0013-7227/91/1294-2212$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 4 Printed in U.S.A.
Nerve Growth Factor Modulates the Activation of the Hypothalamo-Pituitary-Adrenocortical Axis during the Stress Response G. TAGLIALATELA, L. ANGELUCCI, S. SCACCIANOCE, P. J. FOREMAN, AND J. R. PEREZ-POLO Department of Human Biological Chemistry and Genetics (J.R.P., P.J.F.), University of Texas Medical Branch, Galueston, Texas 77550; Institute for Research on Senescence-Sigma Tau (G. T), Pomezia, Italy; and Department of Pharmacology (L.A., S.S.), University of Rome La Sapienza, Rome, Italy
tivation of the HPAA is significantly reduced by pretreatment of the rats with anti-NGF immunoglobulin G. These results suggest that the stimulatory action of NGF on HPAA activity requires the release of ACTH secretagogues from the hypothalamus and that NGF may modulate the HPAA response to stress stimuli. {Endocrinology 129: 2212-2218,1991
ABSTRACT. In the present study, we have investigated the functional relationship between the nerve growth factor protein (NGF) and the hypothalamus-pituitary-adrenocortical axis (HPAA) We have found that while iv injected NGF is able to stimulate the HPAA activity in rats, NGF is not able to stimulate the axis after a block of the hypothalamus produced by chlorpromazine-morphine-Nembutal treatment. Also, the stress ac-
T
HE ACTIVATION of the hypothalamus-pituitaryadrenocortical axis (HPAA) is an integral part of the stress response. In response to stressors, hypothalamic CRFs are released into the portal circulation of the hypophysial stalk and then act on the pituitary where they stimulate the release of ACTH. The ACTH, in turn, stimulates the release of corticosteroid hormones from the adrenal glands. These circulating corticosteroids inhibit further HPAA activity via specific receptors present in the pituitary, hypothalamus, and hippocampus (1-4). The different occupancy levels of hippocampal corticosteroid receptors modulate the release of CRF, arginine vasopressin, and oxytocin from the hypothalamus, particularly in the stress-activated state (5-7). In addition, the binding of glucocorticoid hormones in the hippocampus has been shown to be essential for the acquisition and retention of behavioral tasks in rodents (8, 9). The concomitant interrelated activities of modulating factors render the activity of the HPAA, under basal and stress conditions, as more complex than a simple cascade of hormonal events. The nerve growth factor protein (NGF) is essential for the maturation and maintenance of sympathetic and Received May 28, 1991. Address requests for reprints to: J. R. Perez-Polo, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77550. Supported by NINDS Grant NS-18708, CNR Grant 88.01813.04, and a grant from the Sigma Tau Company, Pomezia, Italy.
sensory innervation (10). NGF and NGF receptors (NGF-R) in the central nervous system (CNS) have been shown to be trophic to cholinergic neurons of the basal forebrain and septum (11, 12). NGF can also influence aspects of rodent behavioral development (13) and act as a cytokine in the immune response (14-17). NGF and hormonal steroids appear to be functionally related. Whereas 17/3-estradiol induces NGF synthesis and secretion of NGF in the C-6 rat glioma cell line (18), the synthetic glucocorticoid dexamethasone decreases NGF binding and NGF-R messenger RNA in the PC12 rat pheochromocytoma cell line (19, 20) as well as blocks NGF-mediated induction of NGF-R mRNA (our unpublished observation). Testosterone appears to decrease the expression of NGF mRNA in rat Sertoli cells (21). During development, local levels of NGF and glucocorticoids determine adrenal medullary chromaffin cell lineage and sympathetic neuron phenotype (22), both derived from neural crest. The stimulation of the HPAA by NGF (23) indicates the possible physiological relationship between NGF and adrenocortical activity. Whereas exposure to cold stress reduces NGF binding in the hippocampus and basal forebrain of young adult rats (24), adrenalectomy decreases NGF levels in the hippocampus while increasing levels of NGF-R in the septum (25). Aggressive behavior, a form of social stress, stimulates the Release of NGF from the submaxillary glands into peripheral blood cir-
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Chlorpromazine HC1 (CPZ) was purchased from Elkins-Sinn Inc. (Cherry Hill, NJ) and morphine sulfate (MS) from Knoll Pharmaceutical Co. (Whippany, NJ); pentobarbital sodium was obtained from Abbott Laboratories (Chicago, IL) and cytochrome c and BSA from Sigma Chemicals (St. Louis, MO). Rat plasma corticosterone RIA kit was from ICN (Costa Mesa, CA). Rat pheochromocytoma (PC12) cells were a generous gift of Dr. Lloyd Greene (Columbia University, New York, NY).
