Luteinizing hormone-releasing hormone and oxytocin response to hyperosmotic stimulation: in vitro study

Brain Research Bulletin, Vol. 52, No. 4, pp. 303–307, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter

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Luteinizing hormone-releasing hormone and oxytocin response to hyperosmotic stimulation: In vitro study Ewa Bojanowska,* Marlena Juszczak, Jan W. Guzek and Ryszard Dabrowski Department of Pathophysiology, Medical University of Lodz, Lodz, Poland [Received 11 January 2000; Revised 3 March 2000; Accepted 3 March 2000] ABSTRACT: It was shown previously that luteinizing hormone-releasing hormone (LHRH) affects the neurohypophysial oxytocin release in water-deprived rats. However, the detailed mechanisms by which LHRH modifies the oxytocin response to hyperosmotic stimulation have not been explained so far. Using the isolated hypothalamo-neurohypophysial explants obtained from euhydrated rats, the effect of LHRH on the oxytocin secretion was studied under conditions of direct osmotic (i.e., Naⴙ- evoked) as well as nonosmotic (i.e., Kⴙ-evoked) stimulation. Additionally, the oxytocin response to LHRH was investigated using the explants obtained from animals drinking 2% saline for eight days (systemic, i.e., both direct and indirect, osmotic stimulation). LHRH significantly enhanced Naⴙ- and Kⴙ-evoked oxytocin release from explants taken from rats drinking tap water, indicating that LHRH could affect the Naⴙ/Kⴙ-dependent depolarization of perikarya of oxytocin neurones. In contrast, LHRH significantly diminished the Kⴙ-stimulated hormone release when the neurohypophysial complex was obtained from previously salt-loaded rats, suggesting that peripheral osmotic stimulation somehow modifies the sensitivity of oxytocinergic neurones to LHRH (possible mechanisms are discussed). It is concluded that LHRH may participate in the regulation of oxytocin secretion via both direct and indirect impact on magnocellular oxytocinergic neurones depending on the current functional status of the hypothalamo-neurohypophysial complex. © 2000 Elsevier Science Inc.

mia was shown to attenuate the oxytocin response to hyperosmotic stimulation [12]. It seems, therefore, that there is a feedback of the plasma sodium concentration on the oxytocin release. Magnocellular neurones can respond to both direct and indirect osmotic stimulation [17,19,22]. Direct osmotic excitation, as brought about by the excess of sodium, produces membrane depolarization and increases the firing rate of neurones of magnocellular nuclei; as a result, the oxytocin release rises [3,17]. The indirect osmotic stimulation involves neuroendocrine reflexes originating both in central and systemic osmoreceptors. Afferent information from these receptors is transmitted to some brain structures that in turn affect the activity of oxytocinergic neurones (for more detailed review see [2]). This “exogenous” control depends on the presence of numerous synaptic inputs to oxytocinergic neurones. Among various neuromodulators known to affect the neuronal activity, peptides represent a distinct and large group of compounds [4,20]. It was shown that luteinizing hormone-releasing hormone (LHRH), a hypothalamic neuropeptide known to modulate the release of luteinizing hormone and follicle-stimulating hormone from the adenohypophysis, can affect the oxytocin release in the rat under conditions of osmotic challenge due to water deprivation [18]. Moreover, the release of LHRH and oxytocin was recently found to undergo different regulatory processes [5]. However, mechanisms that contribute to these effects are not known. In the rat, only few LHRH neurones were found in the neurohypophysis [28] whilst numerous LHRH fibres project from the preoptic area to other brain regions [29]. Some of them, e.g., circumventricular organs, are known to affect the activity of the neurohypophysial system [13]. It may be, therefore, possible that the effect of LHRH on the neurohypophysial hormone release is mediated by some circumventricular organs and/or other hypothalamic structures where LHRH is considered to be a putative neurotransmitter. The present study has been designed to investigate some mechanisms of LHRH action on oxytocinergic neurones. To this end, the effect of LHRH on the oxytocin response to direct or indirect hyperosmotic stimulation as well as nonosmotic activation was studied using the isolated rat hypothalamo-neurohypophysial complex (HNC).

KEY WORDS: Direct osmotic stimulation, Systemic osmotic stimulation, Potassium stimulation.

