Nerve growth factor: effects on d -amphetamine-induced activity and brain monoamines

Brain Research, 176 (1979) 297-310 @ Elsevier/North-Holland Biomedical Press

297

N E R V E G R O W T H F A C T O R : EFFECTS O N D - A M P H E T A M I N E - I N D U C E D ACTIVITY AND BRAIN MONOAMINES

MICHAEL E. LEWIS*, ROGER M. BROWN, MICHAEL J. BROWNSTE1N, TESSA HART** and DONALD G. STEIN (D.G.S.) Department of Psychology, Clark University, Worcester, Mass. 01610 and Department of Neurology, University of Massachusetts Medical Center, Worcester, Mass. 01605 and ( R.M.B.) Laboratory of Neuropsychology, (M.J.B.) Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.)

(Accepted March 1st, 1979)

SUMMARY Adult male rats were given 6-hydroxydopamine lesions of the nucleus accumbens, followed immediately by injections of saline or nerve growth factor ( N G F ; 125 B.U.) near the substantia nigra. Such lesions were previously reported to attenuate the locomotor response to D-amphetamine. NGF-treated rats showed an enhanced response to D-amphetamine (1.5 mg/kg) when tested 15 days postoperatively. Levels of dopamine and norepinephrine in the striatum and nucleus accumbens were equivalently depressed in the two lesion groups, indicating that the apparent recovery of the NGF-treated rats was probably not due to catecholaminergic neuronal regrowth. lntracerebral N G F administration enhanced the response to D-amphetamine l 5 days later in rats without lesions, and also appeared to result in increased turnover of brain norepinephrine and serotonin at 3, but not 15, days postadministration. N G F might increase dopamine turnover at 15 days, but the evidence obtained did not convincingly confirm or negate this possibility. The results show that intracerebral N G F administration can produce similar behavioral changes in brain-damaged and intact rats, and also modify the apparent turnover of brain monoamines.

INTRODUCTION Nerve growth factor ( N G F ) is a protein of known importance for the development and maintenance of peripheral sympathetic neurons 5~, and recent anatomical, behavioral, and biochemical studies have been taken to suggest a possible role for * To whom reprint requests should be addressed at: The Psychological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EB, U.K. ** Currently at the Department of Psychology, University of Houston.

298 N G F in the central nervous system18, a0, as well. For example, Bj6rklund and Stenevi "~,l° found that a single intraventricular injection of N G F in adult rats potentiated axonal growth from transected noradrenergic, dopammergic, and indoleaminergic fibers. Direct intracerebral injections of N G F near noradrenergic cell bodies or axons were found to be more reliable than intraventricular injections for accelerating the growth of damaged noradrenergic neurons 49. The first studies exploring the behavioral effects of N G F administration to brain-damaged rats were performed by Berger, Wise and Stein 6, who found that a single intraventricular injection of N G F at the time of surgery accelerated recovery from the consummatory deficits induced by lateral hypothalamic lesions~ in a study designed to investigate both the behavioral and neurochemical effects of N G F administration, Hart et al. 2z, placed N G F in the vicinity of lesions in the caudate nucleus of' rats. The NGF-treated rats persevered less on a spatial reversal task than buttertreated control rats with identical lesions. Despite the large size of the lesions, the caudate dopamine levels of the two groups, measured 6 months after surgery, were within normal limits. Since caudate nucleus lesions would destroy a large number of dopaminergic fibers, these results can be interpreted to indicate that, by 6 months after surgery, some compensatory process had occurred to restore normal levels of dopamine in NGF-treated and untreated animals. Inasmuch as steady-state caudate dopamine levels appear to reflect the integrity (surviving terminals) of the nigrostriatal dopaminergic system 1, the results might be taken to suggest that sprouling of intact dopaminergic fibers had occurred. Such reinnervation could have been accelerated by N G F 9, but this effect would not have been detected so long after surgery and treatment. It thus appears important to obtain tissue measures shortly alter behavioral recovery has been detected in NGF-treated animals. The present experiments were designed according to this consideration, and attempted to relate a behavioral effect of N G F to changes in the steady-state levels or turnover of brain monoamines. EXPERIMENT1 A recent study by Kelly, Seviour and lversen 27 appeared to provide a useful experimental situation in which to test NGF. Bilateral injections of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens of rats produced a diminished locomotor response to D-amphetamine (1.5 mg/kg), an effect clearly observed at two weeks after surgery. However, a significant recovery of the response to D-amphetamine occurred within 80 days, and was correlated with a rise in dopamine levels in the nucleus accumbens. One interpretation of these findings is that the recovery of the response to Damphetamine was due to dopaminergic reinnervation of the nucleus accumbens. This notion is supported by the finding that behavioral supersensitivity to apomorphine was disappearing while the response to D-amphetamine was recovering 27. The ingrowth of new fibers would be expected to diminish post-denervation supersensitivity15, 50. Furthermore, there is evidence for collateral sprouting from dopaminergic neurons into partially denervated olfactory tubercle 10, suggesting again that these neurons can successfully reinnervate a denervated zone. The hypothesis of the present

