Peter Sotonyi

The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis.

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.

Minireview: Ghrelin and the Regulation of Energy Balance—A Hypothalamic Perspective

The recently discovered hormone, ghrelin, has been recognized as an important regulator of GH secretion and energy homeostasis. Orexigenic and adipogenic ghrelin is produced by the stomach, intestine, placenta, pituitary, and possibly in the hypothalamus. The concentration of circulating ghrelin, principally derived from the stomach, is influenced by acute and chronic changes in nutritional state. To date, most studies focused on the role of ghrelin in GH secretion or its function in complementing leptin action to prevent energy deficits. The potential significance of ghrelin in the etiology of obesity and cachexia as well as in the regulation of growth processes is the subject of ongoing discussions. A large quantity of information based on clinical trials and experimental studies with ghrelin and previously available synthetic ghrelin receptor agonists (GH secretagogues) must now be integrated with a rapidly increasing amount of data on the central regulation of metabolism and appetite. In this overview, we summarize recent findings and strategies on the integration of ghrelin into neuroendocrine networks that regulate energy homeostasis.

Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity.

The neuronal circuits involved in the regulation of feeding behavior and energy expenditure are soft-wired, reflecting the relative activity of the postsynaptic neuronal system, including the anorexigenic proopiomelanocortin (POMC)-expressing cells of the arcuate nucleus. We analyzed the synaptic input organization of the melanocortin system in lean rats that were vulnerable (DIO) or resistant (DR) to diet-induced obesity. We found a distinct difference in the quantitative and qualitative synaptology of POMC cells between DIO and DR animals, with a significantly greater number of inhibitory inputs in the POMC neurons in DIO rats compared with DR rats. When exposed to a high-fat diet (HFD), the POMC cells of DIO animals lost synapses, whereas those of DR rats recruited connections. In both DIO rats and mice, the HFD-triggered loss of synapses on POMC neurons was associated with increased glial ensheathment of the POMC perikarya. The altered synaptic organization of HFD-fed animals promoted increased POMC tone and a decrease in the stimulatory connections onto the neighboring neuropeptide Y (NPY) cells. Exposure to HFD was associated with reactive gliosis, and this affected the structure of the blood-brain barrier such that the POMC and NPY cell bodies and dendrites became less accessible to blood vessels. Taken together, these data suggest that consumption of an HFD has a major impact on the cytoarchitecture of the arcuate nucleus in vulnerable subjects, with changes that might be irreversible due to reactive gliosis.

Sex differences in adult suprachiasmatic nucleus neurons emerging late prenatally in rats

The suprachiasmatic nucleus (SCN) is implicated in the control of circadian rhythms of gonadal function. Although several structures surrounding the SCN are sensitive to the effects of gonadal steroids, similar effects in the SCN remain unclear. For example, there are conflicting data on whether the SCN is sexually differentiated. This study attempted to determine sex differences in the number of SCN cells generated during late gestation, and if testosterone mediates these differences. Pregnant female rats were treated with 5‐bromo‐2′‐deoxyuridine (BrdU; 50 mg/kg) on gestational day 18 (E18), the day when aromatase activity peaks in the developing rat fetus. These animals were also given injections of oil or testosterone propionate (10 mg/0.1 mL peanut oil) from E15 until parturition. Litters were allowed to survive until adulthood and were killed on postnatal day 60 (PN60). Following fixation, brain sections containing the SCN from these rats were processed for BrdU immunocytochemistry. A second set of SCN sections was processed for immunocytochemistry detecting BrdU and some of the cell groups prevalent within the SCN. Data showed that female rats have a higher number of cells labeled with BrdU in the SCN, particularly in the medial and caudal SCN. This sex difference was abolished in animals treated with testosterone during late gestation. Double immunocytochemistry revealed that BrdU‐labeled cells were neurons expressing calbindin‐D28K, vasoactive intestinal peptide and, to a lesser degree, vasopressin. Our results unveiled a previously unknown effect of gonadal steroids on the developing SCN, which may contribute to the emergence of gender‐specific circadian rhythms.

Estrogen Promotes Parvalbumin Expression in Arcuate Nucleus POMC Neurons

We have found that estrogen promotes suppression of feeding and a lean body mass while activating the arcuate nucleus proopiomelanocortin (POMC)-expressing neurons. These neurons, when activated, suppress appetite and increase energy expenditure. Because the increased activation of POMC neurons by estradiol was associated with increased glutamate receptor presence that enable calcium influx, we analyzed the expression of the calcium-binding protein, parvalbumin, in these hypothalamic neurons. We observed that estrogen treatment of female mice resulted in induction of parvalbumin-immunoreactivity in arcuate nucleus neurons, a large number of which was POMC-expressing. These data indicate that the increased excitatory activity induced by estradiol in the arcuate nucleus in support of suppression of appetite is associated with calcium overload of these neurons. Although parvalbumin may protect these cells from calcium overload-associated neuronal degeneration, maintenance of calcium entry may lead to increased vulnerability of POMC neurons during the course of sustained satiety.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications

BackgroundBased on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17β-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations.MethodsCombined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode.ResultsCombined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that γ-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity.ConclusionAltogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.