INTRODUCTION Oxytocin, a peptide neurohormone produced in the supraoptic and paraventricular magnocellular nuclei of the hypothalamus, is released from the neurohypophysis into the general circulation. In females, it stimulates the function of the uterus and mammary glands. In addition, oxytocin receptors were identified in the rat kidney [26], implying a role of this hormone in the regulation of some renal functions. Indeed, oxytocin was demonstrated to control the plasma sodium concentration by induction of natriuresis in water-deprived rats [10]. On the other hand, hypernatraemia and plasma hyperosmolality are known to activate magnocellular oxytocinergic neurones [6,24] and to increase the oxytocin secretion [22] whilst chronic hyponatrae-

* Address for correspondence: Ewa Bojanowska, Department of Pathophysiology, Medical University of Lodz, 60 Narutowicza Street, PL-90-136 Lodz, Poland. Fax: ⫹48-42-632-23-47.

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BOJANOWSKA ET AL. MATERIALS AND METHODS

Animals Male Wistar rats (300 –350 g) were used for experiments. They were kept in a 14:10-h light– dark cycle at 20 –22°C. Food and water were given ad libitum except in the third series of experiments when 2% NaCl was provided instead of tap water for 8 days before decapitation. Experimental Protocol After decapitation, the brain and the pituitary with the stalk still attached were carefully removed from the skull. Then a block of tissue containing the hypothalamus was isolated according to Gregg and Sladek [7]; such an explant contained supraoptic nuclei with intact axonal projections to the neurohypophysis as well as suprachiasmatic, preoptic, ventromedial nuclei and the organum vasculosum of the lamina terminalis. The explant was placed immediately in a vial with 1 ml of the Krebs-Ringer fluid (KRF) containing: 120 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 0.7 mM MgSO4, 22.5 mM NaHCO3, 10 mM glucose, 1 g/l bovine serum albumin and 0.1 g/l ascorbic acid (pH 7.4 –7.6; osmolality ⫽ 285–295 mOsm/kg H2O). The medium was continuously gassed with carbogen (95% O2 and 5% CO2) and maintained at 37°C. The incubation fluid was changed every 20 min. Each study started after a 60-min equilibration period. In the first series of experiments the effect of LHRH on the basal and osmotically stimulated (i.e. sodium-evoked) oxytocin release was studied. The explants isolated from rats drinking tap water ad libitum (euhydrated animals) were incubated successively in: (1) the normal KRF (B1), (2) the modified KRF containing the excess of sodium chloride (S1; osmolality ⫽ 320 –330 mOsm/kg H2O), (3) the incubation fluid as (1) alone or with 4 or 40 nM LHRH (produced by Department of Organic Chemistry, Pedagogical College, Opole, Poland) (B2), and (4) the KRF as (2) alone or with LHRH in the same concentrations (S2). Incubation in each medium proceeded for 20 min. In between the incubation period nos. 2 and 3, the explants were washed in the normal medium and these samples were discarded. In the second series of experiments the effect of LHRH on non-osmotically stimulated (i.e., potassium-evoked) oxytocin release was studied using explants isolated from euhydrated animals. The experimental protocol was similar to that described above. To stimulate oxytocin release, the incubation fluid containing the excess (40 mM) of potassium was administered instead of the hyperosmotic medium. NaCl concentration in the medium was appropriately reduced to maintain the osmolality. In the third series, the effect of LHRH on potassium-stimulated oxytocin release from explants obtained from rats drinking 2% NaCl (osmotically challenged animals) was investigated. The experimental protocol was the same as in the second series. Additionally, the plasma osmolality and the haematocrit were estimated in the salt-loaded rats as well as in animals used for experiments of the second series. Plasma osmolality was measured in duplicate by freezing point depression using semimicroosmometer (Knauer & Co GMBH, Berlin, Germany). The body weight was also determined at the beginning and at the end of the 8-day period of salt loading. Oxytocin concentrations in the medium samples were measured by radioimmunoassay using the method described by Lewandowska et al. [18]. Anti-oxytocin antibody was raised by Dr. Monika Orlowska-Majdak (Department of Physiology, Medical University of Lodz). Cross reactivity with arginine vasopressin (AVP) for these antibodies was 1.1%; with LHRH, thyrotropin releasing hormone (TRH) and angiotensin II—less than 0.002%. The lower limit of detection for the assay was 1.73 pg per tube.

