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(The FASEB Journal. 1998;12:199-207.)
© 1998 FASEB

Inherent glucocorticoid response potential of isolated hypothalamic neuroendocrine neurons

S. Hellbacha, P. Gärtnera, J. Deickea, D. Fischera, A. H. S. Hassana and O. F. X. Almeida1

a Neuroadaptations Group, Department of Neuroendocrinology, Max Planck Institute of Psychiatry,D-80804 Munich, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the broader framework of facilitating investigations into the inherent responses of restricted neuronal phenotypes devoid of their in vivo afferents, serum- and steroid-free cultures enriched in corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), and ß-endorphin (ß-END) peptidergic neurons were prepared from the hypothalamic paraventricular (PVN: CRH and AVP) and/or arcuate (ARC: ß-END) nuclei of juvenile male rats. The functional viability of these ARC/PVN cultures was verified by their ability to synthesize and secrete CRH, AVP, and ß-END under basal and depolarizing (veratridine) conditions in vitro. Peptide secretion was shown to be Ca2+ and Na+ dependent in that it was blocked in the presence of veraspamil and tetrodotoxin, respectively. Exposure of ARC/PVN cocultures to the glucocorticoid dexamethasone (DEX) resulted in a dose-dependent increase of CRH secretion and an inhibition of AVP and ß-END; the CRH responses deviated strikingly from predictions based on in vivo experiments. Steroid withdrawal or treatment with the glucocorticoid receptor antagonist RU38486 reversed these trends. Opposite effects of DEX on CRH secretion were observed in cultures consisting of PVN cells only. Supported by studies using an opioid receptor agonist (morphine) and antagonist (naloxone), these observations demonstrate that ARC-derived (ß-END) neurons modulate the responses of PVN neurons to DEX.—Hellbach, S., Gärtner, P., Deicke, J., Fischer, D., Hassan, A. H. S., Almeida, O. F. X. Inherent glucocorticoid response potential of isolated hypothalamic neuroendocrine neurons. FASEB J. 12, 199–207 (1998)


Key Words: corticotropin-releasing hormone • arginine vasopressin • ß-endorphin • dexamethasone • glucocorticoid feedback


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE HYPOTHALAMIC PARAVENTRICULAR (PVN) and arcuate (ARC) nuclei represent important sites of adrenocorticosteroid action in the brain. In the PVN, corticosteroids act on corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) neurons; in the ARC, they target neurons producing pro-opiomelanocortin (POMC), from which the opioid peptide ß-endorphin (ß-END) is derived (1). A range of neuroendocrine and behavioral processes are subject to the actions of CRH, AVP, and ß-END in the brain, and CRH and AVP neurons that project to the median eminence provide the major central drive on pituitary-adrenal secretion (2–4); adrenocorticosteroids, secreted in response to this central drive, ultimately exert negative feedback actions on the aforementioned endocrine neurons (5, 6), thus completing the homeostatic circuit. Results of numerous in vivo studies show that corticosteroids inhibit CRH, AVP, and ß-END synthesis and secretion, and that their removal enhances the activity of AVP and CRH neurons (7–12). These patterns presumably reflect a summation of 1) direct responses by the peptidergic neurons under consideration, and 2) indirect responses by intra- and extrahypothalamic inputs to CRH, AVP, and ß-END neurons. Although corticosteroids inhibit CRH neuronal activity, much evidence suggests that corticosteroid negative feedback effects seen at the hypothalamic level are primarily manifested in the hippocampus (13–16). It nevertheless remains of interest, especially in the context of pathophysiologies associated with dysfunction in a particular neuronal system, to obtain a more precise definition of the intrinsic steroid response potential of a given neuronal phenotype. We reasoned that this could best be achieved by depriving specific subsets of neurons of their normal afferents. Since there are no methods available to isolate neurons of a given neurochemical phenotype, we performed this work on populations of cells consisting of a restricted number of phenotypes. We examined the secretory responses of cells from the ARC and PVN to the synthetic glucocorticoid dexamethasone (DEX), a type I corticosteroid receptor (CR) -preferring ligand. Since earlier studies (1) have demonstrated mutual in vivo interactions between CRH, AVP, and ß-END neurons, we also examined how cells derived from the PVN only respond to DEX.