Ultrastructural Abnormalities in CA1 Hippocampus Caused by Deletion of the Actin Regulator WAVE-1

By conveying signals from the small GTPase family of proteins to the Arp2/3 complex, proteins of the WAVE family facilitate actin remodeling. The WAVE-1 isoform is expressed at high levels in brain, where it plays a role in normal synaptic processing, and is implicated in hippocampus-dependent memory retention. We used electron microscopy to determine whether synaptic structure is modified in the hippocampus of WAVE-1 knockout mice, focusing on the neuropil of CA1 stratum radiatum. Mice lacking WAVE-1 exhibited alterations in the morphology of both axon terminals and dendritic spines; the relationship between the synaptic partners was also modified. The abnormal synaptic morphology we observed suggests that signaling through WAVE-1 plays a critical role in establishing normal synaptic architecture in the rodent hippocampus.

Gonadotropin-releasing hormone fibers contact POMC neurons in the hypothalamic arcuate nucleus.

The metabolic state has long been shown to affect reproduction. Peripheral signals and hormones from the reproductive organs are also known to regulate energy metabolism and feeding and energy expenditure. Much attention has been paid to determine the signaling flow from key hypothalamic neuronal populations, including those producing the anorexigenic proopiomelanocortin (POMC) derivate, α-melanocyte stimulating hormone (α-MSH), to the medial preoptic area gonadotropin-releasing hormone (GnRH) neurons, cells that are the drivers of ovulation and reproduction in general. In this study, the authors explored whether a reverse signaling modality may also exist. Specifically, the authors analyzed GnRH efferents in the arcuate nucleus with particular emphasis on their anatomical proximity to arcuate nucleus melanocortin perikarya. Using correlated light and electron microscopy, the authors observed direct apposition between GnRH-containing axon terminals and POMC cell bodies. These data provide the first experimental evidence to suggest that GnRH may have a direct influence on feeding, energy expenditure, and glucose homeostasis, independent of the activity of the gonadal axis.

Food restriction modifies ultrastructure of hippocampal synapses.

Consumption of high‐energy diets may compromise health and may also impair cognition; these impairments have been linked to tasks that require hippocampal function. Conversely, food restriction has been shown to improve certain aspects of hippocampal function, including spatial memory and memory persistence. These diet‐dependent functional changes raise the possibility that the synaptic structure underlying hippocampal function is also affected. To examine how short‐term food restriction (FR) alters the synaptic structure of the hippocampus, we used quantitative electron microscopy to analyze the organization of neuropil in the CA1 stratum radiatum of the hippocampus in young rats, consequent to reduced food. While four weeks of FR did not modify the density, size, or shape of postsynaptic spines, the synapses established by these spines were altered, displaying increased mean length, and more frequent perforations of postsynaptic densities. That the number of perforated synapses (believed to be an indicator of synaptic enhancement) increased, and that the CA1 spine population had on average significantly longer PSDs suggests that synaptic efficacy of axospinous synapses also increased in the CA1. Taken together, our ultrastructural data reveal previously unrecognized structural changes at hippocampal synapses as a function of food restriction, supporting a link between metabolic balance and synaptic plasticity.

Three-dimensional visualization of the distribution of melanin-concentrating hormone producing neurons in the mouse hypothalamus.

We present here a new procedure to represent the 3D distribution of neuronal cell bodies within the brain, using exclusively softwares free for research purposes. Our technique is based on digitalized photos of brain slices processed by immunohistochemical technique, and the 3D Slicer software. The technique presented enables transposition of immunohistochemical or in situ hybridization data to the stereotaxic mouse brain atlas (e.g. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. second ed. Academic Press, San Diego). By exporting the finalized models into a popular 3D design software (3DS Max) arbitrary environment and motion simulation can be created to improve the visual understanding of the area studied. Application of this technique provides the possibility to store, analyze and compare data - e.g. on the hypothalamic neuropeptides - across experimental techniques and laboratories. The method is exemplified by visualizing the distribution of immunohistochemically identified melanin-concetrating hormone (MCH) containing perikarya within the mouse hypothalamus.