W. Wuttke

Involvement of preoptic-anterior hypothalamic GABA neurons in the regulation of pituitary LH and prolactin release.

SummaryThe effects of intraventricular injections of the highly specific gamma-amino-butyric acid (GABA) agonist muscimol (5 nmol/animal) on blood LH and prolactin levels were measured in ovariectomized (ovx) and in ovx estrogen-progesterone (OEP) primed rats. While the drug stimulated pituitary prolactin release in both experimental groups, pituitary LH release was significantly inhibited in the ovx animals. Muscimol was without any effect on LH levels in ovx-OEP primed rats.Bilateral implantation of tubes containing a muscimol-mannitol mixture into the medial preoptic/ anterior hypothalamic (MPO/AH) area abolished pulsatile LH release whereas blood prolactin values were elevated.The intraventricular injection of GABA (8 μmol) also reduced LH and increased prolactin levels in the blood. Measurements of catecholamine turnover rates in the MPO/AH and in the mediobasal hypothalamus (MBH) yielded reduced preoptic but unchanged hypothalamic norepinephrine (NE) and stimulated hypothalamic dopamine (DA) turnover.In view of the well known stimulatory involvement of the NE system in the mechanism of pulsatile LH release and the inhibitory effect of GABA and its agonist muscimol on pulsatile LH release, it is suggested that GABA inhibits NE release in the MPO/AH by the mechanism of presynaptic inhibition. The observation that muscimol is unable to suppress LH release in vox OEP-primed rats may indicate that those estrogen receptive neurons in the MPO/AH which mediate the negative feedback action of the steroid may use GABA as neurotransmitter and that they are the neurons which inhibit NE release. The inhibitory effect of locally implanted muscimol into the MPO/AH also supports this hypothesis. The facilitatory action of this implanted GABAergic drug on prolactin release points to the involvement of control mechanisms for the regulation of prolactin secretion which reside in the MPO/ AH. The stimulatory effect of intraventricularly injected GABA on hypothalamic DA turnover makes it likely that other than dopaminergic mechanisms are involved in mediating the stimulatory effect of GABA on prolactin release.

Single unit recordings in the rat pineal gland: evidence for habenulo-pineal neural connections.

SummaryExtracellular potentials were recorded in the pineal gland of urethane-anesthetized rats. Two distinct populations of excitable pineal “cells” were found, the silent “cells” which were driven by habenula stimulation and the spontaneously active cells. In the former case 17 of the responses (median latency of 1.2 ms) showed a positive-negative potential, and 6 (about 1 ms latency) showed only positive potential of 1–2 ms duration. The remaining cells (114), which could not be driven by habenula stimulation, exhibited spontaneous activity with a firing frequency from less than 1 Hz to greater than 100 Hz with a median firing frequency of 10 Hz.These experiments clearly demonstrate a direct habenulo-pineal fiber pathway and furthermore show that there are neuronal elements in the pineal which are only activated by habenula stimulation.

Involvement of Catecholamines and Glutamate in GABAergic Mechanism Regulatory to Luteinizing Hormone and Prolactin Secretion

There is some evidence that a population of estrogen-receptive neurons exists in the preoptic/anterior hypothalamic area which uses gamma-aminobutyric acid (GABA) as neurotransmitter and which is involved in mediating the negative feedback of estrogens on pituitary luteinizing hormone (LH) secretion. These neurons are proposed to be presynaptic inhibitors to norepinephrine (NE) release thereby inhibiting the stimulatory effect of NE on LHRH neurons. Muscimol, a potent GABA agonist, inhibits pituitary LH release in ovariectomized rats after intraventricular injection of 5 nmol. This treatment significantly increased prolactin levels. Catecholamine turnover rates in micropunches of various hypothalamic and mesolimbic structures following intraventricular treatment with muscimol were determined using the method of blocking the activity of tyrosine hydroxylase by alpha-methyl-p-tyrosine. Muscimol did not affect catecholamine, GABA and glutamate concentrations. Turnover rates of NE were significantly reduced in the medial preoptic/anterior hypothalamic area. In this structure as well as in the nucleus accumbens and in the anterior mediobasal hypothalamus turnover rates of dopamine (DA) were also reduced whereas DA turnover in mediocortical amygdalae was increased by muscimol. The selective reduction of NE turnover following muscimol may be explained by a direct or indirect action of the GABA-eric drug on NE axon terminals. The reduced NE and DA turnover in the medial preoptic area may be causally related to reduced serum LH levels whereas the reduced hypothalamic DA turnover may explain increased blood prolactin levels.