To date, all reports on hypothalamic neurons in culture have used growth media supplemented with gonadal and adrenal steroid hormones and/or steroid-rich serum (17, 18), thus precluding studies of the effects of steroid hormones per se. In the present work we circumvented these limitations by developing a chemically defined serum- and steroid-free medium. A further major advance represented in this work is the ability to grow neurons originating from 18- or 19-day-old postnatal rats; based on ontogenetic descriptions for hypothalamic peptidergic and steroid receptor systems (19–23), it is safe to assume that our preculture neurons were fully differentiated. Thus, in contrast to cultures prepared from fetal donors, the responses elicited by the present cultures may be expected to reflect the properties of mature neurons more accurately.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cultures were prepared from 18- to 19-day-old male Wistar rats (housing and procedures approved by local animal welfare authorities) following a protocol deposited with the American Society for Information Science (ASIS), where other experimental details may be also found. Briefly, rats were decapitated and slices containing the PVN and/or ARC were microdissected out in ice-cold Earle's buffered saline solution. After further trimming, slices were dissociated with papain and suspended in neural cell growth medium complemented with a modification of Sato and Bottenstein's N2 and Romijn's supplements, nerve growth factor, and epidermal growth factor.

Cell viability (trypan blue dye exclusion) immediately before plating was usually >90%. Cells were seeded at a density of 1000 cells/mm2 on coverslips precoated with an aqueous solution of gelatine and poly-d-lysine and placed in an incubator (35°C; 99% relative humidity; 93% air:7% CO2). In addition to examination by phase-contrast microscopy, morphobiochemical characterization of cells was carried out using immunocytochemistry with antibodies against neurofilament proteins (68, 160, and 210 kDa), CRH, AVP, and ß-END at various intervals in vitro. In situ hybridizations for CRH, AVP, POMC, and the type II CR (glucocorticoid receptor; GR) were performed on the cultures to ascertain their viability in terms of mRNA synthesis. Veratridine (10-6 M) was applied to demonstrate that the cultures could respond to a depolarizing stimulus by secreting CRH, AVP, and ß-END; peptide concentrations in the medium were measured by specific and sensitive radioimmunoassays.

DEX-induced changes in the release of CRH, AVP, and ß-END from ARC/PVN or PVN-only cultures were measured after 8 days in vitro (div); cells were exposed to the drug for 24 h. In some cases, cells were coexposed to DEX and one of the following compounds: Ca2+-channel blocker verapamil hydrochloride (5.10-5 M); Na+-channel blocker tetrodotoxin (TTX; 10-6 M); type II CR (GR) antagonist mifepristone (RU38486; 10-5 M), or the pan-opioid receptor antagonist naloxone hydrochloride (NAL; 10-6 M). The effects of DEX withdrawal from the culture medium on peptide release were also studied.

Treatment-induced changes in peptide release were compared to amounts of peptide released under basal conditions and/or in time-matched collections from control wells. Because of occasional large between-well variations in the amounts of peptide released, well-by-well (treated vs. basal) comparisons were made and the results summarized in terms of percent change in peptide release (basal levels = 100%). In all instances, data are depicted as means ± SEM, with n = 5–6. Initially, data were subjected to analysis of variance (ANOVA), with between-treatment group differences being identified by the Student-Newman-Keuls method in which the level of significance was preset at P<=0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphobiochemical and pharmacological characterization of cultures
Within 24 h of plating (1 div), cells that had retained their processes after enzymatic treatment were easily recognizable by further extension of their neurites; the latter were often branched and reached lengths of 10- to 20-fold the diameter of the cell body. Gradually (2–5 div), there was a marked increase in the number of neurite extensions, many of which were 5- to 40-fold the diameter of the cell body. Cultures usually contained a small number of glial cells, but despite the omission of anti-mitotic drugs in the culture medium, glial proliferation apparently did not interfere with neuronal survival (judged by neurofilament immunostaining and phase contrast microscopy after at least 3 wk in vitro); positive NF staining was detectable in many cells and neurites for up to 21 div. In situ hybridization histochemistry showed that the neurons in culture had the capacity to transcribe AVP-, CRH-, POMC genes, and the type II CR (GR) gene. The number, size, and shape of cells showing detectable amounts of the three peptide mRNA hybridization signals corresponded well with those of cells immunostained for CRH, AVP, and ß-END peptide immunoreactivity (c.f. Fig. 1, upper panels). A more comprehensive description of the morphological characterization of the cultures is deposited with the ASIS. Cultures containing cells from the ARC and PVN responded to a depolarizing challenge (veratridine; 10-6 M; 8 h) with significant increases in the release of CRH (335%), AVP (193%) and ß-END (458%) as compared to basal secretion of CRH (typically, 19.4 pg/ml), AVP (16.7 pg/ml), and ß-END (49 pg/ml). Despite the extended exposure to the depolarizing stimulus, neurons remained viable for several days thereafter, as judged by phase contrast microscopy. Basal secretion of the three peptides was seen to be Ca2+ dependent: incubation with the Ca2+-channel antagonist verapamil (5.10-5 M) abolished (P<=0.05) the secretion of CRH, AVP, and ß-END. Blockade of Na+-dependent axonal transmission with TTX (10-9 M) also resulted in a significant attenuation of basal peptide secretion (P<=0.05).