299 study was that if N G F accelerates reinnervation of the nucleus accumbens after 6OHDA lesions, treated rats should show a significantly greater locomotor response to D-amphetamine than non-treated rats at two weeks after surgery. In addition, nucleus accumbens dopamine should be higher in the treated than in the untreated rats with lesions. Methods"

The subjects were 25 male rats of the Charles River CD strain, about 95 days old at the time of surgery. The rats were anesthetized with an intraperitoneal injection of Nembutal (35 mg/kg) followed by an intramuscular injection of Vetalar (60 mg/kg), and positioned in a Kopf stereotaxic apparatus. 6-Hydroxydopamine hydrobromide (6-OHDA; Sigma Chemical Co.) was dissolved immediately prior to injection in chilled 0.9% saline containing ascorbic acid (1 mg/ml). Bilateral injections of 6OHDA (8/~g in 2 #1, expressed as base) were made into the nucleus accumbens (AP 3.4, L 1.7 and V --7.2; ref. 40) with a 10 #l Hamilton 701-SN syringe driven by a hydraulic system connected to an electric syringe pump (Sage Instruments, model 341). The rate of injection was 0.42 #l/rain (setting 6), and the syringe was left in position for I rain following completion of the injection. Rats receiving the sham lesion had the syringe cannula lowered to these coordinates, but no solution was injected. Immediately following the bilateral 6-OHDA or sham lesions, the rats were bilaterally injected with 2.5 #1 of 2.5 S NGS (125 B.U., or approximately 1.25 #g, prepared according to the method of Bocchini and Angeletti n, and kindly donated by Dr. Lloyd Greene, Children's Hospital, Boston, Mass.) or 0.9% saline near the substantia nigra (AP --3.0, L 2.0 and V 8.0) using the same injection procedure as above. This locus of injection was selected on the assumption that the nigrostriatal dopamine system would have an important role in the reinnervation process, and also to avoid a direct interaction between N G F and 6-OHDA (ref. 29). The experimental group (6-OHDA/NGF) was given the lesion and N G F (n = 9), the lesion control group (6-OHDA/saline) was given the lesion and saline (n = 8), and the sham control group (sham/saline) was given the sham lesion and saline (n -- 8). Fifteen days after surgery, the animals were given intraperitoneal injections of D-amphetamine (1.5 mg/kg, expressed as the salt, prepared from D-amphetamine sulfate dissolved in 0.9 % saline; Smith, Kline and French Laboratories). Thirty minutes prior to injection, the rats were weighed and individually placed in cages (34 cm wide, 16 cm high, and 56 cm long) transected down the long axis by two photocell beams (Archer Photoelectric Relays). The testing room was darkened, and the light sources were covered by a red gel to minimize transmission of visible light. The number of times the photocell beams were broken was automatically recorded every 10 rain on a print-out (BRS Foringer) in a room adjacent to the testing room. Following the 30 min habituation period, the rats were removed from the cages, injected with D-amphetamine, and immediately replaced. Subsequent locomotor activity (photocell beam interruptions) were recorded in l0 rain blocks for a period of 2 h.