Trunk blood was collected 2 h after iv injection (the HPAA response to NGF still plateaus at this time interval) in order to avoid stress bias due to anesthesia. For experiments in which the pituitary-adrenocortical response to NGF was compared with that to CRF in CPZ-MS-Nb-treated rats, animals were killed 30 min after the NGF injection, a time interval at which the HPAA response to CRF plateaus (23) and the response to NGF has already reached its maximum. In order to study on the same individual the time course activation of the HPAA after iv injection of NGF, rats were surgically implanted with a permanent cannula in the jugular vein and used 4 days afterward. NGF was delivered iv via the cannula, and blood samples were drawn at the time-points shown via the same route. Blood was collected in ice-cold tubes containing trasylol (500 kallikrein-inhibiting units/ml) and after centrifugation at 1,900 X g for 20 min plasma was stored frozen at -70 C for later hormone analyses. Plasma corticosterone was determined by the method described by Murphy (30) and plasma ACTH by RIA (Incstar, MN). The sensitivity and inter- and intraassay variations of the assays are described elsewhere (31). For stress experiments, animals were injected sc with sheep anti-NGF immunoglobulin G (IgG) preparation (250 Ail/rat), or preimmune sheep IgG preparation (250 /tl/rat) in the morning on 2 consecutive days. On the third day, 1 h before the final experimental procedure the animals were given 250 n\ ip of the IgG preparations. A separate set of animals (anti-NGF, preimmune IgG, and blanks) received only one ip injection 1 h before the experiment. Stress consisted in a 1-h exposure to 4 C (cold room). Rats were killed immediately at the end of the stress period and trunk blood processed as above.
Animals
NGF preparation
Male 3-month-old Sprague-Dawley rats (200-250 g) were purchased from Harlan Sprague Dawley Co.(Houston, TX). Animals were housed three per cage, with food and water ad libitum, and a 12-h dark/light cycle (lights on at 0750 h) and 23 ± 1 C temperature.
The /3-NGF subunit was isolated from mouse submaxillary glands according to the method of Mobley et al. (32). Concentrations of NGF were assessed spectrophotometrically at 280 nm wavelength, and biological activity was assayed by the ability to stimulate neurite outgrowth in the PC12 cells.
Experimental procedures
Anti-NGF IgG fraction preparation
Pharmacological block of the hypothalamic secretagogues release was obtained according to the method described by Arimura et al. (29). Briefly, rats were injected with CPZ (10 mg/kg sc) followed 3 h later by MS (20 mg/kg sc) and, after 15 min, by sodium pentobarbital [Nembutal (Nb), 25 mg/kg ip]. Immediately after the Nb injection, the rats were kept under an oxygen tent until death. Forty-five minutes after the last injection, animals were injected via the jugular vein with either NGF or cytochrome c at the concentrations indicated below. Cytochrome c was used as a control for NGF because these two proteins display similar physicochemical characteristics. Before the iv injection, 1 ml aliquot of blood was quickly drawn from the jugular vein to asses basal plasma corticosteroid levels. One hour after iv injection, a time interval at which the pituitaryadrenocortical response to NGF plateaus, rats were killed by decapitation and trunk blood collected in tubes containing 1 mg EDTA as an anticoagulant. Tubes were then centrifuged at 2000 X g for 20 min and the supernate (plasmatic fraction) stored at -20 C until assayed for corticosterone content. Nonblocked rats were injected iv under light ether anesthesia.
Serum was collected from a sheep that had been previously immunized against mouse |8-NGF by venous puncture. Aliquots were also drawn from the same animal before immunization (preimmune fraction). Sera were precipitated with saturated ammonium sulfate solution with continuous stirring. After centrifugation for 20 min at 400 X g, the precipitate was redissolved in borate buffered saline (BBS), pH 7.8, and dialyzed against BBS (4 days; 8 L.). Protein concentration was determined, and 25 mg of the crude IgG fraction were loaded onto a Sephadex-Protein A chromatographic column (Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with 20 ml of 0.2 M sodium citrate buffer. The eluate was dialyzed overnight against ammonium bicarbonate buffer, pH 8.0, and evaporated in a Speed Vac Concentrator (Sarvant Instruments, Hicksville, NY). The IgG fraction was then reconstituted in PBS, pH 7.6, and the IgG concentration determined spectrophotometrically. Biological activity of the IgG preparation was assayed by the ability of antibody to block neurite outgrowth in NGF-treated PC12. When PC12 cells were cultured in serum containing medium in the presence of NGF (1 ^g/ml), there
culation and elevates NGF mRNA and NGF protein levels in the hypothalamus of mice (26-28). The original study demonstrating the action of NGF on the HPAA did not address the question as to the site of NGF action in the CNS or its physiological significance (23). Here, we report that the NGF-induced pituitary-adrenocortical activation is abolished in rats with a pharmacological block of the hypothalamus. Moreover, inactivation of NGF in the peripheral circulation by the injection of antibodies against NGF significantly attenuates the pituitary-adrenocortical stress response. These results indicate that exogenous NGF stimulates the HPAA at hypothalamic or suprahypothalamic levels. These results also support the hypothesis that stress stimulation of NGF release may be a significant component of the response of the HPAA to stress.
Materials and Methods Materials
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was abundant neurite outgrowth by the NGF-treated PC 12 (Fig. 1C). As shown in Fig. ID, NGF activity is completely blocked by the presence of 5 /i\/m\ of the sheep anti-NGF IgG fraction while the anti-NGF IgG itself does not elicit any morphological response by the PC12 (Fig. IB). Statistical analysis
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Statistical differences among groups were assessed by the analysis of variance (ANOVA) followed by the Fisher's least significant difference test for multiple comparisons.