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  		Figure 1. Upper panels: Immunocytochemical localization of CRH-, AVP-, and ß-END-positive neurons in 2-wk-old cultures of the ARC/PVN originating from 19-day-old male rats. Lower panels: Corresponding phase microscopic fields in which the CRH-, AVP-, and ß-END-immunopositive cells shown in upper panels were located. Arrows mark identical cells in each pair of panels, for orientation purposes. Scale bar represents 20 µm.

Secretory responses by ARC/PVN cultures to DEX
As shown in Fig. 2a, the synthetic glucocorticoid DEX (presented continuously over 24 h) produced highly significant changes in ß-END secretion (H=15.3, df=5, P<=0.01): at doses between 10-12 and 10-8 M, DEX caused a significant (P<=0.05) reduction in ß-END secretion. Subsequent withdrawal of DEX from the culture medium (24 h) resulted in graded increases in ß-END secretion (H=28.2, df=5, P<= 0.0001; Fig. 2a). These increases were inversely correlated (F=40.3, df=1,28, P<=0.01) with the dose of DEX to which the cells were exposed during the preceding 24 h period, the first effects being apparent in cultures withdrawn from DEX 10-10 M treatment (P<=0.01). Continued exposure of the cultures to DEX for 24 h resulted in significant dose-related decreases in AVP secretion (H=21.9, df=5, P<=0.001), as depicted in Fig. 2b. The first significant changes (P<=0.05) were seen at a dose of 10-10 M, and there was a significant negative correlation between the DEX dose and AVP release (F=23.5, df=1,28, P<=0.001). The cultures responded to the subsequent withdrawal of DEX over the next 24 h with a highly significant (F=11.5, df=5,30, P<=0.0001) increase in AVP release ( Fig. 2b); however, the latter increase was not related to the dose to which the cells had been previously exposed: response to withdrawal from DEX 10-12 M was not significantly different from responses to withdrawal from DEX 10-11–10-8 M. Compared with basal conditions, DEX at doses between 10-10 and 10-8 M elicited significant increases in CRH secretion (H=31, df=5, P<=0.0001; Fig. 2c), and there was a significant linear correlation between DEX dose and CRH response (F=28.3, df=1,28, P<=0.0001). Withdrawal of DEX from the culture medium for 24 h thereafter resulted in significant effects on CRH release (H=29.8, df=5, P<=0.0001; Fig. 2c), with a tendency for CRH release to be inversely related to the dose of DEX (10-11–10-8 M) to which the cells had been previously exposed.



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  		Figure 2. Effects of increasing doses of dexamethasone (10-12–10-8 M) (•) and subsequent withdrawal ({circ}) on the release of ß-END (a), AVP (b), and CRH (c) from male ARC/PVN cultures. Asterisks indicate significant differences (P<=0.05) as compared to basal release (100%); crosses denote significant differences (P<=0.05) between treatment effects. Each data point (24 h media collection) represents means ± SEM of six independent determinations.