FIG. 1. The comparison of the basal (period B1) and stimulated (period S1) oxytocin release from isolated rat hypothalamo-neurohypophysial explants as estimated from all experiments of each series (mean ⫾ SEM); I, euhydrated rats—sodium stimulation (n ⫽ 27); II, euhydrated rats—potassium stimulation (n ⫽ 33); III, salt-loaded rats—potassium stimulation (n ⫽ 30).

Statistical Analysis Statistical significances were estimated by nonparametric Kruskal and Wallis’ rank test (when more than two groups were compared) followed by two-way Wilcoxon’s test (two means comparison). All the data are expressed as means ⫾ SEM and p ⬍ 0.05 is considered as significant. RESULTS The average basal and stimulated oxytocin release (as calculated from all experiments in each series) is shown in Fig. 1. The effect of LHRH on the oxytocin secretion was estimated using B2/B1 (basal release) and S2/S1 (stimulated release) ratios for each explant. Series I LHRH in a concentration of 4 nM markedly increased both the basal and Na⫹-stimulated oxytocin release whilst 40 nM LHRH increased significantly only the basal oxytocin secretion (Fig. 2). Series II and III The body weight, plasma osmolality and haematocrit in euhydrated and salt-loaded rats are shown in Table 1. The body weight of rats drinking 2% saline for 8 days was reduced and their plasma osmolality and haematocrit were increased as compared with control values obtained from euhydrated rats. LHRH (4 nM) increased both the basal and K⫹-stimulated oxytocin secretion and 40 nM LHRH enhanced significantly only the basal oxytocin output from explants originating from euhydrated rats (Fig. 3). Interestingly, the effect of 4 nM LHRH on

LHRH AND OXYTOCIN RELEASE

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FIG. 2. The effect of luteinizing hormone-releasing hormone (LHRH) on the basal and Na⫹-evoked oxytocin release (as estimated on the basis of the ratio between two incubation periods) from hypothalamo-neurohypophysial explants obtained from euhydrated rats. Each bar represents mean ⫾ SEM and figures in bars indicate the number of animals in each group. *p ⬍ 0.01; **p ⬍ 0.05.

K⫹-evoked oxytocin release (Fig. 3) was less marked than the effect of LHRH on Na⫹-induced oxytocin secretion (Fig. 2). Under basal conditions, however, 4 nM LHRH enhanced similarly the oxytocin release in both series of experiments (Figs. 2 and 3). When explants were taken from salt-loaded rats, LHRH in a concentration of 4 nM markedly reduced the K-stimulated oxytocin release. On the other hand, 40 nM LHRH did not influence markedly either the basal or K⫹-stimulated oxytocin release from the HNC of salt-loaded animals (Fig. 4). DISCUSSION The results of this study show that LHRH may modulate the oxytocin release from the rat HNC. To activate oxytocinergic

TABLE 1 BODY WEIGHT, PLASMA OSMOLALITY AND HAEMATOCRIT IN RATS DRINKING TAP WATER OR 2% SALINE

Rats

Euhydrated (n ⫽ 33) Salt-loaded (n ⫽ 30)

Initial Body Weight (g)

Final Body Weight (g)

Haematocrit (%)

Plasma Osmolality (mOsmol/kg H2O)

335 ⫾ 10

345 ⫾ 10

47 ⫾ 1

282 ⫾ 4

324 ⫾ 6

248 ⫾ 8

52 ⫾ 1

323 ⫾ 6

Values are mean ⫾ SEM.

FIG. 3. The effect of luteinizing hormone-releasing hormone (LHRH) on the basal and K⫹-evoked oxytocin release (as estimated on the basis of the ratio between two incubation periods) from hypothalamo-neurohypophysial explants obtained from euhydrated rats. Each bar represents mean ⫾ SEM and figures in bars indicate the number of animals in each group. *p ⬍ 0.05.

