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NOVEL REGULATORS OF THE IN VITRO RELEASE OF HYPOTHALAMIC CORTICOTROPHIN-RELEASING HORMONE TWO DECADES AFTER ITS DISCOVERY: A REVIEW Alfredo Costa, Rossella E. Nappi*, Antonella Smeraldi, Matteo Bergamaschi, Pierluigi Navarra**, Ashley Grossman*** IRCCS C. Mondino Institute of Neurology, Laboratory of Neuroendocrinology, “Maugeri - Mondino University of Pavia” Research Center, Pavia, University of Pavia; * Department of Obstetrics and Gynaecology, IRCCS S. Matteo Hospital, University of Pavia, Italy; **Institute of Pharmacology, Catholic University Medical School, Rome, Italy; *** Department of Endocrinology, St Bartholomew’s Hospital, London, UK. Reprint requests to: Dr A. Costa, C. Mondino Institute of Neurology, University of Pavia, via Palestro 3, 27100 Pavia, Italy. E-mail: [email protected] Many environmental and occupational chemicals are known to affect the central and/or peripheral nervous system, causing changes that may result in neurological and psychiatric disorders. Because of the limited accessibility of the mammalian nervous tissue, new strategies are being developed to identify biochemical parameters of neuronal cell function, which can be measured in easily obtained tissues, such as blood cells, as potential markers of the chemically-induced alterations occurring in the nervous system. This review includes a comparative analysis of the effects of mercurials on calcium signalling in the neuroadrenergic PC12 cells and rat splenic T lymphocytes in an attempt to characterize this second messenger system as a potential indicator of subclinical toxicity. The suitability of neurotransmitter receptors in blood cells, such as the sigma binding sites, as biological markers of psychiatric disorders is also discussed. KEY WORDS: AVP, CRH, hypothalamus, in vitro, neuropeptides, stress. FUNCT NEUROL 2001;16 (SUPPL.): 205-216 INTRODUCTION Corticotrophin-releasing hormone (CRH) is a 41 amino-acid peptide which was isolated and characterised exactly two decades ago (1). It is distributed heterogeneously throughout the central nervous system (CNS) (2) and, due both to its ability to stimulate the release of adrenocorticotrophic hormone (ACTH) from the pituitary and to its numerous extra-hypophyseal biological effects, is now considered one of the major neuropeptides involved in adaptive processes: CRH indeed coordinates the neuroendocrine, behavioural, autonomic and immune responses to stressors (3-5). The recent cloning of specific CRH receptors (6-8) has further facilitated research on CRH in physiology and in the development of neuropsychiatric diseases, including depression, anxiety and eating disorders. For this reason, and thanks to the availability of specific antagonists and laboratory animal models (such as transgenic mice), CRH is still a peptide under active investigation 20 years after its discovery (9). While CRH-containing neurons are widely distributed within the CNS, the hypothalamic paraventricular nucleus (PVN) is the principal FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 205 A. Costa et al. site of origin of the parvocellular neurosecretory neurons which deliver the neuropeptide into the hypophyseal-portal system, thereby initiating the response of the pituitary-adrenal axis. The process of CRH release from the hypothalamus, which appears to be increasingly complex (10), is under the control of a considerable number of mediators, each of which has a different effect. The PVN is, indeed, at the very centre of several intra- as well as extra-hypothalamic inputs, such that a given amount of CRH released represents the net result of a complex interplay of modulatory (inhibitory or stimulatory) factors. In order to characterise these factors, various techniques have been used, but it would appear that the in vitro hypothalamic preparations have, to date, been among the most productive model systems (11,12). Methods used to study the rat hypothalamus in vitro differ to a considerable extent according to a number of factors, such as the age of the hypothalamus (foetal or adult), the type of incubation system (acute or chronic), and the specific perifusion techniques used (incubation or continuous flow). In this paper, we describe the in vitro acute incubation system of hypothalamic explants that has been in use for several years in our laboratories. The advantages and limitations of this system are compared with other in vitro methods, and some of the data obtained on the effect of various substances on CRH release are reviewed, paying particular attention to observations on novel regulators