In a separate study, the CRH contents of cell lysates were examined after DEX treatment (10-9 M; 24 h) in order to gain an insight into the paradoxical results obtained when CRH secretion was measured after treatment with the steroid: DEX-treated cells had a CRH content of 933 ± 216 pg/mg protein vs. non-treated cells that had a CRH content of 2307 ± 775 pg/mg protein (P<=0.05). The DEX-induced changes in peptidergic secretion were demonstrated to be Ca2+ dependent. Thus, cells treated with verapamil (5.10-5 M) ± DEX (10-9 M) showed a significant reduction (P<=0.05) in their release of CRH. Verapamil significantly potentiated the suppression of CRH secretion from cells that were being withdrawn from DEX (H=29.3, df=5, P<=0.0001). Verapamil also potentiated (P<=0.05) the reduction of AVP and ß-END release caused by exposure to DEX, and markedly attenuated the increase seen in the secretion of both peptides after withdrawal from the steroid (HAVP=29.3, df=5, P<=0.0001; Hß-END=31.8, df=5, P<=0.0001). Changes in peptidergic secretion after treatment with DEX or after withdrawal of the steroid were shown to involve Na+-dependent axonal transmission: irrespective of whether DEX (10-9 M) produced a stimulation (CRH) or inhibition (AVP and ß-END) of peptide release, cells treated with a combination of TTX (10-6 M) and the steroid showed (further) significant reductions in their release of the three peptides investigated (HCRH=18.1, df=3, P<=0.0004; HAVP=17, df=3, P<=0.0007; Hß-END=16.7, df=3, P<=0.0008). Glucocorticoid receptors were shown to influence CRH, AVP, and ß-END secretion. As compared to DEX (10-9 M) alone, the glucocorticoid receptor antagonist RU38486 (10-5 M) significantly altered the secretion of CRH, AVP, and ß-END (HCRH=15.3, df=3, P<=0.0016; HAVP=16.4, df=3, P<=0.0009; Hß-END=14.2, df=3, P<=0.0027; Fig.3a–c ). Treatment with RU38486 resulted in an antagonism of the DEX-evoked stimulation and inhibition of CRH and ß-END secretion (P<=0.05). In the case of AVP, RU38486 (DEX produced further significant reductions (P<=0.05) in secretion, with no differences being observed between the results from the RU38486 only and RU38486/DEX treated cells ( Fig. 3b). Last, treatment of ARC/PVN cells with the panopioid receptor antagonist NAL (10-6 M) ± HCRHDEX (10-9 M) over a period of 24 h resulted in significant changes in the secretion of CRH and ß-END a (HCRH=21.9, df=3, P<=0.0001; Hß-END=20.1, df=3, P<=0.0002), but not AVP ( Fig. 4a–c ). Coadministration of the two drugs further increased the DEX-stimulated secretion of CRH (P<=0.05), and a disinhibition of the DEX-induced suppression of ß-END secretion (P<=0.05). When given alone, NAL significantly increased CRH and ß-END secretion relative to baseline values (P<=0.05), while not displaying any effect on basal AVP secretion.



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  		Figure 3. Effects of the type II CR antagonist RU 38486 (RU; 10;ms5 M) on basal and dexamethasone (DEX; 10;ms9 M) -induced CRH (a), AVP (b), and ;hb-END (c) secretion from male ARC/PVN cultures. Asterisks indicate significant differences (P;cc0.05) as compared to basal secretion (100%), and crosses denote significant differences (P;cc0.05) between effects of dexamethasone treatment (DEX) and simultaneous exposure to dexamethasone and RU 38486 (DEX and RU). Each data point (24 h media collection) represents means ;pm SEM of five independent determinations.



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  		Figure 4. Influence of the pan-opioid antagonist naloxone (NAL; 10;ms6 M) on basal and dexamethasone (DEX; 10;ms9 M) -induced secretion of CRH (a), AVP (b), and ;hb-END (c) from male ARC/PVN cultures. Asterisks indicate significant differences (P;cc0.05) as compared to basal secretion (100%); crosses denote significant differences (P;cc0.05) between effects of dexamethasone treatment (DEX) and coincubation with dexamethasone and naloxone (DEX and NAL). Each data point (24 h media collection) represents means ;pm SEM of six independent determinations. d) Changes in CRH secretion when ARC/PVN cultures are exposed to MOR (10;ms6 M) under basal conditions and in the presence or absence of DEX (10;ms9 M). Means ;pm SEM of four to six independent determinations are depicted.

