Robert L. Moss

The effect of LHRH antagonist analogs and an antibody to LHRH on mating behavior in female rats

The action of two antagonist analogs and an antibody to luteinizing hormone-releasing hormone (LHRH) on sexual receptivity was studied in avariectomized, estrogen-progesterone primed female rats. Small amounts of each LHRH substance or saline was infused through a cannula positioned in either the third ventricle or arcuate-ventromedial (ARC-VMH) area of the hypothalamus. Infusions were carried out at the time of progesterone priming, which was 42 hrs post-estrogen treatment, and sexual receptivity, as denoted by the lordosis-to-mount ratio, was measured six hrs later. One antagonist analog, [D-pGlu1, D-Phe2, D-Trp3,6]-LHRH[1], had little or no effect on sexual receptivity when tested in either site. Similarly, an antibody to LHRH, tested only in the ARC-VMH, had no observable effect on lordotic behavior. However, the second and the most potent antagonist analog, [Ac-dehydro-Pro1, pCl-D-Phe2, D-Trp3,6]-LHRH[2], produced a marked and significant decrement in lordotic behavior when infused into either the third ventricle or ARC-VMH. These results suggest that this potent and long-acting, competitive antagonist analog of LHRH prevented endogenous LHRH from exerting its normal role in the induction of sexual receptivity and provide evidence to support the contention that the role of LHRH in mediating receptivity in the female rat is physiologically relevant.

Action of estrogen and mechanical vaginocervical stimulation on the membrane excitability of hypothalamic and midbrain neurons

The effect of vaginocervical stimulation (VCS) and estrogen iontophoresis on the electrical activity recorded in urethane-anesthetized female rats from medial preoptic-septal (MPO-S) and midbrain central gray (MCG) neurons was studied during two phases of the estrous cycle, namely metestrus (M) and late proestrus-estrus (LP-E). The spontaneous discharge rate of both MPO-S and MCG neurons varied over the two stages of the estrous cycle. The spontaneous electrical activity of the MPO-S neurons was higher during M than during LP-E whereas MCG unit activity was low during M and higher during LP-E. The VCS-evoked changes in unit activity were specific, in that they were observed in response to mechanical genital stimulation and not in response to painful stimuli and/or nonspecific arousal. These responses were not dependent on the stage of estrous cycle. Finally, the iontophoresis of 17 beta-estradiol hemisuccinate evoked electrophysiological responses from MPO-S and MCG neurons. More MPO-S neurons were responsive to estrogen in LP-E than in M, while fewer MCG neurons were responsive to estrogen in LP-E than in M. The results clearly show that ongoing electrical activity of hypothalamic and midbrain nerve cells can fluctuate between M and LP-E phases of the estrous cycle and change with vaginocervical probing as well as iontophoretically applied estrogen. Furthermore, the results suggest the existence of a reciprocal relationship between the membrane activity of MPO-S and MCG neurons which may be related CNS control of reproductive activities.

Midbrain LHRH infusions enhance lordotic behavior in ovariectomized estrogen-primed rats independently of a hypothalamic responsiveness to LHRH

Lordotic behavior of ovariectomized estrone-primed rats was measured after infusions of luteinizing hormone-releasing hormone (LHRH), thyrotropin (TRH) or saline into the ventrolateral midbrain central gray (VL-MCG), or after LHRH or saline infusions into a lateral control site, the dorsolateral reticular formation (DL-RF). Infusion of LHRH, but not TRH, into the VL-MCG increased lordotic behavior. LHRH had no effect in the DL-RF. In a second experiment, rats were fitted with cannulae in the VL-MCG and in the arcuate-ventromedial nucleus area (ARC-VM). Serotonin was infused into the ARC-VM, and LHRH was infused into either the ARC-VM or the VL-MCG. Serotonin blocked the behavioral effect of LHRH infusion into the ARC-VM, but did not prevent enhancement of lordosis by LHRH infusions into the VL-MCG. These results suggest that LHRH infusions into the midbrain do not require hypothalamic responsiveness to LHRH for their effect, and are therefore unlikely to act by diffusion of LHRH rostrally. The effect of LHRH in the midbrain is site-specific, since lateral infusions (1.75 mm away) were ineffective.