recently made by our groups. ASSESSING HYPOTHALAMIC CRH RELEASE IN VITRO Cellular processes underlying hormone secretion from the hypothalamus are relatively difficult to study in the whole animal. On the other hand, knowledge of the nature of the mechanisms that regulate hypothalamic func- 206 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 tion is indispensable for a complete understanding of endocrine homeostasis. Consequently, over the past few years, several investigators have begun to use isolated hypothalamic preparations to elucidate the physiological, pharmacological, and biochemical characteristics of the hypophysiotropic neurons. However, different techniques have been used by various research groups to maintain the hypothalamus in vitro. These techniques can be roughly divided into three major groups: synaptosomal and other subcellular preparations, neuronal cell cultures, and tissue cultures (including short- and long-term maintenance of hypothalamic preparations). Hypothalamic explants, coupled with indirect measurements (bioassays) and subsequently with direct measurements [radioimmunoassay (RIA) and immunoradiometric assay (IRMA)] of peptides, have been used over the past 30 years by several authors, and have included both incubated and perifused hypothalami (minced or whole) and median eminence fragments (13-16). Prior to the development of these techniques, relatively little work was undertaken with a view to improving understanding, through the use of direct measurements, of the regulation of hypothalamic hormone release. The system used in our laboratory was developed by Lengyel et al. (17), based on a technique originally devised by Jones et al. (18), in order to: a) establish the characteristics of peptide secretion in vitro, b) identify the nature of the immunoreactive forms released in vitro (e.g., by means of chromatographic techniques), c) investigate the direct/indirect effects of various classic neurotransmitters, putative neurotransmitters and neuromodulators on hypothalamic secretion, and d) characterise the particular receptors involved, by using specific agonist and antagonist drugs. The characteristics of the system have been previously described in full detail (11). Briefly, the freshly dissected rat hypothalamic halves are placed in sterile polyethylene vials containing Release of CRH in vitro Earle's balanced salt solution (EBSS), supplemented with 0.2% human serum albumin, 60 ug of ascorbic acid/ml to prevent oxidation, and 100 kallikrein-inactivating units (kIU) of aprotinin/ml to prevent peptide degradation by hypothalamic peptidases. Two hypothalami (four halves) are incubated in one vial, in 500 ul of EBSS, in order for the basal release of CRH to be measurable by RIA. Vials are kept in a water bath at 37°C and gassed in an atmosphere of O2/CO2 (95%/5%), at a 1 litre/min flow rate. An 80-min preincubation period is allowed for peptide stabilisation, during which time the medium is aspirated every 20 min and replaced with fresh medium. After preincubation, the tissue is usually incubated in fresh medium for a 20-min control period, followed by another 20-min period in either medium alone (control group) or in medium containing test substances (test group). Viability of the tissue is always tested by incubation in the presence of 56 mM KCl at the end of each experiment. Media collected throughout the studies are stored at -20°C until assay. L I M I TATIONS AND ADVA N TAGES OF THE SYSTEM A system that provides information regarding neuroendocrine regulation in the rat hypothalamus should simulate the physiological situation as closely as possible. Indeed, the biggest criticism of is that investigations are performed on the isolated hypothalamus, which is a structure removed from its natural environment; thus, its function in vitro does not necessarily mirror what actually occurs in vivo. Although the incubation devices (temperature, oxygen and nutrient supplies) recreate, as far as possible, the physiological conditions in a CSF-like milieu, inputs from the supra- and extrahypothalamic brain areas are inevitably lacking. Moreover, by their very nature, the preparations are no longer subject to the diverse and complex neuronal feedback controls that are known to operate in vivo. Although such characteristics may be advantageous when assessing putative modulatory substances, the true physiological relevance of the in vitro set-up should be considered when investigating the control of neuropeptide release. In other words, results obtained from this technique can define intrinsic biological mechanisms in a reliable