In another study designed to clarify the inferred causal role of opioids in the stimulation of CRH secretion by DEX, cultures were also treated with the opioid receptor agonist MOR (10-6 M) ± DEX (10-9 M) over a period of 24 h, and their CRH secretory responses were measured as before. One-way ANOVA demonstrated significant between-treatment effects (F=578.2, df=3, 83, P<=0.0001). As shown in Fig. 4d, DEX induced an increase in the secretion of CRH (P<=0.05), and MOR alone significantly reduced CRH secretion (P<=0.05). When MOR treatment was combined with DEX, the secretion of CRH was significantly reduced (P<=0.05) compared to control levels, although CRH levels were still significantly higher than those found after exposure to MOR alone (P<=0.05).

DEX-induced changes in CRH and AVP secretion from PVN cultures
Exposure of PVN cells to DEX (10-9 M) for 24 h had highly significant effects on CRH and AVP secretion (HCRH=15.7, df=2, P<=0.0004; HAVP=15.7, df=2, P<=0.0004), with a significant reduction being seen in the secretion of both peptides (P<=0.05; Fig. 5 a, b ). During a subsequent 24 h period of withdrawal from the steroid, CRH secretory rates returned toward those found under basal conditions (P<=0.05), whereas those of AVP were significantly increased as compared to basal values (P<=0.05). No effect on CRH secretion was observed when PVN cells were treated with NAL ±DEX (data not shown).



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  		Figure 5. Effects of dexamethasone (DEX; 10;ms9 M) and subsequent withdrawal (DEX/wd) on the secretion of CRH (a) and AVP (b) AVP from male PVN cultures. Asterisks indicate significant differences (P;cc0.05) as compared to basal secretion (100%); crosses denote significant differences between effects of DEX treatment and DEX/wd (P;cc0.05). Each data point (24 h media collection) represents means ;pm SEM of six independent determinations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Intricate morphological and functional interactions exist between hypothalamic CRH, AVP, and ß-END neurons in vivo; all three neuronal phenotypes are glucocorticoid sensitive and are involved in the regulation of the HPA axis (1, 2, 4, 5). However, with the exception of one study in which hypothalamic slice cultures were used (24), studies of their intrinsic steroid responsiveness (e.g., when disconnected from their intra- and extrahypothalamic inputs) have hitherto been hampered by difficulties in growing them in defined, steroid-free media. This paper describes a first step in overcoming this limitation through the establishment and morphological and pharmacological characterization of a novel procedure for culturing dissociated hypothalamic CRH, AVP, and ß-END neurons from 18- to 19-day-old male rats. Although fetal hypothalamic cultures have been described previously (17), the rat HPA axis is neither fully functional nor sexually differentiated until around 2.5 wk postpartum (20, 25, 26). To assess the functional activity of our cultured neurons, we monitored the secretion of CRH, AVP, and ß-END after treatment with the type II CR-selective ligand DEX (27).

In vivo studies have shown that glucocorticoids inhibit CRH, AVP, and ß-END secretion. The present findings, showing differential responses by cultures derived from either the ARC/PVN or the PVN alone, however, indicate that the direction of response is governed by the cellular phenotypes present. Thus, exposure of ARC/PVN cultures to DEX resulted in an inhibition of AVP and ß-END secretion and a stimulation of CRH secretion; in contrast, PVN cultures responded to DEX with a suppression of CRH and AVP secretion. These effects of DEX may be considered specific in that they displayed 1) dose dependency and 2) reversibility on withdrawal of the steroid, with the secretion of CRH returning to basal and that of AVP and ß-END showing rebound effects. They apparently reflected secretion from axonal terminals in that they were abolished by the Ca2+ and Na+ channel blockers verapamil and TTX, respectively. Last, the DEX effects on CRH and ß-END secretion were attenuated by the type II CR antagonist RU38486, an indication that the observed effects were receptor mediated. Paradoxically, AVP secretion was reduced by RU38486 (±DEX), a finding possibly explained by the conditional agonistic activity of RU38486 (28) and the exquisite sensitivity of AVP neurons to glucocorticoids (11, 12, 29, 30). The observation that AVP secretion plateaued maximally after withdrawal from a DEX dose as low as 10-11 M indeed supports the latter view.