neurones, two various stimuli have been administered. Sodium (osmotic) stimulation enhanced the oxytocin secretion moderately whilst potassium (non-osmotic) stimulation increased markedly the hormone release. These results are in accordance with an earlier report where similar stimuli were compared as to the effect on the neurohypophysial hormone release in vitro [30]. The differentially potentiated oxytocin response of euhydrated rat explants to sodium and potassium stimulation during the incubation period S1 may account for the relatively lower increment of oxytocin secretion induced by both LHRH and the excess of potassium as compared with that induced by LHRH and the excess of sodium (the incubation period S2). Apparently, the large increase in the oxytocin release as evoked by potassium stimulation under S1 conditions attenuated the responsiveness of oxytocinergic neurones to LHRH during the successive incubation period S2. It is well established that only the perikarya of magnocellular neurones can respond to the direct osmotic stimulation in vitro [7], whereas potassium-induced depolarization stimulates both cell bodies in the magnocellular nuclei and axon terminals in the neurohypophysis. In euhydrated rats, LHRH enhanced both sodium- and potassium-evoked oxytocin release. This suggests that LHRH might act either at the level of the hypothalamic magnocellular nuclei or of the neurohypophysis. The latter, however, seems not to be the case since only sparse LHRH fibres were shown to project to the neurohypophysis in the rat [28]. Hence, it seems that LHRH could act as a neuromodulator to activate primarily perikarya of oxytocinergic neurones. What is more, neurones containing LHRH were found to be located quite close to

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FIG. 4. The effect of luteinizing hormone-releasing hormone (LHRH) on the basal and K⫹-evoked oxytocin release (as estimated on the basis of the ratio between two incubation periods) from hypothalamo-neurohypophysial explants obtained from rats drinking 2% saline for 8 days. Each bar represents mean ⫾ SEM and figures in bars indicate the number of animals in each group. *p ⬍ 0.05.

the supraoptic nuclei [8] thus indicating that such interaction is possible. We also found that previous chronic salt loading of donor animals completely changed the oxytocin response to LHRH in vitro. Firstly, LHRH did not affect the basal oxytocin release from explants originating from osmotically challenged rats whereas it clearly stimulated the oxytocin secretion when the explants were obtained from euhydrated rats. Similarly, the oxytocin response of salt-loaded rat explants to potassium excitation was significantly reduced as compared with results obtained from euhydrated animals. These observations indicate that the prolonged osmotic stimulation diminishes the sensitivity of oxytocinergic neurones to stimulatory agents under basal conditions probably due to the depletion of neurohypophysial hormone stores. This finding is also in concert with results obtained by Larsen et al. [16] as to the inhibitory effect of chronic salt loading on the vasopressin release from the isolated neurohypophysis. Secondly, the stimulatory effect of LHRH on the potassiumevoked oxytocin release found in euhydrated rats was reversed, i.e., 4 nM LHRH markedly decreased the oxytocin release from the HNC explants obtained from salt-loaded animals. Interestingly, similar results were described when the effect of intracerebroventricularly injected LHRH was investigated in vivo in dehydrated animals [18]. Although the mechanism of this phenomenon remains to be elucidated, it may be hypothesized that altered sensitivity of oxytocinergic neurones to neurotransmitters regulating their activity could account for the present finding. It should be noted that intact synaptic transmission was demonstrated to be necessary to maintain the sensitivity of supraoptic neurones to osmotic stimulation [27]. Chronic osmotic stimulation is known to