manner, but only to a limited extent, and without taking into account their systemic consequences. Combination of these data with those from complementary in vivo studies is clearly indispensable, and may make it possible to relate in vivo expression to in vit ro changes. Another major point regarding this kind of tissue incubation is the actual survival time of the tissue in the medium. Histological studies of oxygen consumption and maintainance of cellular integrity have shown that incubation (or perifusion) in CSF-like media allows the whole hypothalamus to survive for at least 3 hours (19). The main limiting factor in the adequate maintainance of the hypothalamus is the size, and possibly the shape, of the tissue examined. There appears to be a gradient of diffusion of nutrients into, and of toxic metabolites out of, the central part of the explant. Although some investigators may continue to use the isolated hypothalamus whole, many others have tried to circumvent this major problem by employing fragmented materials, such as quartered hypothalami, or slices of various widths. These preparations are advantageous in terms of nutrient diffusion, but the procedures inevitably lead to a loss of interneuronal connections. In this respect, we have found the use of halved hypothalami more satisfactory, since the volume of tissue is actually decreased but the neuronal projections remain largely unaffected (17). It should be added, however, that there is still controversy regarding data obtained from whole tissue as opposed to those obtained from hypothalamic halves. The bisection of the tissue, in our experience, allows a larger surface to be exposed to the FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 207 A. Costa et al. medium, thus prolonging cell survival and facilitating the access of test substances to the neuronal .nuclei close to the midline. This is supported by the observation that the average survival of hypothalamic explants in our system exceeds 4 hours, as indicated by the good responses to incubation with 56 mM KCl seen to date (11). One of the advantages of this system is the fact that minimal trauma to the tissue can be ensured through the taking of extreme care in brain removal and dissection procedures, as well as thanks to the CSF-like fluid surrounding the preparations in the vials. Then, during the experiments themselves, damage to the hypothalami can be avoided providing the experimenter collects and renews the incubation medium with care. Moreover, the system guarantees the retention of a high degree of cellular integrity and the preservation of the constituents of the tissue, such as the intrinsic neuronal networks, the supporting glia, and other neuronal cells. Therefore, compared to other systems, such as synaptosomes or incubated slices, this method seems to mirror more closely the previous physiological state. Another advantage is that the technique allows precise control of the composition of the medium, and thus the study of the effects of various neuroactive substances. Finally, the day-to-day running of the system is relatively inexpensive. As mentioned above, the use of this incubation system has provided a considerable amount of data on the mechanisms of the hypothalamic release of CRH. The effects of classical neurotransmitters (such as acetylcholine, catecholamines, serotonin, GABA), excitatory amino-acids, endogenous opioids, various neuropeptides, cytokines, bacterial lipopolysaccharide and gaseous transmitters – nitric oxide (NO) and carbon monoxide (CO) – have been extensively reviewed by ourselves and other groups (11,12,20-24). This review will focus on recent data on the in vitro regulation of CRH release by two different agents: the prod- 208 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 uct of the obese gene, leptin, and the human immunodeficiency virus-1 (HIV-1)-related protein, gp120. R E G U L ATION OF CRH RELEASE BY LEPTIN Increasing evidence suggests that energy balance is regulated at central level by brain circuitry controlling both energy production (food intake) and energy expenditure (thermogenesis) (25). Peripheral factors, produced by changes in adipose tissue mass, can signal to the brain, informing it of the level of accumulated fat depots in order to regulate energy input and output. The obese (Ob) gene product, leptin, is a 16kDA peptide synthesised and secreted by adipose tissue. While it appears to have various neuroendocrine functions, being responsible, for example, for biological effects in reproduction, adaptive responses of the newborn, and control of GH, TRH-TSH and PRL secretion (26), leptin is currently considered as the main afferent signal in the regulatory mechanisms of energy homeostasis. Its effect, which is probably generated at the level of the hypothalamus, an area particularly rich in leptin (Ob) receptors (see 26 for review), appears to be, predominantly, the suppression of appetite (27), although its activation of the sympathetic nervous system may also be important (28). Several neuropeptides are known to be involved in eating behavior: CRH is a potent appetite-suppressant agent, acting at least in part by inhibiting neuropeptide Y (NPY) release (29,30), and it also increases energy expenditure (30,31). Due to its presence in several brain areas, including the adrenergic nuclei, CRH forms a network of neuronal pathways, and can function in concert with another peptide, urocortin, to coordinate anorectic and thermogenic actions and to optimise energy losses (25). In the light of previous studies demonstrat- Release of CRH in vitro ing that during the response to fasting leptin may signal to the rat hypothalamus and thus alter the expression of genes encoding for both NPY and CRH (32), we speculated that the primary effect of leptin in the hypothalamus is via stimulation of CRH secretion. To evaluate this hypothesis, we tested the effect of murine leptin on CRH release from hypothalamic explants in our in vitro system. As expected, biologically-active leptin (but not the heat-denatured peptide) was found to stimulate CRH release in a dose-dependent manner, and via a non-adrenergic mechanism (33). These findings were thus consistent with the view that leptin, through the reported presence of specific leptin receptors in the brain, participates in the feedback regulation of body adiposity by acting directly in the brain to reduce food intake. CRH appears to be one key mediator of the central effects of leptin, a fact supported by several other later studies. Indeed, in the mouse leptin was found to elicit CRH release from superfused brain slice preparations containing hypothalamus or amygdala (34), although it was apparently unable to alter NPY or CRH secretion in another in vitro study (35). The intracerebroventricular administration of leptin in mice was found to stimulate CRH and to reduce NPY synthesis in the PVN, and this effect is restrained by the presence of endogenous corticosterone (34). In the same experimental conditions, leptin markedly increased hypothalamic CRH mRNA, but did not reduce NPY mRNA (36), while in another study it increased hypothalamic CRH content and inhibited food intake at 2h in food-deprived rats (37). Consistently, central leptin administration in the rat increased the expression of CRH mRNA and AVP mRNA in the PVN, and plasma AV P, ACTH and corticosterone levels (38). Furthermore, using primary cultures of rat hypothalamic cells, other authors also observed a stimulatory role of leptin on CRH secretion (39). Finally, the systemic (i.p.) administration of leptin in the rat was found to acutely increase the expression of CRH mRNA in the PVN as well as CRH receptor-2 (CRHR-2) mRNA in the ventromedial hypothalamus (40). There is still uncertainty over the site of leptin action in the brain. A highly-specific saturable transport system by means of which the peptide crosses the blood-brain barrier (BBB) has been described (41), but the CNS regions where it primarily acts are unclear. Leptin receptors are present in several hypothalamic nuclei, but apparently not in the PVN (25). This may imply that leptin acts indirectly, other factors mediating its effect on CRH. Classical neurotransmitters may be involved, although we were unable to show any modulatory role of adrenergic pathways in vitro (33). Interleukins, which are known to stimulate CRH release directly (see 11, for review), are other possible candidates. A recent in vitro study reported that NO-synthase (NOS)- or cyclooxygenase (COX)-mediated mechanisms were not involved, whereas leptin was able to enhance the production of prostaglandin E 2 and F 2α, which can themselves stimulate CRH release (39). However, it is extremely likely that other peptides are also involved in the central regulation of appetite, such as NPY, urocortin, orexin, glucagon-like peptide-1, galanin, ghrelin and melanin concentrating hormone, which may mediate some of the effects of leptin at hypothalamic level. It would therefore appear that leptin suppresses appetite and stimulates the hypothalamo-pituitary-adrenal (HPA) axis via CRH release, which is consistent with the idea that high circulating leptin levels may be involved in the HPA axis activity changes generally