The robust response of AVP and ß-END neurons to DEX withdrawal shows that suppression of their secretory activity by DEX did not result from mere exhaustion of releasable peptide stores (c.f. refs 8 and 31, which describe glucocorticoid inhibition of AVP and ß-END synthesis). Our observations that DEX does not appreciably diminish the cellular content of AVP, together with recent measurements of AVP in hypophysiotropic nerve endings in corticosteroid-treated rats (30), accord with this interpretation. However, a similar explanation cannot be applied to the paradoxical (but see ref 32) responses of CRH neurons to DEX treatment and withdrawal: in keeping with previous reports on DEX-induced changes in CRH content and gene transcription (33, 34), measurements of CRH in lysates from DEX-treated cells were markedly decreased, a finding that might account for the observed DEX withdrawal-induced decrease in CRH secretion. Further studies on the kinetics of, and relationship between, CRH synthesis and stimulus-secretion coupling from releasable pools are clearly warranted. The existence of ultrashort negative feedback of CRH upon its own secretion (35), and newer evidence that corticoids may also act by increasing neuronal excitability (see refs 36–39) in addition to their established genomic actions (40), are also worth considering.

Complex intrahypothalamic interactions between CRH, AVP, and ß-END neurons have been gleaned from morphological and pharmacological investigations (2, 41). The latter obervations, showing that CRH stimulates ß-END secretion secondary to an increase in AVP release, do not, however, readily fit with observations made in the present study: DEX treatment caused a decline in AVP and ß-END secretion and a concomitant increase in CRH secretion; conversely, DEX withdrawal resulted in a stimulation of AVP and ß-END secretion and a reinstatement of basal levels of CRH secretion. These findings suggest that, in the absence of other neural inputs, the secretion of AVP and ß-END may not entirely depend on stimulation by CRH. On the other hand, since AVP neurons are particularly sensitive to glucocorticoids (11, 12, 30), the parallelism between the secretory profiles of AVP and ß-END is explicable on the basis of the mandatory role of AVP in ß-END secretion (see ref 1).

Published data would suggest that CRH-producing neurons are less sensitive to glucocorticoid feedback than AVP neurons (12, 24, 30). The present data, obtained from CRH neurons dissociated from their in situ (e.g., hippocampal) neural inputs, strongly suggest the direct stimulation of CRH neurons by DEX (but see following text). An alternative hypothesis, based on the demonstration that opioids bimodally influence CRH secretion (35, 42–44) with low (10-11–10-10 M) and high (10-7–10-5 M) doses of ß-END, respectively, stimulating and inhibiting CRH secretion, deserves consideration. Together with the known glucocorticoid-induced suppression of hypothalamic POMC gene expression (8), it is conceivable that DEX treatment results in a reduced opioidergic (inhibitory) tone on CRH neurons, thus leading to a net increase in CRH secretion. Conversely, the DEX withdrawal-induced increase in ß-END secretion (primarily, due to glucocorticoid removal and/or secondarily, due to the disinhibition of AVP release) may be considered among the causes for the subsequent decline in CRH secretion. This view is partially supported by the observation that the opioid antagonist NAL itself produced an increase in CRH secretion under basal conditions and potentiated the DEX-induced increase in CRH secretion. The latter may not be as paradoxical as it seems at first, since NAL (±DEX) also stimulated ß-END secretion, probably by antagonizing inhibitory presynaptic opioid autoreceptors (45). The proposed scenario, in which DEX stimulates CRH secretion secondarily to an inhibition of ß-END neurons, is supported by our finding that coincubation of DEX-treated ARC/PVN cultures with the prototypic opioid receptor agonist MOR results in a diminution of CRH release.