300 Eight days after testing, the animals were stunned by a blow to the head and decapitated. The brains were rapidly removed and frozen on dry ice. Serial frontal sections of 300/zm thickness were made in a cryostat at --10 °C. Tissue from the nucleus accumbens and striatum was removed from the sections using the micropunch dissection method of Palkovits 3s. Dopamine and norepinephrine concentrations (ng/mg protein) were determined in the tissue samples with a modification 39 of the enzymatic-isotopic method of Coyle and Henry ~2. The statistical significance of the predicted higher values of locomotor activity and catecholamine levels in 6-OHDA/NGF rats, compared to 6-OHDA/saline rats, was evaluated with one-tailed Exact Randomization Tests a6. Results

Figure 1 shows the changes in locomotor activity of the groups after administration of o-amphetamine. The 6-OHDA/NGF operates were significantly more active than the 6-OHDA/saline during time blocks 3, 6, 8, 10, I l, and 12 (P < 0.05), indicating that the predicted ameliorative effect of N G F on locomotor responding to o-amphetamine occurred primarily during the later part of the test period. As expected 27, the sham/saline operates showed a greater response over the whole period than the 6OHDA/saline operates (P < 0.05). The results of the catecholamine assays, shown in Table 1, indicated that in comparison to controls, dopamine was reduced in the nucleus accumbens by 83 ~ in the 6O H D A / N G F operates and by 88.8 ~ in the 6-OHDA/saline operates (P < 0.000l for each). Although the difference between the 6-OHDA groups was in the expected direction, it was not statistically significant (P > 0.1). In comparison to controls, striatal dopamine was depleted by 39.4 ~ in the 6-OHDA/NGF operates (P <:; 0.01) and by 47.6 ~ in the 6-OHDA/saline operates (P <~ 0.001). Again, the difference is in favor of the NGF-treated operates, but is not statistically significant. Norepinephrine in the

LOCOMOTOR RESPONSES TO D-AMPHETAMINE 1.5 mg/kg

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Fig. 1. Changes in locomotor activity over time after administration of 1.5 mg/kg D-amphetamineto the rats 15 days after surgery.

301 TABLE

1

Catecholamines (ng/mg protein) in nucleus accumbens and striatum (mean ± S.E.) Catecholamine

Brain r e g i o n

Dopamine Norepinephrine

Sham/Saline

6-OHDA/Saline

6-OHDA/NGF

Nucleusaccumbens 69.5 ± 3.4 Striatum 109.3 ÷L 13.6

7.8 zL 2.3 58.4 i 5.5

11.8 :L 2.9 66.2 ± 9.4

Nucleusaccumbens Striatum

0.14 ± 0.05 0.22 ~ 0.04

0.16 :~ 0.06 0.21 ± 0.03

0.85 ~ 0.29 0.17 ± 0.05

nucleus accumbens was significantly reduced in both groups with lesions (P < 0.001), while striatal norepinephrine, normally very low, was not affected. Discussion

In rats with 6-OHDA lesions of the nucleus accumbens, administration of N G F near the substantia nigra was followed, 15 days later, by an apparent recovery of the locomotor response to D-amphetamine. However, the NGF-treated operates did not show the predicted significant increase in dopamine in the nucleus accumbens, contradicting the hypothesis that dopaminergic reinnervation of that structure would be the basis of NGF-induced recovery (see ref. 28). If the remaining dopaminergic neurons were important for the recovery of the locomotor response to D-amphetamine, these neurons must have functioned more effectively in the NGF-treated than untreated rats. Stricker and Zigmond 51 have suggested that recovery of function after the massive catecholamine depletions induced by lateral hypothalamic lesions depends on compensatory processes within residual neurons of the damaged catecholaminergic pathways. In particular, they emphasized the probable role of increased tyrosine hydroxylase activity and turnover in recovery. N G F has been shown to facilitate recovery after lateral hypothalamic lesions 6, and has been shown to stimulate tyrosine hydroxylase activity in superior cervical ganglia s4,as,aG. Two further experiments were performed to attempt to evaluate the Stricker and Zigmond al model of recovery in relation to this first experiment. EXPERIMENT 2 If N G F facilitated recovery of the locomotor response to D-amphetamine in brain-damaged rats by increasing the synthesis of dopamine in remaining dopaminergic neurons, N G F should have the same effect on synthesis in a fully intact dopaminergic system. Thus, N G F administration should facilitate the locomotor response to D-amphetamine in rats without lesions. This possibility is interesting since the behavioral effects of N G F in normal rats have never been explored. Methods

