Janice H. Urban

Neuroendocrine Regulation of the Luteinizing Hormone-Releasing Hormone Pulse Generator in the Rat

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.

Steroid Modulation of Neuropeptide Y-Induced Luteinizing Hormone Releasing Hormone Release from Median Eminence Fragments from Male Rats

Neuropeptide Y (NPY) has been shown to stimulate hypothalamic release of luteinizing hormone-releasing hormone (LHRH) both in vitro and in vivo. In female rats, NPY facilitation of LHRH release is greatly augmented in advance of preovulatory LHRH surges, likely via the actions of ovarian steroids. However, the role of NPY in regulating LHRH release in male rats and the effects of testicular hormones on LHRH responses to NPY in males are not well understood. The objective of the present studies was to determine whether NPY stimulates LHRH release in vitro from hypothalamic tissue of male rats, and whether these effects could be modulated by testosterone (T). Mediobasal hypothalamic (MBH) or median eminence (ME) fragments from either sham-operated or castrated male rats (7 days) were placed in superfusion chambers and superfused with M199 for a 30-min baseline, 30-min challenge with NPY (10(-7)M), and a final 30-min challenge with 56 mM KCl. One-milliliter fractions were collected every 10 min and average LHRH release values over the 30-min periods were compared among groups. NPY (10(-7)M) produced a significant increase in LHRH release from the MBH and ME from intact animals. In contrast, the same dose of NPY did not stimulate LHRH release from tissues from castrated animals; only with a higher dose of NPY (10(-6)M) were the effects of NPY on LHRH release significant. Potassium challenge (56 mM KCl) significantly stimulated LHRH release from the ME of both intact and castrate male rats suggesting that all tissues were able to respond to a stimulus, and that castration did not alter the responsiveness of the LHRH neuron to a nonspecific secretagogue. To determine the extent to which T regulates the sensitivity of LHRH responses to NPY, male rats were castrated and implanted with T capsules that maintained either low (1.24 +/- 0.06 ng/ml) or high (2.17 +/- 0.31 ng/ml) physiological plasma levels of T. Treatment with the higher dose of T restored the ability of NPY to stimulate LHRH release from the ME tissues. These results demonstrate that NPY stimulates LHRH release from the hypothalamus in vitro, and that gonadal steroids, in this case T and/or its metabolites, modulate the responsiveness of the LHRH neuron to NPY. Based on these data from intact and castrate-derived tissues, it appears that steroids are necessary to maintain LHRH responsiveness to NPY receptor stimulation.

REM sleep deprivation induces galanin gene expression in the rat brain

Rats were deprived of REM sleep for 24 h by keeping them on small platforms that were placed in a water bath (the platform method). Galanin coding mRNA was visualized using in situ hybridization, and cells expressing galanin mRNA were counted. In REM sleep-deprived animals the cell count was higher in the preoptic area and periventricular nucleus. Lesions of this area have been reported to induce wakefulness in cats and rats. Galanin administered into the lateral ventricle had no effect on sleep. We conclude that REM sleep deprivation can induce galanin gene expression in some brain areas, but galanin alone does not modify spontaneous sleep.

Rapid photoperiod-induced increase in detectable GnRH mRNA-containing cells in Siberian hamster

To determine whether changes in gonadotropin-releasing hormone (GnRH) neurons are early indicators of photostimulation, Siberian hamsters were placed in short days (6:18-h light-dark) at 3 (experiment 1) or 6 (experiment 2) wk of age where they were held for 3 (experiment 1) or 4 (experiment 2) wk. Hamsters were then moved to long photoperiod (16:8-h light-dark). In experiment 1, brains were collected 1-21 days after transfer from short to long days. In experiment 2, brains were collected only on the second morning of long day exposure. Long and short day controls were included in both experiments. Cells containing GnRH mRNA, as visualized by in situ hybridization, were counted. As expected, there were no differences in the number of detectable GnRH mRNA-containing cells among animals chronically exposed to long or short photoperiods. However, on the second morning after transfer from short to long photoperiod, a positive shift in the distribution of GnRH mRNA-containing cells occurred relative to the respective controls in the two experiments. Increases in follicle-stimulating hormone secretion and gonadal growth occurred days later. In conclusion, a rapid but transient increase in the distribution of detectable GnRH mRNA-containing cells is an early step in the photostimulation of the hypothalamic-pituitary-gonadal axis.

Quantification of mRNA in Discrete Cell Groups of Brain by in Situ Hybridization Histochemistry

Publisher Summary This chapter focuses on the methods found useful in the evaluation of the regulation of neuropeptide gene expression in discrete cell groups of the rat brain. Vasopressin-containing neuronal systems provides an interesting model for several reasons, including the fact that the vasopressin gene is expressed both in well-defined nuclear groupings of cells in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and in scattered, low-density cells in extrahypothalamic brain regions, including the bed nucleus of the stria terminalis (BNST) and medial amygdaloid nucleus (MA). When performing in situ hybridization, it is important to remember that RNA in the tissue is vulnerable to degradation by RNases. When establishing an in situ hybridization assay, there are a number of methodological considerations. The sequences of particular genes can be checked by computer for homology with other known sequences. In situ hybridization, when appropriately applied, can provide important information, which cannot presently be obtained using other methods.