Experiments were also performed on cultures excluding the ARC, the major ß-END-producing site in the brain. The results from studies in which PVN cells were exposed to DEX, and subsequently deprived of the steroid, were concordant with expectations based on earlier in vivo and in vitro reports (4, 5, 35, 46, 47): DEX exposure produced a significant suppression of CRH and AVP secretion, whereas its withdrawal produced a ‘rebound’ response in the secretion of AVP and restored CRH secretion to basal levels. Since ß-END may not represent the sole opioid responsible for the observed DEX-stimulated secretion of CRH (PVN neurons also secrete two other opioid peptides, dynorphin and enkephalin, and the receptors for these peptides are also NAL sensitive; see ref 7), we also incubated these PVN cultures with DEX in the presence of NAL. The finding that the opioid antagonist did not influence the DEX-evoked pattern of CRH and AVP secretion demonstrated that dynorphinergic and enkephalinergic contributions may be dismissed.

The findings summarized above show that PVN neurons, which display abundant numbers of type II CR (41, 48), can respond directly to glucocorticoids; this is consistent with previous assertions made on the basis of studies involving microimplantation of corticoids in the vicinity of the PVN (33, 49). Further, the present data obtained from isolated PVN neurons provide strong evidence CRH and AVP neurons can be negatively regulated by DEX without the intermediation of other brain nuclei. This interpretation obviously conflicts with a view expressed earlier in this discussion: that CRH neurons may not be subject to negative regulation by glucocorticoids. However, inasmuch as the second (PVN) series of results were obtained from cultures devoid of ARC neurons, we conclude that whereas CRH neurons are themselves glucocorticoid sensitive (see refs 35, 50), modulation of their secretory output by glucocorticoids is profoundly influenced by the presence of factors originating in the ARC. Even though evidence was obtained that ß-END is one of these regulatory factors, the possible role of other ARC-derived neuromodulators should not be neglected.

In vitro approaches obviously call for cautious extrapolation of results to physiological mechanisms; for example, our inability to exclude magnocellular AVP-producing neurons from the cultures and our 24 h block sampling procedure (necessitated by the present lack of more sensitive detection systems) may appear to limit the interpretational value of the data obtained. However, both potential criticisms are contestable. The first may be dismissed on the grounds that magno- and parvocellular AVP cells resemble each other in their response to the corticoid milieu (24, 51). With respect to the second argument, although one group has reported pulsatile CRH secretion in vivo (52), there is no a priori reason to assume that our sampling schedule should lead to more compromising results than those obtained in most neuro- endocrinological experiments, when single-point determinations appear to accurately reflect the activity of a given axis (e.g., ref 53). Further, it remains questionable whether dissociated cells can be expected to display synchronous bursts of peptide release, a prerequisite for the detection of pulsatile secretion. Calogero et al. (35, 54) have elegantly demonstrated the utility of in vitro systems for defining the role of neurotransmitters in the control of CRH secretion. Nevertheless, because of the multiple stimulatory and inhibitory afferents to a given neuronal system, studies like the present one may inadvertently oversimplify the real situation in vitro; the physiological response to an agent will ultimately be determined by the state of activation of the input and target neurons. These caveats aside, the utility of the above-described approaches to dissect the physiological and pharmacological properties of, mechanisms used by, and interactions among, the fully differentiated components of complex neural networks appears promising.


   ACKNOWLEDGMENTS
 
This work was supported by a Deutsche Forschungsgemeinschaft grant (SFB220/TPC8). The authors gratefully acknowledge the advice and encouragement of Professor B. A. Demeneix (Paris) and Dr. V. K. Patchev, and all colleagues who provided antisera.


   FOOTNOTES
 
1 Correspondence: Neuroadaptations Group, Department of Neuroendocrinology, Max Planck Institute of Psychiatry, Kraepelinstrasse 2–10, D-80804 Munich, Germany. E-mail: osa{at}mpipsykl.mpg.de

2 Abbreviations: CR, corticosteroid receptor; PVN, hypothalamic paraventricular nucleus; ARC, arcuate; CRH, corticotropin-releasing hormone; AVP, arginine vasopressin; POMC, pro-opiomelanocortin; ß-END, ß-endorphin; DEX, dexamethasone; ASIS, American Society for Information Science; GR, glucocorticoid receptor; NAL, naloxone hydrochloride; TTX, tetrodotoxin; ANOVA, analysis of variance.

Received for publication August 4, 1997. Accepted for publication October 27, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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