Dopaminergic neurons in the mediobasal hypothalamus of old rats: Evidence for decreased affinity of tyrosine hydroxylase for substrate and cofactor

The effect of aging on the activity of tyrosine hydroxylase (TH) and on the number of TH-positive perikarya in the hypothalamus was studied in old and young female rats. The activity of TH in the mediobasal hypothalamus (MBH) of old rats was significantly (P less than 0.025) less than that in young rats. In old rats, the Km of TH for tyrosine as well as cofactor, 6-methyl-5,6,7,8-tetrahydropterine (6MPH4), was markedly greater than the Km in young rats. The maximal velocity was only slightly reduced in old animals. Contiguous coronal sections of the brain of an old and a young female rat were immunocytochemically stained for TH, and the TH-positive perikarya in the hypothalamus were counted. In the circumventricular region, 6793 TH-positive perikarya were present in the young brain and 6632 in the old brain. In the arcuate region, 2868 and 2760 TH-positive perikarya were counted in the young and old brain, respectively. It is concluded that the reduced TH activity in the MBH of old rats is not a consequence of a reduction in the number of TH-positive perikarya in the arcuate or circumventricular regions of the hypothalamus but is due to a reduction in the affinity of TH for its substrate and cofactor.

LHRH and mating behavior: Sexual receptivity versus sexual preference

Sexual behavior and sexual preference measurements were obtained from ovariectomized female rats treated with estrogen, estrogen and progesterone, or estrogen followed by third ventricular infusion of luteinizing hormone releasing hormone (LHRH) or saline. The lordosis-to-mount ratio and the occurrence of receptive and proceptive behaviors were scored to assess total sexual receptivity. Sexual preference was determined by placing the test female in the center of a four-winged choice box apparatus in which each of the outer wings contained one of the following incentive animals: a sexually active male (SM), a castrate male (CM), a female in proestrus (PF), and an ovariectomized female (OF). Time spent in close proximity to the incentive animals was measured as an index of sexual preference. Estrogen and progesterone treatment resulted in high sexual receptivity and a marked preference for SM. Estrogen alone or in combination with saline also produced a significant preference for SM. Animals treated with estrogen and LHRH exhibited high levels of sexual receptivity compared to estrogen saline treated controls, but no enhancement of preference for SM was detected. The results indicate that fractionation of sexual receptivity and sexual motivation occurs following estrogen-LHRH treatment.

Differential effects of morphine, dopamine and prolactin administered iontophoretically on arcuate-ventromedial hypothalamic neurons

The effect of iontophoretically applied morphine, dopamine and prolactin on the spontaneous, extracellular electrical activity of arcuate-ventromedial hypothalamic neurons was studied in urethane-anesthetized female rats. The deposition of morphine and dopamine suppressed, while prolactin was found to enhance, the rate of spontaneous firing of more than half of the neurons recorded. It is suggested that the present findings may be of significance in the understanding of the interaction of the opioids, dopamine, and prolactin in the regulation of prolactin secretion.

Relationship between the Central Regulation of Gonadotropins and Mating Behavior in Female Rats

Elsewhere in this volume Drs. McCann (pp. 1–21) and Clemens (pp. 23–53) report on the baffling array of hormones and other biologically active substances produced by the hypothalamus, the gonads, and the pituitary gland which are involved in the control of reproductive activities. The causal relationship in the female rat between the discharge of pituitary gonadotropins, the release of ovarian hormones, and the subsequent onset of sexual receptivity is fairly well understood. In the normal cyclic female rat on the morning of proestrus, the secretion of estrogens from the developing graafian follicle reaches peak values and, in turn, acts on the hypothalamus to trigger the ovulatory surge of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) via the release of the hypothalamic releasing factors, LH-releasing factor (LRF) and possible FSH-releasing factor (FRF). The surge in LH and FSH occurs on the afternoon of proestrus; ovulation takes place in the early hours of the following morning (estrus).