The subjects were 12 male rats of the Chalres River CD strain, about 95 days old at the time of surgery. The rats were prepared for surgery as described in Experiment

302 1, and given bilateral intracerebral injections of N G F (125 B.U, in 2.5 !~t/side: n -:: 6) of 0.9 ~ saline (2.5 #l/side; n -- 6). Of the 6 rats in the N G F group, 4 were given injections near the substantia nigra, using the same coordinates as in t(xperiment i. while two rats were given injections near the mesolimbic (A 10) cell group (AP -3.9, L 0.5 and V --8.5). The supply of N G F was exhausted with these 6 animals, so the groups could not be enlarged as planned. The 6 NGF-treated rats were matched by 6 other rats given saline injections near the substantia nigra (n 4) or mesolimbic cell region (n =: 2). Fifteen days after surgery, the animals were given intraperitoneal injections of D-amphetamine (1.5 mg/kg), and were tested for locomotor activity as described in Experiment 1. The NGF-treated rats were predicted to show greater activity than the saline-treated rats. The results were evaluated statistically with one-tailed Exact Randomization Tests a6.

Results The activity scores of the rats injected near the substantia nigra or neighboring mesolimbic cell area were not different within treatment groups, and were thus combined for further analysis. As Fig, 2 shows, the NGF-treated rats were more responsive to D-amphetamine than the saline-treated rats. The differences were statistically significant during the latter part of the test period, i.e., for time blocks 6, 7, 8, 10, and 12, P ~. 0.05. Discussion lntracerebral administration of N G F to rats without lesions resulted, 15 days later, in an augmented locomotor response to D-amphetamine. This effect occurred during the later part of the test period, similar to the result obtained with brain-damaged rats in Experiment 1. The results may be taken to indicate that N G F exerts

LOCOMOTOR RESPONSESTO D-AMPHETAMINE 1.5 mg/kg 140, ' !GF 100 ~ U

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Fig. 2. Changes in locomotor activity over time after administration of 1.5 mg/kg D,amphetamine to the rats 15 days after intracerebral injections of NGF or saline.

303 similar actions in the damaged and intact brain. Since the locomotor response to Damphetamine depends primarily upon dopaminergic neurons 13,27,41, it is reasonable to hypothesize that the effects of N G F were upon these neurons. The time course of the facilitation of the amphetamine response by N G F is consistent with the hypothesis that N G F increases dopamine synthesis. Since amphetamine acts to release dopamine from nerve terminals and block its reuptake 7,~3, continued responding to the drug should depend on the functional (i.e. releasable) pool size of dopamine and its rate of replenishment. As amphetamine progressively exhausts the functional pool, the response to the drug must diminish. However, if the rate of synthesis of dopamine was accelerated, the pool would be exhausted more slowly, prolonging the response to amphetamine. The NGF-treated rats in this and the previous experiment showed relative increased activity during the last half of the test session. In other words, the response to amphetamine was not heightened uniformly over time, but appeared to be prolonged. Thus, the metabolic functioning of dopaminergic neurons may somehow be responsive to NGF. EXPERIMENT 3 This experiment was designed to examine the question of whether intracerebral administration of N G F could affect the turnover of dopamine and the other monoamines, norepinephrine and serotonin. The per cent depletion of each catecholamine after 2 h of synthesis inhibition was used to provide a relative measure of turnover for dopamine and norepinephrine 54. Levels of serotonin and its deaminated metabolite, 5hydroxyindoleacetic acid, were measured in the absence of synthesis inhibition to obtain an index of serotonin turnover2°, 45. Since N G F is known to increase tyrosine hydroxylase activity in peripheral adrenergic neurons, increases in both dopamine and norepinephrine turnover (if N G F diffused to noradrenergic neurons) were predicted. Changes in serotonin turnover could not be predicted, since an effect of N G F on indoleamine synthesis (or relevant enzymes) had not been reported when the experiment was performed. Methods" The subjects were 36 male rats of the Charles River CD strain, about 95 days old at the time of surgery. The rats were prepared for surgery as described previously, and given bilateral intracerebral injections of 2.5S N G F (1.25 #g/side; generously donated by Dr. Gordon Guroff, National Institutes of Health, Bethesda, Md.) or a control solution of 0.05 M sodium acetate, both of which were neutralized with 0.1 M sodium phosphate buffer (pH 7.4). The injections were made near the substantia nigra using the same volumes and procedures as before. Sixteen rats each were given N G F or buffer solution, while 4 rats served as unoperated control subjects. Half of each group of operates were given intraperitoneal injections of a-methyl-p-tyrosine methyl ester (a-MpT; 250 mg/kg, dissolved in 0.9 ~ saline; Sigma Chemical Co.) at either 3 or 15 days after surgery. Two hours after the injections, these rats were stunned by a blow to the head and then decapitated. The brains were rapidly removed and frozen on dry ice.