Results NGF injection in CPZ-MS-Nb-treated rats Intravenous injection with either 10 or 100 pmol/g body weight (bw) of NGF in 4-month-old male rats produced a significant increase in plasma corticosterone levels at either NGF dose, while no effect was observed after a control injection of cytochrome c (Fig. 2). Circulating levels of corticosterone in both groups of rats injected with NGF were more than 10-fold higher than
5 U ro CO
a Control
10
1OO
Dose (pmol/g bw) FIG. 2. Plasma corticosterone levels in rats 1 h after injection (i.v.) with NGF or cytochrome c. Control corticosterone values have been obtained from blood samples drawn from each rat immediately before the injection. Mean values ± SEM; n = 6-10 animals per group.
those observed before the NGF injection or in the cytochrome c-treated animals, indicating that both doses of NGF were saturating. The time-course response of the HPAA to NGF is shown in Fig. 3. The plasma corticosterone levels reached a maximum 30 min after the NGF injection and remained elevated for up to 3 h, while plasma ACTH titers peaked 30 min after NGF injection and returned to basal values 120 min later. No effect on plasma corticosterone was observed in cytochrome cinjected rats. These results were quantitatively and qualitatively similar to those obtained by Otten et al. (23). There was no elevation of plasma corticosterone levels after the injection of NGF (10 pmol/g bw iv) into hypothalamus-blocked rats (Fig. 4). Hypothalamus-blocked rats, although insensitive to NGF, were still capable of a full pituitary-adrenocortical activation when challenged with an iv injection of 2 ng CRF (Table 1). Effect of anti-NGF treatment effect on the stress response
FIG. 1. Microphotograph of PC 12 cells treated for 24 h with NGF (1 Mg/ml) in presence of sheep anti-NGF IgG fraction (5 ml/ml). A, Control cells; B, anti-NGF treated cells; C, NGF-treated cells; D, NGF antiNGF treated cells. Reference bar, 150 /xm.
Treatment of rats with anti-NGF IgG fraction for 3 consecutive days significantly reduced the response of the HPAA to cold stress, as determined by measurements of plasma corticosterone levels (Fig. 5). A partial response to cold stress in the anti-NGF-treated rats might have been present in that the plasma corticosterone concentration was somewhat higher, although not statistically significant, compared with the treatment-matched basal animals. There was also a small but statistically not significant effect of the injection procedure on plasma corticosterone levels in basal animals. A single injection of the anti-NGF IgG fraction (250
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60 r
FIG. 3. Plasma corticosterone (upper panel) and plasma ACTH {lower panel) values in rats injected with 10 pmol/g bw of either NGF (O) or cytochrome c (•). Animals were freely moving and injected via a permanent cannula in the jugular vein. Blood samples were collected at the time-points shown via the same route. Mean values ± SEM; n = 35 animals per group.
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Time after injection (min.)
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Time after injection (min.) /Lil/rat ip) 1 h before exposure to a cold environment did not attenuate the subsequent stress response of the HPAA. However, this event was not statistically different from the stress-induced increase in circulating corticosterone levels observed in the control IgG-injected rats or in the untreated animals. In this experiment the corticosterone levels were: untreated stressed, 11.99 ± 1.29; control IgG stressed, 11.90 ± 1.11; anti-NGF stressed, 8.20 ± 2.24. (ANOVA: F = 1.92; degrees of freedom = 2,12; P = 0.2).
Discussion In the rat, pharmacological block of the hypothalamus completely abolishes its capability to release humoral secretagogues of pituitary ACTH (29) in response to a variety of stressors, as measured by the absence of any stress-induced elevation of serum corticosterone levels.
However, in this condition the ability of the pituitary to release ACTH and the consequent release of corticosteroids from the adrenals in response to an iv injection of CRF are not abolished (29, 33). Therefore this pharmacological manipulation provides a functional inactivation of the HPAA at the hypothalamic level, while leaving the remaining components of the HPAA responsive to appropriate stimuli. In the unblocked control rats, iv injection of NGF fully activated the HPAA, as measured by elevation of plasma ACTH and corticosterone titers. Since we did not observe any rise in plasma corticosterone levels after NGF injection in pharmacologically (CPZ-Ms-Nb) blocked rats, but did observe a full response to CRF, we conclude that the site of action of NGF in stimulating the HPAA is either the hypothalamus or some suprahypothalamic structure that regulates the HPAA or both. Given that in this condition the pituitary is responsive to CRF stimulation, any activity
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NGF
FIG. 4. Plasma corticosterone levels in CPZ-MS-Nb-treated rats before (PRE) or 1 h after (POST) the iv injection of either NGF (10 pmol/g bw) or cytochrome c (10 pmol/g bw). Mean values ± SEM; n = 4-6 animals per group.
POST-INJECT
PRE-INJECT TABLE 1. Plasma corticosterone values in CPZ-MS-Nb-treated rats before or 30 min after iv injection of cytochrome c (10 pmol/g bw), NGF (10 pmol/gr bw), or CRF (2 fig/rat) Plasma corticosterone (fig/100 ml)
Cytochrome c NGF CRF
Pre-injection
Post-injection
3.84 ± 0.29 4.05 ± 0.52 3.40 ± 0.82
2.59 ± 0.32 2.81 ± 0.19 18.91 ± 1.93°
Values are mean ± SEM of four rats per group. °P< 0.001 (ANOVA).
