Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area

The ultrastructural appearance of luteinizing hormone-releasing hormone (LHRH) immunoreactive elements was studied in the medial preoptic area (MPOA) of adult male Fischer 344 rats. The purpose of the study was to determine the distribution and morphology of innervation of the LHRH neuron. Although not numerous, both axo-somatic and axo-dendritic synapses were present and generally of the asymmetric (Gray's II) category. Analyses of 56 profiles of 11 separate perikarya revealed only 7 axo-somatic terminals. The synaptic input to LHRH dendrites was a fraction of that to non-identified dendrites in the same electron micrographic fields; 0.4% of LHRH dendritic membrane was in synaptic contact compared to 6.6% of nonidentified dendritic membrane. In addition to receiving an input, LHRH processes were also seen to make synapses onto non-immunoreactive elements. Close examination of this material for evidence of contact between LHRH elements revealed two clear examples of synaptic interaction and several instances of close association in which no other elements intervened.     

Role of voltage-dependent modulation of store Ca2+ release in synchronization of Ca2+ oscillations

Slow waves are rhythmic depolarizations that underlie mechanical activity of many smooth muscles. Slow waves result through rhythmic Ca(2+) release from intracellular Ca(2+) stores through inositol 1,4,5-trisphosphate (IP(3)) sensitive receptors and Ca(2+)-induced Ca(2+) release. Ca(2+) oscillations are transformed into membrane depolarizations by generation of a Ca(2+)-activated inward current. Importantly, the store Ca(2+) oscillations that underlie slow waves are entrained across many cells over large distances. It has been shown that IP(3) receptor-mediated Ca(2+) release is enhanced by membrane depolarization. Previous studies have implicated diffusion of Ca(2+) or the second messenger IP(3) across gap junctions in synchronization of Ca(2+) oscillations. In this study, a novel mechanism of Ca(2+) store entrainment through depolarization-induced IP(3) receptor-mediated Ca(2+) release is investigated. This mechanism is significantly different from chemical coupling-based mechanisms, as membrane potential has a coupling effect over distances several orders of magnitude greater than either diffusion of Ca(2+) or IP(3) through gap junctions. It is shown that electrical coupling acting through voltage-dependent modulation of store Ca(2+) release is able to synchronize oscillations of cells even when cells are widely separated and have different intrinsic frequencies of oscillation.     

Galanin-like peptide and ghrelin increase cytosolic Ca2+ in neurons containing growth hormone-releasing hormone in the arcuate nucleus

Galanin-like peptide (GALP), discovered in the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC), a feeding-controlling center. Intracerebroventricular injection of GALP has been shown to stimulate food intake in the rats. However, the mechanisms underlying the orexigenic effect of GALP are unknown. The present study aimed to determine the target neurons of GALP in the ARC. We investigated the effects of GALP on cytosolic free Ca2+ concentration ([Ca2+]i) in the neurons isolated from the rat ARC, followed by neurochemical identification of these neurons by immunocytochemistry using antisera against growth hormone-releasing hormone (GHRH), neuropeptide Y (NPY) and proopiomelanocortin (POMC), the peptides localized in the ARC. GALP at 10(-10) M increased [Ca2+]i in 11% of single neurons of the ARC, while ghrelin, an orexigenic and GH-releasing peptide, at 10(-10) M increased [Ca2+]i in 35% of the ARC neurons. Some of these GALP- and/or ghrelin-responsive neurons were proved to contain GHRH. In contrast, NPY- and POMC-containing neurons did not respond to GALP. These results indicate that GALP directly targets GHRH neurons, but not NPY and POMC neurons, and that ghrelin directly targets GHRH neurons in the ARC. The former action may be involved in the orexigenic effect of GALP and the latter in the GH-releasing and/or orexigenic effects ghrelin.     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone during estradiol-induced luteinizing hormone surges in the ovariectomized ewe