Evidence for vasoactive intestinal polypeptide (VIP) altering the firing rate of preoptic, septal and midbrain central gray neurons

The effect of the iontophoretic application of vasoactive intestinal polypeptide (VIP) on the extracellular electrical activity (neuronal firing rate) of 91 neurons localized in the preoptic (PO), septal (S) region and midbrain central gray (MCG) was studied in urethane-anesthetized female rats. When applied in minute quantities, VIP induced both excitatory (N = 14) and inhibitory (N = 8) changes in the membrane excitability of PO and S neurons (total N = 58), while only inhibitory (N = 9) changes were observed in the MCG neurons (total N = 33; thus 24 MCG neurons were found to be unresponsive to VIP). The latency and duration of the VIP-induced response was, for the most part, characterized by a rapid onset and persisted for the duration of the ejecting pulse. However, five out of the 58 PO and S neurons and one out of the 33 MCG neurons did show responses that were longer and more variable in latency and duration. Of 26 PO neurons recorded and tested with VIP, only five neurons were determined to be antidromically identified (AI) as having their axons in the median eminence. The application of VIP increased the neuronal firing rate in two AI PO neurons, decreased the activity in one, and was ineffective in altering the activity in two other AI PO neurons. The VIP-induced changes in the neuronal firing rate appear to be specific and reproducible, and not related to the ejecting current nor pH of the solution. The results suggest that VIP, a gastrointestinal hormone that is also localized in the brain, can alter the neuronal firing rate of hypothalamic and midbrain neurons, thus providing additional evidence of its possible influence on brain and neuroendocrine function.

Immunocytochemical localization of hypothalamic luteinizing hormone-releasing hormone in male ferrets

LHRH-containing neurons within the hypothalamus were immunocytochemically identified in adult male ferrets that were either gonadally intact, castrated, or castrated and treated with testosterone. The distribution of LHRH-immunopositive neuron cell bodies was similar in the three treatment groups. The majority of these cells was located mediobasally in the retrochiasmatic area, including some within the ventrolateral aspects of the arcuate nucleus. These soma were associated with a dense basal LHRH fiber plexus which extended to the median eminence. A smaller number of cell bodies was found slightly more dorsal and lateral to the major concentration at the base and midline. Isolated LHRH perikarya were occasionally observed in dorsal areas of the hypothalamus. There were no differences in the mean total number of hypothalamic LHRH cell bodies identified in the three treatment groups. These results indicate that the documented negative feedback effects of testosterone on LH secretion in male ferrets are not the result of an alteration in the absolute number of neurons capable of synthesizing LHRH.

Electrophysiological Studies into Circuitry Involved in LHRH-Mediated Sexual Behavior

Luteinizing hormone-releasing hormone (LHRH) has two well-documented neu-roendocrinological functions; the decapeptide acts on the pituitary gland to stimulate the release of LH (Redding et al., 1972; Ishikawa and Nagayama, 1973; Liu et al., 1976; Blake, 1978), and it acts in the brain to enhance mating behavior in the female rat (Moss and McCann, 1973, 1975; Pfaff, 1973; Foreman and Moss, 1977, 1978a,b; Moss and Foreman, 1976; Sakuma and Pfaff, 1980; Riskind and Moss, 1979). Previous investigations indicated that the ability of LHRH to enhance mating behavior was not dependent on its effect on the pituitary-ovarian-adrenal axis. The decapeptide was shown to be capable of facilitating mating behavior in the ovar-iectomized, adrenalectomized (Moss and McCann, 1975) and ovariectomized, hypophysectomized female rat (Pfaff, 1973). Recent research efforts in our laboratory have been directed toward distinguishing the neuronal circuitry involved in the LHRH enhancement of mating behavior from the circuitry involved in LHRH-induced luteinizing hormone (LH) release. A combination of behavioral, endocrine, and electrophysiological techniques has been employed in an attempt to separate LHRH pathways participating in LH release from those contributing to the initiation of mating behavior.