304 The operates which were not injected with a-MpT were also decapitated at 3 or 15 days after surgery. The whole brains were weighed and then homogenized in centrifuge tubes containing 10 ml of 0.4 N perchloric acid, 0.2 ml of 10~ EDTA, and 0.1 ml of 5°~ NazS2Os. The homogenates were processed and the supernatants passed through strong cation exchange columns as described by Atack 3. 5-HydroxyindoJeacetic acid, norepinephrine, dopamine, and serotonin were sequentially eluted :',:~1 and then assayeda, T M via oxidation of these amines into their fluorescent derivatives. The percentage ofdopamine and norepinephrine depleted in the a-MpT-treated rats relative to the non-a-MpT-treated rats was calculated, and these values were compared for the N G F and buffer groups. Since greater depletions reflect a more rapid turnover rate, the per cent depletions were predicted to be greater in the NGF-treated rats. The statistical significance of the predicted differences was evaluated with one-tailed t-tests. Possible differences between the groups in serotonin and 5-hydroxyindoleacetic acid levels were statistically evaluated with two-tailed t-tests. Results The per cent depletions of dopamine and norepinephrine of the ~-MpT-treated rats are shown in Fig. 3. The depletions of dopamine in the N G F and buffer rats are equivalent at 3 days after treatment. By 15 days, the N G F rats appear to show greater dopamine depletions, but the difference is not statistically significant. Three of the 4 N G F rats showed greater dopamine depletions than any of the buffer rats ( N G F : 56.8, 47.4, and 44.4 ~ ; buffer: 39.1,38.7, 35.6, and 35.0 ~o), while one possibly aberrent N G F rat showed less depletion than any of the buffer rats (29,3 %). However, this animal cannot be eliminated using statistical criteria because of the small n. PERCENT DECREASE IN BRAIN CATECHOLAMINE LEVELS TWO HOURS AFTER aMpT ADMINISTRATION 3 Days Post-injection

15 Days Post-injection

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Fig. 3. Per cent decrease in brain levels of dopamine (DA) and norepinephrine (NE) at 2 h after

synthesis inhibition with a-methyl-p-tyrosine in rats given intracerebral injections of NGF Or buffer (B) 3 or 15 days previously (means :k S.E. ; n .... 4 in each group).

305 EFFECT OF NGF ON BRAIN 5-HT AND 5-HIAA LEVELS •

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Fig. 4. Change over time of brain levels of 5-hydroxytryptamine (5-HT;serotonin)and its metabolite 5hydroxyindoleacetic acid (5-HIAA) after intracerebral injections of NGF or buffer (B); data from non-treated control animals (N) may be regarded as 'day 0' values (means S.E.).