characteristic of human obesity and of most animal models of obesity (42). However, the interactions between leptin, CRH and the HPA axis are still poorly understood and are likely to be complex. Since leptin and cortisol show an inverse circadian rhythm, it has been suggested that a regulatory feedback is present (43). Several studies have reported that FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 209 A. Costa et al. high-dose exogenous glucocorticoids increase circulating leptin concentrations in humans; conversely, leptin has been shown to exert inhibitory effects on the HPA axis responses in some studies, both at hypothalamic and at adrenal levels (44,45). However, there is now general agreement on the existence of a closed, bi-directional, circuit linking HPA axis function and adipose tissue metabolism, with different types of stress possibly being associated with many phenotypes of obesity (43). As these mechanisms are still far from clear, studies are in progress to further characterise the biological mediators of leptin effects in vitro. REGULATION OF CRH RELEASE BY gp120 Another substance that we have recently studied as a possible mediator of CRH release is the HIV coat protein gp120, a viral component which has been associated with neuropathological changes (46). A broad spectrum of in vitro studies has documented that gp120 causes excitotoxic, glutamate-mediated death of neuronal cells (47). In addition, many of the effects of gp120 appear to involve the production of NO and cytokines (48-50), as well as activation of the inducible COX pathway (51). These observations have led to speculation that gp120 may be the primary pathogenic agent in the loss of brain cortical neurons (52), accounting for the typical neurological deficits occurring in AIDS (53). Our interest in gp120 was prompted by the observation that patients with HIV-1 infection exhibit deranged activity of the HPA axis (54), which may have important pathogenetic and therapeutic implications from the point of view of both CNS damage and of the clinical progression of disease (55). Several studies in HIV patients indeed showed increased plasma cortisol and ACTH levels, blunted ACTH and cortisol responses to CRH administration, and 210 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 blunted cortisol responses to ACTH administration (56-59). Interestingly, CNS expression of gp120 in transgenic mice has been reported to activate the HPA axis, as indicated by increased plasma ACTH and corticosterone levels and pituitary ACTH content (60). It has also been observed that infusion of gp120 into the rat brain increases pituitary-adrenal activity via interleukin-1 (IL-1), and decreases peripheral cellular immune responses (48). In addition, in vitro studies on non-transgenic mouse hypothalamic preparations have shown that gp120 directly stimulates AVP release, suggesting that HIV-induced activation of HPA function may be due to an increased hypothalamic drive (60). We therefore speculated that the primary effects of gp120 in the hypothalamus may occur via stimulation of both CRH and AVP secretion, and tested the direct effect of gp120 on the release of CRH and AVP in our in vitro system. As previous studies had shown that NO is involved in some of the effects of gp120, we also considered the possible mediation by NO pathways of any observed effect of gp120. The addition of gp120 to hypothalamic explants indeed resulted in a clear stimulation of both CRH and AVP release, while in the presence of the NOS inhibitor N G-methyl-L-arginine (LNMMA) the stimulatory effect of gp120 on the release of both peptides was completely lost (61). These observations were found to be entirely consistent with the effects seen in the hypothalamic tissue from rats administered gp120 systemically (i.p.). Indeed, gp120 induced the expression of Fos protein (the product of the early gene c-Fos, an index of neuronal activation) in both the parvo- and the magnocellular PVN, while the phenomenon was significantly attenuated by the co-administration of L-NMMA. In addition, double immunocytochemistry showed increased co-staining for Fos protein and CRH or AVP in the PVN following gp120, the number of double-labelled CRH and AVP cells for Fos protein being markedly reduced Release of CRH in vitro by co-administration of L-NMMA (61). These data, which also appear to support those of another recent study (62), lend further support to the hypothesis that HIV-related hyperactivity of the HPA axis is secondary to increased hypothalamic drive, and that gp120 activates the HPA axis via both the principal ACTH