BOJANOWSKA ET AL. increase the number of ␥-aminobutyric acid (GABA) synapses in the oxytocin-containing neurones [21]. Moreover, GABA was shown to inhibit the electrical activity of rat supraoptic neurones [11] and, in turn, the oxytocin release in the rat (for review see [25]). It may be, therefore, predicted that, in osmotically challenged rats, the effect of LHRH on oxytocinergic neurones is mediated mainly via GABA-ergic neurones. Such impact might originate in some extra-HNC structures which modulate the function of supraoptic neurones. It seems that the preoptic nucleus could be the appropriate candidate as it was shown to be sensitive to both the systemic [1] and direct [9] hyperosmotic stimulation and to have inhibitory, GABA-ergic inputs to supraoptic oxytocinergic neurones [23]. Moreover, dehydration was demonstrated to increase the number of LHRH-containing neurones in the preoptic nuclei [15]. Hypothalamic explants employed in this study contained these nuclei with, most likely, intact pathways to supraoptic nuclei. Thus, the above described interaction could occur under conditions of our experiment. Our findings remain also in concert with a recent report where LHRH was shown to modulate synaptic transmission in the rat brain [31]. In contrast to the effect produced by 4 nM LHRH, the higher dose of LHRH (40 nM) did not affect significantly the oxytocin release from explants obtained from salt-loaded animals under potassium stimulation. Interestingly, similar observation was made by Juszczak et al. [14] as to the effect of melatonin on sucklinginduced oxytocin secretion. Melatonin was shown to affect the oxytocin secretion only when administered at doses comparable with its physiological levels. It seems that similar mechanism could account for the lack of effect of the higher LHRH dose used in our study. Alternatively, the higher dose of LHRH could affect not only the activity of the magnocellular oxytocinergic system but also the activity of other hypothalamic structures, especially under conditions of generalised stimulation due to the excess of potassium. This interaction might finally result in the suppression of oxytocinergic neurone function. Moreover, such interaction could also account for the lack of effect of 40 nM LHRH on K⫹-evoked oxytocin release from explants obtained from euhydrated rats. Taken together, the various (i.e., enhanced or inhibited) oxytocin responses to LHRH in euhydrated and osmotically challenged rats would confirm the hypothesis as to the presence of both direct and indirect effect of LHRH on the magnocellular oxytocinergic neurones. ACKNOWLEDGEMENTS

The authors are very grateful to Dr. Bozena Stempniak for her kind assistance with radioimmunoassay technique. This study was supported by Medical University of Lodz, contract no. 502-11-615 (211).

REFERENCES 1. Aradachi, H.; Honda, K.; Negoro, H.; Kubota, T. Median preoptic neurones projecting to the supraoptic nucleus are sensitive to haemodynamic changes as well as to rise in plasma osmolality in rats. J. Neuroendocrinol. 8:35– 43; 1996. 2. Bourque, C. W.; Oliet, S. H. R.; Richard, D. Osmoreceptors, osmoreception, and osmoregulation. Front. Neuroendocrinol. 15:231–274; 1994. 3. Brimble, M. J.; Dyball, R. E. J.; Forsling, M. L. Oxytocin release following osmotic activation of oxytocin neurones in the paraventricular and supraoptic nuclei. J. Physiol. (Lond.) 278:69 –78; 1978. 4. Chowdrey, H. S.; Lightman, S. L. Role of central amino acids and peptide-mediated pathways in neurohypophysial hormone release. Ann. NY Acad. Sci. 689:183–193; 1993. 5. Evans, J. J.; Janmohamed, S.; Forsling, M. L. Gonadotrophin-releasing hormone and oxytocin secretion from the hypothalamus in vitro during

LHRH AND OXYTOCIN RELEASE

6.

7. 8.

9.

10. 11.

12. 13. 14. 15. 16.

17. 18.

pro-estrus: The effects of time of day and melatonin. Brain Res. Bull. 48:93–97; 1999. Fenelon, V. S.; Poulain, D. A.; Theodosis, D. T. Oxytocin neuron activation and Fos expression: A quantitative immunocytochemical analysis of the effect of lactation, parturition, osmotic and cardiovascular stimulation. Neuroscience 53:77– 89; 1993. Gregg, C. M.; Sladek, C. D. A compartmentalized, organ-culture hypothalamo-neurohypophysial system for the study of vasopressin release. Neuroendocrinology 38:397– 402; 1984. Herbison, A. E.; Theodosis, D. T. Localization of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone releasing hormone in the male and female rat. Neuroscience 50:283–298; 1992. Honda, K.; Negoro, H.; Dyball, R. E. J.; Higuchi, T.; Takano, S. The osmoreceptor complex in the rat: Evidence for interactions between the supraoptic and other diencephalic nuclei. J. Physiol. (Lond.) 431: 225–241; 1990. Huang, W.; Lee, S. L.; Arnason, S. S.; Sjoquist, M. Dehydration natriuresis in male rats is mediated by oxytocin. Am. J. Physiol. 270:R427–R433; 1996. Ibrahim, N.; Shibuya, I.; Kabashima, N.; Setiadji, V. S.; Ueta, Y.; Yamashita, H. GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro. Brain Res. 813:88 –96; 1998. Ivanyi, T.; Dohanics, J.; Verbalis, J. G. Effect of chronic hyponatremia on central and peripheral oxytocin and vasopressin secretion in rats. Neuroendocrinology 61:412– 420; 1995. Johnson, A. K.; Gross, A. M. Sensory circumventricular organs and brain homeostasis pathways. FASEB J. 7:678 – 686; 1993. Juszczak, M.; Stempniak, B. The effect of melatonin on sucklinginduced oxytocin and prolactin release in the rat. Brain Res. Bull. 44:253–258; 1997. Krisch, B. The distribution of LHRH in the hypothalamus of the thirsting rat. A light and electron microscopic immunocytochemical study. Cell Tissue Res. 186:135–148; 1978. Larsen, P. J.; Jukes, K. E.; Chowdrey, H. S.; Lightman, S. L.; Jessop, D. S. Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland. Endocrinology 134: 1635–1639; 1994. Leng, G.; Mason, W. T.; Dyer, R. G. The supraoptic nucleus as an osmoreceptor. Neuroendocrinology 34:75– 82; 1982. Lewandowska, A.; Bojanowska, E.; Stempniak, B.; Guzek, J. W. Luliberin inhibits the release of oxytocin from the hypothalamoneurohypophysial system in dehydrated but not in euhydrated or haemorrhaged rats. Endocrinol. Regul. 29:225–231; 1995.