Sequential bleeding and push-pull perfusion of the hypothalamus were used to characterize luteinizing hormone (LH) and LH-releasing hormone (LHRH) release in ovariectomized (OVX) ewes after injection of corn oil or estradiol benzoate (EB). Push-pull cannulae were surgically implanted into the stalk median eminences of 24 OVX ewes. Seven to 14 days later each of 20 animals was given an i.m. injection of 50 micrograms EB. Blood samples and push-pull perfusate were collected at 10-min intervals for 6-12 h beginning 12-15 h after EB injection. Four OVX ewes were given i.m. injections of corn oil 7 days after implantation of push-pull cannulae. Blood samples and push-pull perfusate were collected at 10-min intervals for 4 h between 18 and 22 h after injection of corn oil. Luteinizing hormone remained below 2 ng/ml throughout most of the sampling periods in 9 of 20 EB-treated ewes. In 5 of these 9 LHRH also was undetectable, whereas in 4 LHRH was detectable (1.84 +/- 0.29 pg/10 min), but did not increase with time. Preovulatory-like surges of LH occurred in 11 EB-treated ewes, but LHRH was undetectable in 5. In 4 of 6 ewes showing LH surges and detectable LHRH, sampling occurred during the onset of the LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of Ca2+ in heavy metal cytotoxicity.

1. Organic xenobiotic metabolism often results in oxidative stress, involving GSH depletion, alteration of thiol/disulphide balance and peroxidation of membrane lipids. These events can lead to the disruption of Ca2+ homeostasis, through impairment of the Ca2+ translocases present in cellular membranes. Inhibition of the activity of Ca,Mg-ATPases due to oxidation of their SH groups would lead to uncontrolled rises in cytosolic Ca2+ levels resulting in loss of cell viability. 2. These observations seem to be of interest when interpreting the biochemical mechanisms of heavy metal cytotoxicity. Since these cations (such as Hg2+, Cu2+, Cd2+ and Zn2+) have an extremely high affinity for SH groups, they may affect the function of SH containing proteins, such as the Ca,Mg-ATPases, as in the case of oxidative stress. 3. Results are reported indicating that Hg2+ may stimulate Ca2+ influx through voltage-dependent channels in different experimental systems. Moreover, evidence is presented that heavy metals can inhibit Ca,Mg-ATPase activity and affect mitochondrial functions in the cells of different organisms. 4. The possibility that heavy metal cytotoxicity is mediated through disruption of Ca2+ homeostasis is discussed.     

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.     

Hypothalamic deafferentation and luteinizing hormone-releasing hormone effects on secretion of luteinizing hormone in prepubertal pigs

The control of luteinizing hormone (LH) secretion was investigated in ovariectomized, prepubertal Yorkshire pigs by comparing the effects of anterior (AHD), complete (CHD), and posterior (PHD) hypothalamic deafferentation to sham-operated controls (SOC). Gilts (n = 16) were assigned randomly to treatments, fitted with an indwelling jugular catheter, and ovariectomized 2 days before deafferentation or sham-operation (Day 0). Blood for radioimmunoassay (RIA) of LH was collected sequentially at 20-min intervals for a period of 2 h before and 24, 48, 72, and 96 h after hypothalamic deafferentation or SOC. Episodic LH release after AHD or CHD was abolished (p less than 0.01), but not after PHD or SOC. Concentrations of serum LH in AHD and CHD dropped (p less than 0.01) at 24 and 48 h after surgery. Levels of LH before and after surgery in PHD and SOC were similar (p greater than 0.05). Infusion of 25 micrograms LH-releasing hormone (LHRH) i.v. at 72 and 96 h after hypothalamic deafferentation and SOC increased (p less than 0.01) serum LH to peak levels within 15 min. after infusion; LH returned to basal levels 60-80 min later. By 96 h after surgery, LH response to LH-releasing hormone (LHRH) was less in AHD and CHD as compared with the response at 72 h postinjection. Concentrations of LH in PHD and SOC were similar (p greater than 0.05) at 72 and 96 h, respectively. The results from this study clearly indicate that neural stimuli originating or traversing the neural areas rostral to the median eminence are required for secretion of LH in the pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible physiological role of guanosine triphosphate and inositol 1,4,5-trisphosphate in Ca2+ release in macrophages stimulated with chemotactic peptide.