The N G F rats showed a significantly greater (t-MpT-induced depletion of norepinephrine than the buffer rats at three days after treatment (t ~ 1.946, df = 6, P < 0.05). As Fig. 3 shows, this effect had disappeared by 15 days after treatment. No difference between groups in brain serotonin (5-HT) levels was found at 3 or 15 days after the intracerebral injections (Fig. 4). However, N G F treatment resulted in a significant increase inthe levels of the 5-HT metabolite 5-hydroxyindoleacetic acid (5HIAA) by three days (t = 3.109, df = 6, P < 0.025). The difference in 5-HIAA levels was no longer statistically sifnificant by 15 days after treatment. Observation of the rats killed at 15 days after treatment indicated that most of the N G F rats lost some body weight (about 1 0 ~ over a period of several days), but recovered their preoperative weight before 15 days. An effect of N G F on body weight was previously found in rats with lateral hypothalamic lesions ~. In addition to body weight loss, most of the N G F rats showed a transient abnormality of posture and gait, characterized by a hunched spine and splayed rear feet. This effect had entirely disappeared within 15 days. Discussion

This experiment attempted to evaluate the hypothesis, from Experiments l and 2, that intracerebral administration of N G F would increase dopamine turnover at 15 days after treatment. Such an effect could be used to explain the prolonged locomotor response to D-amphetamine observed in NGF-treated rats. Unfortunately, this experiment did not provide clear evidence either for or against the hypothesis. Future

306 studies of this type should employ methods allowing measurement of dopamine turnover in discrete regions of both the nigrostriatal and mesolimbic systems. N G F treatment appeared to result in increased norepinephrine turnover at three days, as indicated by the greater a-MpT-induced depletion of this amine in the N G F group. N G F administration also resulted, three days later, in heightened 5-HIAA levels with no change in the steady-state level of 5-HT, indicating a probable increase in 5-HT turnover20, 45. The relationship of these apparent changes in monoamine turnover to the behavioral effects of N G F remains unclear. GENERAL DISCUSSION The goal of the experiments reported here was to obtain a behavioral effect of N G F which could be interpreted with neurochemical data. The locomotor response to D-amphetamine, which has been shown to depend on dopaminergic neurons 13,~7,4~, appeared to provide a useful experimental model for exploring the behavioral effects of NGF. Following 6-OHDA-induced depletions of dopamine in the nucleus accumbens, administration of N G F near the substantia nigra appeared to promote recovery of the locomotor response to D-amphetamine (Experiment I). However, the apparent recovery could not be related to increased dopaminergic neuronal reinnervation of the nucleus accumbens, as determined by a very sensitive dopamine assay, This finding raised the possibility that N G F somehow enhanced the functioning of the remaining dopaminergic neurons. Administration of N G F to rats without lesions resulted in an augmented locomotor response to o-amphetamine similar to that found in the rats with 6-OHDA lesions (Experiment 2). Unfortunately, clear evidence for an effect of N G F on dopaminergic neuronal functioning, as indicated by a measure of turnover, was not obtained (Experiment 3). Nevertheless, transient increases in apparent turnover of norepinephrine and serotonin were detected after N G F treatment in rats without lesions. Thus, while the goal of the experiments was not achieved, the results provide interesting new evidence for effects of N G F on behavioral and neurometabolic indices. Further studies will be required to determine whether the effect of N G F on the locomotor response to a low dose of D-amphetamine can be extended to the stereotyped responding after higher doses 2~, and whether such behavioral effects can be related to more sensitive measures of dopaminergic neuronal functioning (e.g. refs. 8, 54) than employed in this study. A theoretical perspective which has arisen from this and other studies of N G F is that the effects of this protein on brain measures and behavior (including behavioral recovery) can be understood as 'trophic responses" of the brain to NGF. The concept of trophism refers to long-term influences of nerves on target tissues, such as regulation of metabolism, in contrast to short-term events such as neurotransmission 2~'a7. On the basis of studies of a variety of mammalian cellular systems, Russell, Byus and Manen 4~ have proposed a general model of the sequential biochemical events of a trophic response. In brief, cells respond to trophic factors (e.g. certain hormones and drugs) with a cyclic AMP-mediated transfer of the trophic stimulus from the cell membrane to the nucleus. The stimulation results in gene activation, with the consequent