307 19. Ludwig, M.; Callahan, M. F.; Morris, M. Effects of tetrodotoxin on osmotically stimulated central and peripheral vasopressin and oxytocin release. Neuroendocrinology 62:619 – 627; 1995. 20. Marcos, P.; Covenas, R.; Narvaez, J. A.; Aguirre, J. A.; Tramu, G.; Gonzalez-Baron, S. Neuropeptides in the cat amygdala. Brain Res. Bull. 45:261–268; 1998. 21. Marzban, F.; Tweedle, C. D.; Hatton, G. J. Reevaluation of the plasticity in the rat supraoptic nucleus after chronic dehydration using immunogold for oxytocin and vasopressin at the ultrastructural level. Brain Res. Bull. 28:757–766; 1992. 22. Neuman, I.; Ludwig, M.; Engelmann, M.; Pittman, Q. J.; Landgraf, R. Simultaneous microdialysis in blood and brain: Oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat. Neuroendocrinology 58:637– 645; 1993. 23. Nissen, R.; Renaud, L. P. GABA receptor mediation of median preoptic nucleus-evoked inhibition of supraoptic neurosecretory neurones. J. Physiol. (Lond.) 479:207–216; 1994. 24. Poulain, D. A.; Wakerley, J. B. Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin. Neuroscience 4:773– 808; 1982. 25. Renaud, P.; Bourque, C. W. Neurophysiology and neuropharmacology of hypothalamic magnocellular neurons secreting vasopressin and oxytocin. Prog. Neurobiol. 36:131–169; 1991. 26. Schmidt, A.; Jard, S.; Dreifuss, J. J.; Tribollet, E. Oxytocin receptors in rat kidney during development. Am. J. Physiol. 259:F872–F881; 1990. 27. Sladek, C. D.; Fisher, K. Y.; Sidorowicz, H. E.; Mathiasen, J. R. Osmotic stimulation of vasopressin mRNA content in the supraoptic nucleus requires synaptic activation. Am. J. Physiol. 268:R1034 – R1039; 1995. 28. Stopa, E. G.; LeBlanc, V. K.; Hill, D. H.; Anthony, E. L. A general overview of the anatomy of the neurohypophysis. Ann. NY Acad. Sci. 689:6 –15; 1993. 29. Witkin, J. W.; Paden, C. M.; Silverman, A.-J. The luteinizing hormone releasing hormone (LHRH) systems in the rat brain. Neuroendocrinology 35:429 – 438; 1982. 30. Yagil, C.; Sladek, C. D. Osmotic regulation of vasopressin and oxytocin release is rate sensitive in hypothalamoneurohypophysial explants. Am. J. Physiol. 258:R492–R500; 1990. 31. Yang, S. N.; Lu, F.; Wu, J. N.; Liu, D. D.; Hsieh, W. Y. Activation of gonadotropin-releasing hormone receptors induces a long-term enhancement of excitatory postsynaptic currents mediated by ionotropic glutamate receptors in the rat hippocampus. Neurosci. Lett. 260:33– 36; 1999.