Hyperpolarization of hypothalamic parvocellular neurons by 17 β-estradiol and their identification through intracellular staining with procion yellow

SummaryIntracellular recordings and injections of procion yellow (PY) were made in parvocellular neurons in hypothalamic slices of female guinea pigs. Eighty-five neurons, with an average resting membrane potential of -35 mV, were recorded in the arcuate (ARC) ventromedial (VM), and in the cellpoor zones between the ARC and VM. Eleven of the ARC neurons and four neurons from the cell-poor zone could be driven antidromically by median eminence (ME) stimulation, nine other neurons from the three areas could be driven orthodromically by stria terminalis (ST) stimulation.Twenty-eight parvocellular neurons were tested with 17 β-estradiol (E2), which was applied in the bathing medium as the free steroid. Eleven neurons (nine ARC and two cell-poor-zone neurons) were hyperpolarized 2 to 24 mV by 10−10 M E2 concentrations. 10−8 M estrone concentration was without effect on three of these cells. Through the intracellular injection of PY, the estrogen-sensitive neurons (N = 11) were identified as small fusiform cells with few dendrites. Spine-like appendages were found on only one of these cells. None of the larger pyramidal-like neurons of these areas responded to the application of E2.

In vivo GABA release from the medial preoptic area of diestrous and ovariectomized rats

SummaryThe push-pull cannula technique was used to examine the endogenous release of GABA from the medial preoptic area (MPO) of unanesthetized rats. In diestrous females the mean resting release of GABA was 27.1±2.0 pmol/min. GABA release was significantly elevated by increasing the potassium concentration in the perfusion solution to 50 mM, whereas it was dramatically inhibited by mercaptoproprionic acid (1.0 mM), a glutamic acid decarboxylase inhibitor. A comparison between diestrous females and chronically castrated animals indicated that endogenous GABA release in OVX animals was only 60–70% of that in diestrous animals. A model for the presynaptic inhibition of NE by estrogen receptive GABAergic neurons in the MPO is proposed.

Effects of chemical lesion of the ventral noradrenergic bundle or of the medial preoptic area on preovulatory LH release in rats

SummaryThe noradrenergic innervation of the medial preoptic area (MPO) and the hypothalamus is provided by mesencephalic neurons via the ventral noradrenergic tract. Fibers of these neurons emerge through the MPO. Bilateral microinjections of 6-OHDA into the ventral noradrenergic bundle (VNB) depletes large parts of the diencephalon of norepinephrine (NE) and dopamine (DA). Since the total hypothalamic DA content is of intrahypothalamic origin, 6-OHDA injection into the VNB does not reduce hypothalamic DA content. Similarly microinjections of 6-OHDA into the MPO reduces hypothalamic and preoptic NE content without altering NE concentrations in other diencephalic structures. Microinjections of 6-OHDA and of the carrier solution of 6-OHDA into the VNB or into the MPO of female rats with regular estrous cycles results in a slight disturbance of the cyclic activity for few days. Within 1–4 days normal cyclic activity is resumed. Preovulatory LH release is substantially reduced 8–12 days after injection of 6-OHDA into the VNB or into the MPO. On the basis of these and previous results it is concluded that the availability of NE in the MPO is an important factor in determining the hight of the preovulatory LH surge.