of NGF on the pituitary is excluded. This is consistent with the reported absence of NGF-R on the ACTHsecreting anterior lobe of the pituitary (34). It has been demonstrated that NGF is released from the submaxillary glands into the circulation during aggressive behavior in rodents (26, 27). Surgical removal of the submaxillary glands abolishes the stress-induced
increase of circulating NGF but has no effect on serum NGF basal levels (26). There remain uncertainties as to measurements of NGF in serum in part due to adherence properties of NGF and the presence of NGF binding proteins in serum (35, 36). This would suggest that NGF, in addition to its paracrine effects on basal forebrain cholinergic neurons, can also act as a classical endocrine hormone, in that it can be released from a discrete organ, the submaxillary glands, into the circulation in response to physiological relevant stimuli. Our finding that treatment of rats with antibodies directed against NGF significantly diminishes the response of the HPAA to cold stress, further suggests a role for circulating NGF as a component of the HPAA response to stress. Further studies are needed to clarify unequivocally the site of NGF action in the stimulation of the HPAA activity. Our finding in hypothalamic-blocked rats would suggest that NGF acts at the hypothalamus, stimulating
BLANK
CONTROL
ANT I-NGF
FIG. 5. Plasma corticosterone levels in rats treated for 3 days with anti-NGF IgG fraction or preimmune IgG fraction (CONTROL). Stress consisted of 1 h exposure to a cold (4 C) environment. *, P < 0.05 vs. blank-stressed or controlstressed animals (ANOVA). Mean values ± SEM; n = 4 per group.
BASAL
COLD STRESS
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NGF AND PITUITARY-ADRENOCORTICAL ACTIVITY
the release of CRF. However, NGF-R have not been reported to be present in those hypothalamic areas in which the CRF-secreting neurons are localized (37). This interpretation does not exclude the possibility that NGF may facilitate the release of other hypothalamic releasing factors, such as arginine vasopressin. Another possibility is that NGF can affect those inhibitory mechanisms that normally are involved in the control of HPAA activity. The hippocampus, a limbic structure that possesses high levels of NGF-R (24, 38), is a likely candidate for this NGF effect since it is known to inhibit hypothalamic activity during basal and, especially, stress conditions (39-43). The NGF released during stress might modulate these events by acting at the hippocampus, as is suggested by the reduced activation of the HPAA in the anti-NGF-treated rats. This is consistent with the finding that after cold stress there is a reduction of NGF binding in the rodent hippocampus (24), possibly due to an increased ligand-receptor complex internalization. Also, the data presented here show a reduced stressinduced activation of the HPAA in the anti-NGF-treated animals, suggesting that after an initial response to stress, the HPAA, in the absence of sufficient circulating NGF, becomes unable to undergo full activation. This can be a likely mechanism of action, provided that the hippocampus is affected by circulating NGF levels. In fact, a selective uptake of NGF takes place in the hippocampus of rats injected iv with 10 pmol/g bw of [125I] NGF, while there is no significant uptake into hypothalamus (Loy, R., G. Taglialatela, L. Angelucci, and J.-R. Perez-Polo, in preparation). Although NGF has effects on adrenal chromaffin cells, there is no evidence of interference with corticosteroid function by the adrenals in NGF-treated rodents. In conclusion, peripheral NGF activates the HPAA by acting within the CNS. There is the possibility that the hippocampus and the hypothalamus participate in NGF action in the CNS. The NGF released into the circulation during stress may play a role during activation of the HPAA.
5. 6.
7.
8.
9.
10. 11. 12.
13. 14. 15. 16.
17. 18. 19. 20.
Acknowledgments Thanks to Mrs. D. Masters for manuscript preparation and K. Werrbach-Perez for excellent technical assistance.
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26. Lakshmanan J 1986 Aggressive behavior in adult male mice elevates serum nerve growth factor levels. Am J Physiol 250:E386E392 27. Aloe L, Alleva E, Bohm A, Levi-Montalcini R 1986 Aggressive behavior induces release of nerve growth factor from mouse salivary glands into the bloodstream. Proc Natl Acad Sci USA 83:61846187 28. Spillantini MG, Aloe E, Alleva E, De Simone R, Goedert M, LeviMontalcini R 1989 Nerve growth factor mRNA and protein increase in hypothalamus in a mouse model of aggression. Proc Natl Acad Sci USA 86:8555-8559 29. Arimura A, Saito T, Schally AV 1967 Assays for corticotropinreleasing factor (CRF) using rats treated with morphine, chloropromazine, dexamethasone and nembutal. Endocrinology 81:235245 30. Murphy BEP 1967 Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive proteinbinding radioassay. J Endocrinol Metab 27:973-990 31. Scacciancoe S, Navarra D, Di Sciullo A, Angelucci L, Endroczi E 1989 Adenosine and pituitary-adrenocortical axis activity in the rat. Neuroendocrinology 50:464-468 32. Mobley WC, Schenker A, Shooter EM 1981 Characterization and isolation of proteolytically modified nerve growth factor. Biochemistry 15:5543-5551 33. Rivier C, Brownstein M, Spiess J, Rivier J, Vale W 1982 In vivo corticotropin-releasing factor-induced secretion of adrenocorticotropin, (8-endorphin, and corticosterone. Endocrinology 110:272278 34. Yan Q, Clark HB, Johnson Jr EM 1990 Nerve growth factor