307 synthesis of new RNA and protein. One of the new proteins synthesized is ornithine decarboxylase, the rate-limiting enzyme in the formation of polyamines, compounds extensively implicated in the regulation of cellular metabolism and growth 52. For example, embryos, tumors, and regenerating liver all possess heightened ornithine decarboxylase activity 4a. The above model of trophic regulation is relevant to the understanding of the effects of NGF. In superior cervical ganglia from young rats, stimulation with N G F results in the following sequence of events : increased formation of intracellular cyclic AMP (ref. 37); increased activity of nuclear protein kinase3a; increased ornithine decarboxylase activity3a; increased RNA polymerase activity24; and induction of tyrosine hydroxylasea4,55,a6. lntraventricular administration of N G F to adult rats is followed 8 h later by a 15-fold increase in brain ornithine decarboxylase activity a°. This effect could be a critical early event in the mediation of the effects of N G F on axonal regrowthg, 10.49 and behavioral recovery6 after brain damage in adult rats. In this context, it is relevant that administration of polyamines has been reported to accelerate recovery of hindlimb reflexes after sciatic nerve crush in adult rats 16. Since these compounds exert potent central actions2,44,5 a, their possible effects on behavioral recovery after brain damage should be explored. The present study has demonstrated that the behavioral effects of N G F are not restricted to brain-damaged rats, a not surprising finding if the name of the protein is not taken literally as a complete description of its biological effects. The behavioral effects of N G F could be related, in part, to increased activity of tyrosine hydroxylase, although an effect of N G F on this enzyme in the brain is still undemonstrated. Nevertheless, the present study has provided evidence for a transient effect of N G F on norepinephrine turnover, which is consistent with an increase in tyrosine hydroxylase activity. In addition, the possibility of a later increase in dopamine turnover cannot be excluded. A delayed effect on dopamine turnover could be due to the time required for axoplasmic flow to deliver newly synthesized tyrosine hydroxylase to nerve terminals aS. In any case, a direct determination of the effect of N G F on regional tyrosine hydroxylase activity will be necessary. An effect on tryptophan hydroxylase activity should also be explored, since N G F administration resulted in an apparent increase in serotonin turnover (Experiment 3). Indeed, a recent study found that N G F administration to newborn rats resulted in an increased level of tryptophan hydroxylase in superior cervical ganglia s`). In addition to alterations in neurotransmitter-synthesizing enzymes, N G F could possibly exert effects on behavior through anatomical changes. The effect of N G F on the activity of ornithine decarboxylasea0, an enzyme intimately associated with cellular growth "52, is consistent with this possibility. Effects on growth need not be limited to the experimentally damaged brain, as Sotelo and Palay as have suggested that a continual process of ~remodelling' of neuronal connections occurs in the undamaged brain. This process could involve endogenous growth factors, perhaps including N G F or NGF-like moleculesa6 acting at cell surface binding sites 17 or via retrograde transport 14. The experiments reported here have demonstrated previously unknown behav-

308 ioral a n d n e u r o m e t a b o l i c changes after intracerebral a d m i n i s t r a t i o n of" N G F . T h e extent to which these a n d o t h e r effects o f N G F can be related to one a n o t h e r , o r u n d e r s t o o d within the ' t r o p h i c response' f r a m e w o r k , remains to be d e t e r m i n e d . M e a n while, this perspective should be a useful guide to future research. ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Ms. V. K. Weise a n d Dr. D. A. Stevens for technical assistance, Dr. N. U r e t s k y for advice d u r i n g the experiments, Dr. T. W. R o b b i n s for helpful c o m m e n t s on the m a n u s c r i p t , a n d Ms. A. Bassett for secretarial assistance. This research was s u p p o r t e d in p a r t by N IA G r a n t A G 00295 and by a Research C a r e e r D e v e l o p m e n t A w a r d M H 70177 and in part by funds f r o m the Research F u n d at C l a r k University a w a r d e d to the first author. We gratefully a c k n o w l e d g e this assistance.

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