The release of Ca2+ induced by inositol 1,4,5-trisphosphate (InsP3) in the presence of GTP was examined by using saponin-permeabilized macrophages. The origin and the amount of mobilized Ca2+ in intact macrophages stimulated with chemotactic peptide were also examined to assess the physiological significance of GTP and InsP3 on Ca2+-releasing activities. The total amount of Ca2+ released by 20 microM-A23187 from the unstimulated intact macrophages was 1.4 nmol/4 x 10(6) cells, and the mitochondrial uncoupler did not cause an efflux of Ca2+ from the cells. The Ca2+ accumulation by the non-mitochondrial pool(s) was inhibited by the presence of GTP, and the total amount of releasable Ca2+ (1.4 nmol/4 x 10(6) cells) was comparable with that accumulated by the non-mitochondrial pool(s) in the presence of GTP at a free Ca2+ concentration of 0.14 microM. The mobilized and subsequently effluxed Ca2+ in cells stimulated with chemotactic peptide was estimated to be 0.3 nmol/4 x 10(6) cells. Much the same amounts were released by about the half-maximal dose of InsP3 from the non-mitochondrial pool(s) of saponin-treated macrophages that had accumulated Ca2+ at a free concentration of 0.14 microM in the presence of GTP. These results suggest that the Ca2+-releasing activity induced by GTP may play a role in the long-term regulation of Ca2+ content in the non-mitochondrial pool(s) of macrophages, and that released by InsP3 can explain, quantitatively, the chemotactic-peptide-induced mobilization of Ca2+.     

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Immunocytochemical investigation of luteinizing hormone-releasing hormone neurons in Syrian hamsters maintained under long or short days

Light-microscope immunocytochemistry was used to investigate the LHRH system of adult male Syrian hamsters. Half of the animals were transferred from long to short photoperiods (14L:10D to 6L:18D) for 10 wk, causing plasma gonadotropin levels and the testes to revert to a prepubertal condition. In spite of the marked differences in the reproductive axis between the two groups of hamsters, the number of immunopositive LHRH neurons observed in the preoptic-medial septal area and diagonal band of Broca was approximately 400 in both cases; of these, 87-91% were monopolar and 9-13% were bipolar, regardless of whether the brains were sectioned in a coronal or sagittal plane. These results, therefore, fail to support the hypothesis that photoperiodic changes in the number of LHRH neurons play a major role in controlling the seasonal regression and recrudescence of the reproductive system in the hamster. However, morphometric analysis of the perikarya using an IBAS 2000 automatic image analyzer revealed a photoperiod-related difference. Surprisingly, the perikarya of both monopolar and bipolar LHRH neurons were significantly larger in hamsters that had been maintained on short days, as opposed to long days. These findings, therefore, are in harmony with the view that the inhibitory effect of short days on the reproductive axis is mediated through a suppression of LHRH secretion, which in turn is reflected as an increase in the net content of LHRH within the brain.     

Thermal stress reduces serum luteinizing hormone and bioassayable hypothalamic content of luteinizing hormone-releasing hormone in hens

Studies were conducted to evaluate the effects of acute (24 h) thermal stress on anterior pituitary function in hens. Circulating levels of luteinizing hormone (LH) were measured and the ability of the pituitary to respond to luteinizing hormone-releasing hormone (LHRH) challenge was determined. Moreover, bioassayable hypothalamic LHRH content was assessed by using dispersed anterior pituitary cells. In two separate experiments, circulating levels of LH were reduced in hens exposed to acute thermal stress (35 degrees C). Injection of LHRH did not result in significant differences in release of LH between normothermic and hyperthermic hens. However, the hypothalamic content of bioassayable hypothalamic releasing activity from hyperthermic hens were significantly reduced compared with normothermic hens. Taken together, these data suggest that the reproductive decline in the acutely heat-stressed hen is mediated by reduced LH releasing ability of the hypothalamus.     

The distribution of luteinizing hormone-releasing hormone (LHRH) neurons and fibers in the Formosan rock-monkey

The anatomical distribution of neurons and fibers containing Luteinizing Hormone Releasing Hormone-Immunoreactivity (LHRH-IR) in the brain of the Formosan Rock-Monkey was investigated employing immunohistochemical techniques. LHRH-IR neurons were observed in an area demarcated rostrally by the diagonal band of Broca and caudally by the mammillary area. The majority of these neurons were principally localized in the preoptic area, periventricular zone, and the arcuate nucleus. The supraoptic nucleus, septal area, triangular septal nucleus, nucleus of the diagonal band of Broca, suprachiasmatic nucleus, retrochiasmatic area, mammillary area, and the amygdala also exhibited neuronal LHRH immunoreactivity. LHRH-IR fibers appeared to originate in all of the above areas of the hypothalamus, project caudally, and subsequently terminate in the median eminence (ME). In addition to the above, LHRH-IR fibers were also detected in the organum vasculosum of the lamina terminalis (OVLT). A scattering of LHRH-IR fibers were also observed in several extrahypothalamic regions, notably the subfornical organ, indusium griseum, habenular complex, septohypothalamic nucleus, and amygdala.     

Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area

Previous studies from many laboratories have failed to demonstrate a significant synaptic input to luteinizing hormone-releasing hormone (LHRH) neurons in the rodent or primate hypothalamus/preoptic area. Having now developed immunocytochemical procedures that result in excellent ultrastructural preservation as well as in retention of antigenicity (Silverman AJ: J Comp Neurol 227:452, 1984), we have reinvestigated the question of the organization of the synaptic arrangements of LHRH neurons in the medial preoptic area of the guinea pig. Afferent inputs to these LHRH neurons include several varieties of axo-somatic and axo-dendritic synapses. Presynaptic terminals contain either round clear vesicles or a mixture of round and flattened vesicles. Most of these terminals, especially when serial sections are examined, contain dense-core granules. Well-defined synaptic clefts are evident and postsynaptic densities can be identified for asymmetrical connections. However, the presence of reaction product in the postsynaptic structure makes it difficult to categorize symmetrical terminals. In addition to these classical inputs, LHRH neurons also enter into complex heterodox synaptic relationships with their neighbors, including somato-dendritic and dendro-dendritic synapses in which the LHRH neuron can be either the pre- or postsynaptic element. These results suggest that complex synaptic relationships might account for the multiple levels of regulation of neurohormone release.     

The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations

There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.     

Possible role of Na+ ions in intracellular Ca2+ release in frog auricular trabeculae

Membrane potential-current and mechanical tension of frog atrial muscle were studied in a Ca and Mg-free solution containing 1 mmol/l EGTA (Ca-free solution). Exposure to Ca-free solution resulted in a shortening of action potential duration within 1.5 min and a subsequent lengthening which were paralleled by changes in magnitude and duration of the contraction. Similarly, the slow inward current quickly disappeared and progressively reappeared with a quite slower inactivation time-course. Its reversal potential varied with [Na]0 as for a pure Na current. By 12 min in Ca-free solution, the tension-voltage relation could be interpreted as the sum of two components correlated with the slow inward current and the membrane potential respectively. Contractures in response to sustained large depolarizations had similar time courses in Ca-free solution and Ringer's containing Na-Ca exchange blockers (Mn2+ 15 mmol/l or La3+ 3 mmol/l). Intracellular Na loading by voltage-clamp depolarizations (40 mV from the resting potential for 100 ms, at 0.2 Hz) in the presence of Veratrine (7.5 X 10(-6) g/ml) caused a large progressive increase in tonic tension. An intracellular Ca2+ release is invoked, partly related to Na+ entry and partly to membrane potential changes. The potential dependent part could be influenced by intracellular Na+.     

Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates

Summary 1. The pulsatile release of luteinizing hormone-releasing hormone (LHRH) is critical for reproductive function. However, the exact mechanism of LHRH pulse generation is unclear. The purpose of this article is to review the current knowledge on LHRH pulse generation and to discuss a series of studies in our laboratory.2. Using push-pull perfusion in the stalk-median eminence of the rhesus monkey several important facts have been revealed. There is evidence indicating that LHRH neurons themselves have endogenous pulse-generating mechanisms but that the pulsatility of LHRH release is also modulated by input from neuropeptide Y (NPY) and norepinephrine (NE) neurons. The release of NPY and NE is pulsatile, with their pulses preceding or occurring simultaneously with LHRH pulses, and the neuroligands NPY and NE and their agonists stimulate LHRH pulses, while the antagonists of the ligands suppress LHRH pulses.3. The pulsatile release of LHRH increases during the estrogen-induced LH surge as well as the progesterone-induced LH surge. These increases are partly due to the stimulatory effects of estrogen and progesterone on NPY neurons.4. An increase in pulsatile LHRH release occurs at the onset of puberty. This pubertal increase in LHRH release appears to be due to the removal of tonic inhibition from aminobutyric acid (GABA) neurons and a subsequent increase in the inputs of NPY and NE neurons to LHRH neurons.5. There are indications that additional neuromodulators are involved in the control of the LHRH pulse generation and that glia may play a role in coordinating pulses of the release of LHRH and neuromodulators.6. It is concluded that the mechanism generating LHRH pulses appears to comprise highly complex cellular elements in the hypothalamus. The study of neuronal and nonneuronal elements of LHRH pulse generation may serve as a model to study the oscillatory behavior of neurosecretion.