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receptor in neural lobe of rat pituitary gland: immunohistochemical localization, biochemical characterization and regulation. J Neurocytol 19:302-312 Beck CE, Perez-Polo JR 1982 Human /? nerve growth factor does not cross-react with antibodies to mouse /3-nerve growth factor in a two-site radioimmunoassay. J Neurosci Res 8:137-152 Perez-Polo JR (ed) 1987 Handbook of Nervous System Factors. CRC Press, Inc., Boca Raton, FL Sofroniew MV, Isacson O, O'Brien TS 1989 Nerve growth factor receptor immunoreactivity in the rat suprachiasmatic nucleus. Brain Res 476:358-362 Koh S, Oyler GA, Higgins GA 1989 Localization of nerve growth factor receptor messenger RNA and protein in the adult rat brain. Exp Neurol 10:209-221 Fendler K, Karmos G, Teledgy M 1961 The effect of hippocampal lesion on pituitary-adrenal function. Acta Physiol Scand 20:2293297 Feldman D, Conforti N 1980 Participation of the dorsal hippocampus in the glucocorticoid feedback effect on the adrenocortical activity. Neuroendocrinology 30:52-55 Patacchioli FR, Scaccianoce S, Taglialatela G, Chiappini P, Angelucci L 1984 Selective suppression of psychic stress response may derive from interaction of serotonin with the glucocorticoid receptor in the hippocampus. J Pharmacologie 15:456-457 Sapolsky RM, Krey LC, McEwen BS 1984 Glucocorticoid-sensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proc Natl Acad Sci USA 81:6174-6177 Sapolsky FM, Krey LC, McEwen BS 1983 The adrenocortical stress response in the aged male rat: impairment of recovery from stress. Exp Gerontol 18:55-63
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Vol. 129, No. 4 Printed in U.S.A.
Nerve Growth Factor Modulates the Activation of the Hypothalamo-Pituitary-Adrenocortical Axis during the Stress Response G. TAGLIALATELA, L. ANGELUCCI, S. SCACCIANOCE, P. J. FOREMAN, AND J. R. PEREZ-POLO Department of Human Biological Chemistry and Genetics (J.R.P., P.J.F.), University of Texas Medical Branch, Galueston, Texas 77550; Institute for Research on Senescence-Sigma Tau (G. T), Pomezia, Italy; and Department of Pharmacology (L.A., S.S.), University of Rome La Sapienza, Rome, Italy
tivation of the HPAA is significantly reduced by pretreatment of the rats with anti-NGF immunoglobulin G. These results suggest that the stimulatory action of NGF on HPAA activity requires the release of ACTH secretagogues from the hypothalamus and that NGF may modulate the HPAA response to stress stimuli. {Endocrinology 129: 2212-2218,1991
ABSTRACT. In the present study, we have investigated the functional relationship between the nerve growth factor protein (NGF) and the hypothalamus-pituitary-adrenocortical axis (HPAA) We have found that while iv injected NGF is able to stimulate the HPAA activity in rats, NGF is not able to stimulate the axis after a block of the hypothalamus produced by chlorpromazine-morphine-Nembutal treatment. Also, the stress ac-
T
HE ACTIVATION of the hypothalamus-pituitaryadrenocortical axis (HPAA) is an integral part of the stress response. In response to stressors, hypothalamic CRFs are released into the portal circulation of the hypophysial stalk and then act on the pituitary where they stimulate the release of ACTH. The ACTH, in turn, stimulates the release of corticosteroid hormones from the adrenal glands. These circulating corticosteroids inhibit further HPAA activity via specific receptors present in the pituitary, hypothalamus, and hippocampus (1-4). The different occupancy levels of hippocampal corticosteroid receptors modulate the release of CRF, arginine vasopressin, and oxytocin from the hypothalamus, particularly in the stress-activated state (5-7). In addition, the binding of glucocorticoid hormones in the hippocampus has been shown to be essential for the acquisition and retention of behavioral tasks in rodents (8, 9). The concomitant interrelated activities of modulating factors render the activity of the HPAA, under basal and stress conditions, as more complex than a simple cascade of hormonal events. The nerve growth factor protein (NGF) is essential for the maturation and maintenance of sympathetic and Received May 28, 1991. Address requests for reprints to: J. R. Perez-Polo, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77550. Supported by NINDS Grant NS-18708, CNR Grant 88.01813.04, and a grant from the Sigma Tau Company, Pomezia, Italy.
sensory innervation (10). NGF and NGF receptors (NGF-R) in the central nervous system (CNS) have been shown to be trophic to cholinergic neurons of the basal forebrain and septum (11, 12). NGF can also influence aspects of rodent behavioral development (13) and act as a cytokine in the immune response (14-17). NGF and hormonal steroids appear to be functionally related. Whereas 17/3-estradiol induces NGF synthesis and secretion of NGF in the C-6 rat glioma cell line (18), the synthetic glucocorticoid dexamethasone decreases NGF binding and NGF-R messenger RNA in the PC12 rat pheochromocytoma cell line (19, 20) as well as blocks NGF-mediated induction of NGF-R mRNA (our unpublished observation). Testosterone appears to decrease the expression of NGF mRNA in rat Sertoli cells (21). During development, local levels of NGF and glucocorticoids determine adrenal medullary chromaffin cell lineage and sympathetic neuron phenotype (22), both derived from neural crest. The stimulation of the HPAA by NGF (23) indicates the possible physiological relationship between NGF and adrenocortical activity. Whereas exposure to cold stress reduces NGF binding in the hippocampus and basal forebrain of young adult rats (24), adrenalectomy decreases NGF levels in the hippocampus while increasing levels of NGF-R in the septum (25). Aggressive behavior, a form of social stress, stimulates the Release of NGF from the submaxillary glands into peripheral blood cir-
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Chlorpromazine HC1 (CPZ) was purchased from Elkins-Sinn Inc. (Cherry Hill, NJ) and morphine sulfate (MS) from Knoll Pharmaceutical Co. (Whippany, NJ); pentobarbital sodium was obtained from Abbott Laboratories (Chicago, IL) and cytochrome c and BSA from Sigma Chemicals (St. Louis, MO). Rat plasma corticosterone RIA kit was from ICN (Costa Mesa, CA). Rat pheochromocytoma (PC12) cells were a generous gift of Dr. Lloyd Greene (Columbia University, New York, NY).