Response of medial preoptic neurons to electrical stimulation of the mediobasal hypothalamus, amygdala and mesencephalon in normal, serotonin or catecholamine deprived female rats

SummarySingle cell activity from preoptic neurons was extracellularly recorded in normal female rats and the effects of electrical stimulation of the mediocortical amygdala (AMY), the N. med. raphes (MES) and the mediobasal hypothalamus (MBH) was tested.1.One type of preoptic neurons (55%) reacted with primary excitation to a single electrical stimulus of AMY, MES or MBH, which was usually followed by a period of postexcitatory inhibition. Another type of neurons (29%) were first inhibited after stimulation of AMY, MES or MBH and then showed a period of postinhibitory excitation. The discharge rate of a third type was not affected by electrical stimulation of these structures (16%). If a neuron was affected by a stimulus in a given area it predictably reacted in the same fashion to stimulation of the other areas.2.The postexcitatory inhibition of the majority of the primarily excited neurons was strong enough to prevent the stimulus response to a second stimulus. Occasional recordings from two neighboured cells simultaneously indicate that they can be inhibitory to each other.3.By pulse train stimulation of the AMY or MES with varying frequencies it could be demonstrated that low frequencies (10 Hz) had a facilitatory action whereas higher frequencies (100 Hz) were inhibitory to preoptic discharge rates.4.No obvious alteration of neuronal properties we found neither in rats treated intraventricularly with 5,6-dihydroxytryptamine, which strongly reduces central nervous system serotonin content, nor in 6-hydroxydopamine treated rats, which had low central nervous system catecholamine levels.

Preoptic unit activity and gonadotropin release

SummaryMultiple unit activity (MUA) in freely moving, alert rats and single cell activity (SCA) in acutely prepared rats were recorded from the medial preoptic area (MPO). The recordings were made on the afternoon of proestrus and after cervical stimulation on the following night. Cortical EEG was continuously monitored. A correlation between long lasting (20–150 min), spontaneously increasing firing rate and increased serum LH and prolactin levels was found on the afternoon of proestrus. Cervical stimulation resulted in increased MUA and SCA for 10–30 min with subsequent elevation in serum LH values. Neural activity then dropped to levels lower than observed before cervical stimulation although serum LH levels were still high. This decreased neural activity in the MPO might indicate a negative feedback of LH. To test this hypothesis highly purified LH (NIH-LH-S 17) was injected during an early phase of neural activation after cervical stimulation. Within 10–60 sec the activated neural activity decreased, thus indicating that the injected LH indeed exerted a direct or indirect negative feedback on neurones located in the MPO. It was also found that most neurones influenced by both genital stimulation and LH injection also responded to other sensory stimuli.

Effects of Electrochemical Stimulation of Medial Preoptic Area on Prolactin and Luteinizing Hormone Release in Old Female Rats

SummarySerum prolactin and LH levels in old constant estrous rats were higher than in old pseudopregnant rats. Electrochemical stimulation of the medial preoptic area in old constant estrous rats resulted in significant increases in serum concentration of prolactin and LH, and subsequent ovulation. Old pseudopregnant rats showed only a small increase in serum prolactin, no increase in serum LH and no ovulation. The high circulating levels of estrogen in old constant estrous rats, and the relative lack of estrogen and the presence of progesterone in the old pseudopregnant rats, may account for the differences observed in response of the medial preoptic area to electrochemical stimulation.

Morphological features of physiologically identified hypothalamic neurons as revealed by intracellular marking

SummaryIn the in vitro slice preparation intracellular recordings and injections of procion yellow (PY) were made in neurons of the hypothalamus. Of these neurons, one medial preoptic-anterior hypothalamus (MPO-AH) and four arcuate-ventromedial hypothalamus (ARC-VM) neurons were driven by electrical stimulation of the median eminence area (ME). Two other MPO-AH and five other ARC-VM neurons were driven by stimulation of the stria terminalis (ST). On the basis of the PY injections two morphologically distinct cell types were delineated: a larger multipolar cell type with a polygonal perikaryon was found with equal frequency in the MPO-AH and the ARC-VM. A smaller fusiform cell type was encountered mainly in the ARC-VM. On the secondary dendrites of both cell types spine-like appendages were seldom seen, but dendritic swellings were common. Some of the dendrites projected to capillaries in both areas and presumably contacted them. The axons were usually tortuous and could only be traced a short distance.