olney, J.W., Fuller, T. and DeGubareff, T., Acute dendrotoxic changes in the hippocampus of kainate treated rats, Brain Research, 176 (1979) 91-100. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. Rose, G., Lynch, G. and Cotman, C., Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus, Brain Res. Bull., 1 (1976) 87-92. Schonfeld, A.R., Heacock, A.M. and Katzman, R., Enhancement of central cholinergic sprouting by prior injury: correlation with endogenous trophic content of hippocampus, Brain Research, 321 (1984) 377-380.

34 Schonfeld, A.R., Heacock, A.M. and Katzman, R., Neurotrophic factors: effects on central cholinergic regeneration in vivo, Brain Research, 336 (1985) 297-301. 35 Steward, O. and Vinsant, S.L., The process of reinnervation in the dentate gyrus of the adult rat: a quantitative electron microscopic analysis of terminal proliferation and reactive synaptogenesis, J. Comp. Neurol., 214 (1983) 370-386. 36 Varon, S. and Bunge, R.P., Trophic mechanisms in the peripheral nervous system, Ann. Rev. Neurosci, 1 0978) 327-361. 37 Varon, S. and Adler, R., Nerve growth factors and control of nerve growth, Curr. Top. Dev. Biol., 16 (1980) 207-252. 38 Varon, S., Manthorpe, M. and Adler, R., Cholinergic neuronotrophic factors: 1. Survival, neurite outgrowth and choline acetyltransferase activity in monolayer cultures from chick embryo ciliary ganglia, Brain Research, 173 (1979) 29-45. 39 Varon, S., Manthorpe, M. and Williams, L.R., Neuronotrophic and neurite-promoting factors and their clinical potentials, Dev. Neurosci., 6 (1983/84) 73-100.