Trunk blood was collected 2 h after iv injection (the HPAA response to NGF still plateaus at this time interval) in order to avoid stress bias due to anesthesia. For experiments in which the pituitary-adrenocortical response to NGF was compared with that to CRF in CPZ-MS-Nb-treated rats, animals were killed 30 min after the NGF injection, a time interval at which the HPAA response to CRF plateaus (23) and the response to NGF has already reached its maximum. In order to study on the same individual the time course activation of the HPAA after iv injection of NGF, rats were surgically implanted with a permanent cannula in the jugular vein and used 4 days afterward. NGF was delivered iv via the cannula, and blood samples were drawn at the time-points shown via the same route. Blood was collected in ice-cold tubes containing trasylol (500 kallikrein-inhibiting units/ml) and after centrifugation at 1,900 X g for 20 min plasma was stored frozen at -70 C for later hormone analyses. Plasma corticosterone was determined by the method described by Murphy (30) and plasma ACTH by RIA (Incstar, MN). The sensitivity and inter- and intraassay variations of the assays are described elsewhere (31). For stress experiments, animals were injected sc with sheep anti-NGF immunoglobulin G (IgG) preparation (250 Ail/rat), or preimmune sheep IgG preparation (250 /tl/rat) in the morning on 2 consecutive days. On the third day, 1 h before the final experimental procedure the animals were given 250 n\ ip of the IgG preparations. A separate set of animals (anti-NGF, preimmune IgG, and blanks) received only one ip injection 1 h before the experiment. Stress consisted in a 1-h exposure to 4 C (cold room). Rats were killed immediately at the end of the stress period and trunk blood processed as above.
Animals
NGF preparation
Male 3-month-old Sprague-Dawley rats (200-250 g) were purchased from Harlan Sprague Dawley Co.(Houston, TX). Animals were housed three per cage, with food and water ad libitum, and a 12-h dark/light cycle (lights on at 0750 h) and 23 ± 1 C temperature.
The /3-NGF subunit was isolated from mouse submaxillary glands according to the method of Mobley et al. (32). Concentrations of NGF were assessed spectrophotometrically at 280 nm wavelength, and biological activity was assayed by the ability to stimulate neurite outgrowth in the PC12 cells.
Experimental procedures
Anti-NGF IgG fraction preparation
Pharmacological block of the hypothalamic secretagogues release was obtained according to the method described by Arimura et al. (29). Briefly, rats were injected with CPZ (10 mg/kg sc) followed 3 h later by MS (20 mg/kg sc) and, after 15 min, by sodium pentobarbital [Nembutal (Nb), 25 mg/kg ip]. Immediately after the Nb injection, the rats were kept under an oxygen tent until death. Forty-five minutes after the last injection, animals were injected via the jugular vein with either NGF or cytochrome c at the concentrations indicated below. Cytochrome c was used as a control for NGF because these two proteins display similar physicochemical characteristics. Before the iv injection, 1 ml aliquot of blood was quickly drawn from the jugular vein to asses basal plasma corticosteroid levels. One hour after iv injection, a time interval at which the pituitaryadrenocortical response to NGF plateaus, rats were killed by decapitation and trunk blood collected in tubes containing 1 mg EDTA as an anticoagulant. Tubes were then centrifuged at 2000 X g for 20 min and the supernate (plasmatic fraction) stored at -20 C until assayed for corticosterone content. Nonblocked rats were injected iv under light ether anesthesia.
Serum was collected from a sheep that had been previously immunized against mouse |8-NGF by venous puncture. Aliquots were also drawn from the same animal before immunization (preimmune fraction). Sera were precipitated with saturated ammonium sulfate solution with continuous stirring. After centrifugation for 20 min at 400 X g, the precipitate was redissolved in borate buffered saline (BBS), pH 7.8, and dialyzed against BBS (4 days; 8 L.). Protein concentration was determined, and 25 mg of the crude IgG fraction were loaded onto a Sephadex-Protein A chromatographic column (Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with 20 ml of 0.2 M sodium citrate buffer. The eluate was dialyzed overnight against ammonium bicarbonate buffer, pH 8.0, and evaporated in a Speed Vac Concentrator (Sarvant Instruments, Hicksville, NY). The IgG fraction was then reconstituted in PBS, pH 7.6, and the IgG concentration determined spectrophotometrically. Biological activity of the IgG preparation was assayed by the ability of antibody to block neurite outgrowth in NGF-treated PC12. When PC12 cells were cultured in serum containing medium in the presence of NGF (1 ^g/ml), there
culation and elevates NGF mRNA and NGF protein levels in the hypothalamus of mice (26-28). The original study demonstrating the action of NGF on the HPAA did not address the question as to the site of NGF action in the CNS or its physiological significance (23). Here, we report that the NGF-induced pituitary-adrenocortical activation is abolished in rats with a pharmacological block of the hypothalamus. Moreover, inactivation of NGF in the peripheral circulation by the injection of antibodies against NGF significantly attenuates the pituitary-adrenocortical stress response. These results indicate that exogenous NGF stimulates the HPAA at hypothalamic or suprahypothalamic levels. These results also support the hypothesis that stress stimulation of NGF release may be a significant component of the response of the HPAA to stress.