secretagogues. Studies are currently under way to elucidate the precise site of gp120 effects in the brain, as well as the relevant peptide-receptor interactions. Although gp120 is a large molecule, it has been found to cross the BBB by inducing adsorptive endocytosis (63). However, alternative/additive mechanisms to explain the effects of circulating gp120 within the hypothalamus may be that gp120 enters the CNS via the circumventricular regions which lack the BBB (such as the organum vasculosum of the lamina terminalis, OVLT). In this case, the effect of gp120 in the PVN would imply the involvement of other mediators. NO pathways are certainly involved, and this is in agreement with previous studies reporting that gp120 stimulates NO production in glial cultures (64,65). With regard to NO modulation of CRH and AVP release, experimental evidence indicates that NO plays in fact a predominant inhibitory role in CRH and AVP secretion in conditions of increased inflammatory-induced secretory drive in vitro (66,67). However, recent in vivo evidence suggests that, in some circumstances (68), NO stimulates ACTH secretion and the transcription of the genes encoding for CRH and AVP in the rat hypothalamus. Thus, NO may have a dual excitatory/inhibitory role in the control of the pituitaryadrenal axis, depending on the stressor (24). On the other hand, gp120 is also known to stimulate the production of cytokines (4850,64,65), some of which, particularly IL-1, are recognized activators of the HPA axis (69) and are able to potently stimulate CRH and AVP release in vitro (70,71). Excitatory aminoacids may be other possible mediators of the central effects of gp120 (60,72,73); in addition, gp120 has been shown to stimulate the inducible isoform of COX (51), and thus the production of prostanoids, which are themselves able to elicit CRH release (70). Finally, another possibility which should be taken into account is that gp120 may act, at least in part, at the level of nerve terminals in the median eminence. The central effects of gp120, along with those of inflammatory agents (such as cytokines) released into the circulation by activated immune cells, may lead to excessive activation of the HPA axis. This endocrine derangement may critically influence the clinical evolution of HIV patients (55,74), since glucocorticoids can cause brain damage in particular circumstances (75) and worsen gp120 neurotoxicity in various brain regions (76,77); they may also potentiate the immunosuppression characteristic of AIDS. Thus, in HIV infection, excess of glucocorticoids induced by a central effect of gp120 may in turn act synergistically with gp120 to cause the variety of neuropathological and cognitive disturbances seen in the AIDS-dementia complex (78). CONCLUDING REMARKS The in vitro system described above has proved to be a valid model for assessing the activity of the isolated hypothalamus, providing insight into the role played in CRH release by traditional neurotransmitters as well as by many other regulatory factors. Indeed, a panoply of neuromodulators appear to influence this process, the main results obtained by our groups over the last few years being summarised in Table I (see over). The biological agents leptin and gp120 have recently joined the growing list of mediators that signal in the hypothalamus, but their effects require further characterisation. Recently, other factors have also been reported by dif- FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001 211 A. Costa et al. Table 1 - The role of different substances in the regulation of CRH secretion from in vitro hypothalamic explants (The receptors involved and/or the mediating factors are reported in parentheses). Excitatory Noradrenaline (β) Serotonin Acetylcholine (M) NPY Interleukin-1 (PGs) Interleukin-2 Interleukin-6 (PGs) LPS (PGs) NO, CO Leptin (PGs) gp120 (NO) Inhibitory No effect GABA opioids (µ,κ) ANP substance P NO, CO MCH EGF NGF melatonin EAA Abbreviations: GABA = gamma-aminobutyric acid; MCH = melanin concentrating hormone; ANP = atrial natriuretic peptide; NPY = neuropeptide Y; EGF = epidermal growth factor; NGF = nerve growth factor; LPS = bacterial lipopolysaccharide; NO = nitric oxide; CO = carbon monoxide; EAA = excitatory amino acids; M = muscarinic receptor; PGs = prostaglandins. ferent groups as being involved in CRH release: some of these, such as activators of protein kinases A and C, appear to be stimulatory, while others, such as glucocorticoids, negatively regulate CRH release from the hypothalamus as part of a feedback control loop (12). 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