Materials and Methods Materials
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was abundant neurite outgrowth by the NGF-treated PC 12 (Fig. 1C). As shown in Fig. ID, NGF activity is completely blocked by the presence of 5 /i\/m\ of the sheep anti-NGF IgG fraction while the anti-NGF IgG itself does not elicit any morphological response by the PC12 (Fig. IB). Statistical analysis
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O O
Statistical differences among groups were assessed by the analysis of variance (ANOVA) followed by the Fisher's least significant difference test for multiple comparisons.
Results NGF injection in CPZ-MS-Nb-treated rats Intravenous injection with either 10 or 100 pmol/g body weight (bw) of NGF in 4-month-old male rats produced a significant increase in plasma corticosterone levels at either NGF dose, while no effect was observed after a control injection of cytochrome c (Fig. 2). Circulating levels of corticosterone in both groups of rats injected with NGF were more than 10-fold higher than
5 U ro CO
a Control
10
1OO
Dose (pmol/g bw) FIG. 2. Plasma corticosterone levels in rats 1 h after injection (i.v.) with NGF or cytochrome c. Control corticosterone values have been obtained from blood samples drawn from each rat immediately before the injection. Mean values ± SEM; n = 6-10 animals per group.
those observed before the NGF injection or in the cytochrome c-treated animals, indicating that both doses of NGF were saturating. The time-course response of the HPAA to NGF is shown in Fig. 3. The plasma corticosterone levels reached a maximum 30 min after the NGF injection and remained elevated for up to 3 h, while plasma ACTH titers peaked 30 min after NGF injection and returned to basal values 120 min later. No effect on plasma corticosterone was observed in cytochrome cinjected rats. These results were quantitatively and qualitatively similar to those obtained by Otten et al. (23). There was no elevation of plasma corticosterone levels after the injection of NGF (10 pmol/g bw iv) into hypothalamus-blocked rats (Fig. 4). Hypothalamus-blocked rats, although insensitive to NGF, were still capable of a full pituitary-adrenocortical activation when challenged with an iv injection of 2 ng CRF (Table 1). Effect of anti-NGF treatment effect on the stress response
FIG. 1. Microphotograph of PC 12 cells treated for 24 h with NGF (1 Mg/ml) in presence of sheep anti-NGF IgG fraction (5 ml/ml). A, Control cells; B, anti-NGF treated cells; C, NGF-treated cells; D, NGF antiNGF treated cells. Reference bar, 150 /xm.
Treatment of rats with anti-NGF IgG fraction for 3 consecutive days significantly reduced the response of the HPAA to cold stress, as determined by measurements of plasma corticosterone levels (Fig. 5). A partial response to cold stress in the anti-NGF-treated rats might have been present in that the plasma corticosterone concentration was somewhat higher, although not statistically significant, compared with the treatment-matched basal animals. There was also a small but statistically not significant effect of the injection procedure on plasma corticosterone levels in basal animals. A single injection of the anti-NGF IgG fraction (250
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60 r
FIG. 3. Plasma corticosterone (upper panel) and plasma ACTH {lower panel) values in rats injected with 10 pmol/g bw of either NGF (O) or cytochrome c (•). Animals were freely moving and injected via a permanent cannula in the jugular vein. Blood samples were collected at the time-points shown via the same route. Mean values ± SEM; n = 35 animals per group.
60
90
120
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210
Time after injection (min.)
9
250
B
200
--
T 0
/ 150
-
\ J
/
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\T o 100
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•
T •
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Time after injection (min.) /Lil/rat ip) 1 h before exposure to a cold environment did not attenuate the subsequent stress response of the HPAA. However, this event was not statistically different from the stress-induced increase in circulating corticosterone levels observed in the control IgG-injected rats or in the untreated animals. In this experiment the corticosterone levels were: untreated stressed, 11.99 ± 1.29; control IgG stressed, 11.90 ± 1.11; anti-NGF stressed, 8.20 ± 2.24. (ANOVA: F = 1.92; degrees of freedom = 2,12; P = 0.2).
Discussion In the rat, pharmacological block of the hypothalamus completely abolishes its capability to release humoral secretagogues of pituitary ACTH (29) in response to a variety of stressors, as measured by the absence of any stress-induced elevation of serum corticosterone levels.
However, in this condition the ability of the pituitary to release ACTH and the consequent release of corticosteroids from the adrenals in response to an iv injection of CRF are not abolished (29, 33). Therefore this pharmacological manipulation provides a functional inactivation of the HPAA at the hypothalamic level, while leaving the remaining components of the HPAA responsive to appropriate stimuli. In the unblocked control rats, iv injection of NGF fully activated the HPAA, as measured by elevation of plasma ACTH and corticosterone titers. Since we did not observe any rise in plasma corticosterone levels after NGF injection in pharmacologically (CPZ-Ms-Nb) blocked rats, but did observe a full response to CRF, we conclude that the site of action of NGF in stimulating the HPAA is either the hypothalamus or some suprahypothalamic structure that regulates the HPAA or both. Given that in this condition the pituitary is responsive to CRF stimulation, any activity
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NGF
FIG. 4. Plasma corticosterone levels in CPZ-MS-Nb-treated rats before (PRE) or 1 h after (POST) the iv injection of either NGF (10 pmol/g bw) or cytochrome c (10 pmol/g bw). Mean values ± SEM; n = 4-6 animals per group.
POST-INJECT
PRE-INJECT TABLE 1. Plasma corticosterone values in CPZ-MS-Nb-treated rats before or 30 min after iv injection of cytochrome c (10 pmol/g bw), NGF (10 pmol/gr bw), or CRF (2 fig/rat) Plasma corticosterone (fig/100 ml)
Cytochrome c NGF CRF
Pre-injection
Post-injection
3.84 ± 0.29 4.05 ± 0.52 3.40 ± 0.82
2.59 ± 0.32 2.81 ± 0.19 18.91 ± 1.93°
Values are mean ± SEM of four rats per group. °P< 0.001 (ANOVA).
of NGF on the pituitary is excluded. This is consistent with the reported absence of NGF-R on the ACTHsecreting anterior lobe of the pituitary (34). It has been demonstrated that NGF is released from the submaxillary glands into the circulation during aggressive behavior in rodents (26, 27). Surgical removal of the submaxillary glands abolishes the stress-induced
increase of circulating NGF but has no effect on serum NGF basal levels (26). There remain uncertainties as to measurements of NGF in serum in part due to adherence properties of NGF and the presence of NGF binding proteins in serum (35, 36). This would suggest that NGF, in addition to its paracrine effects on basal forebrain cholinergic neurons, can also act as a classical endocrine hormone, in that it can be released from a discrete organ, the submaxillary glands, into the circulation in response to physiological relevant stimuli. Our finding that treatment of rats with antibodies directed against NGF significantly diminishes the response of the HPAA to cold stress, further suggests a role for circulating NGF as a component of the HPAA response to stress. Further studies are needed to clarify unequivocally the site of NGF action in the stimulation of the HPAA activity. Our finding in hypothalamic-blocked rats would suggest that NGF acts at the hypothalamus, stimulating
BLANK
CONTROL
ANT I-NGF
FIG. 5. Plasma corticosterone levels in rats treated for 3 days with anti-NGF IgG fraction or preimmune IgG fraction (CONTROL). Stress consisted of 1 h exposure to a cold (4 C) environment. *, P < 0.05 vs. blank-stressed or controlstressed animals (ANOVA). Mean values ± SEM; n = 4 per group.
BASAL
COLD STRESS
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NGF AND PITUITARY-ADRENOCORTICAL ACTIVITY
the release of CRF. However, NGF-R have not been reported to be present in those hypothalamic areas in which the CRF-secreting neurons are localized (37). This interpretation does not exclude the possibility that NGF may facilitate the release of other hypothalamic releasing factors, such as arginine vasopressin. Another possibility is that NGF can affect those inhibitory mechanisms that normally are involved in the control of HPAA activity. The hippocampus, a limbic structure that possesses high levels of NGF-R (24, 38), is a likely candidate for this NGF effect since it is known to inhibit hypothalamic activity during basal and, especially, stress conditions (39-43). The NGF released during stress might modulate these events by acting at the hippocampus, as is suggested by the reduced activation of the HPAA in the anti-NGF-treated rats. This is consistent with the finding that after cold stress there is a reduction of NGF binding in the rodent hippocampus (24), possibly due to an increased ligand-receptor complex internalization. Also, the data presented here show a reduced stressinduced activation of the HPAA in the anti-NGF-treated animals, suggesting that after an initial response to stress, the HPAA, in the absence of sufficient circulating NGF, becomes unable to undergo full activation. This can be a likely mechanism of action, provided that the hippocampus is affected by circulating NGF levels. In fact, a selective uptake of NGF takes place in the hippocampus of rats injected iv with 10 pmol/g bw of [125I] NGF, while there is no significant uptake into hypothalamus (Loy, R., G. Taglialatela, L. Angelucci, and J.-R. Perez-Polo, in preparation). Although NGF has effects on adrenal chromaffin cells, there is no evidence of interference with corticosteroid function by the adrenals in NGF-treated rodents. In conclusion, peripheral NGF activates the HPAA by acting within the CNS. There is the possibility that the hippocampus and the hypothalamus participate in NGF action in the CNS. The NGF released into the circulation during stress may play a role during activation of the HPAA.
5. 6.
7.
8.
9.
10. 11. 12.
13. 14. 15. 16.
17. 18. 19. 20.
Acknowledgments Thanks to Mrs. D. Masters for manuscript preparation and K. Werrbach-Perez for excellent technical assistance.
21.
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