Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Temporal regulation by estrogen of beta-preprotachykinin mRNA expression in the rat ventromedial nucleus of the hypothalamus.

In the rat, reproduction and sexual behavior are controlled by the gonadal steroid regulation of synaptic interactions within the sexually dimorphic limbic-hypothalamic system. The effects of estrogen on the ventromedial nucleus of the hypothalamus, one nucleus within the circuit, are central to the modulation of this behavior. Involvement of the neuropeptide substance P, a member of the tachykinin family of neuropeptides, has been implicated in the regulation of both lordosis behavior and gonadotropin release. However, previous studies have provided conflicting evidence as to whether levels of substance P in the ventromedial nucleus of the hypothalamus are modulated by circulating estrogens. To study this question further, in situ hybridization histochemistry was used to examine levels of beta-preprotachykinin mRNA, which encodes substance P and other tachykinins, in the ventrolateral subdivision of the ventromedial hypothalamus at 10 consecutive timepoints over a 4 day period subsequent to an acute administration of estrogen. Following estrogen treatment, beta-preprotachykinin mRNA expression was increased in cells of the ventrolateral portion of the ventromedial nucleus of the hypothalamus which constitutively express beta-preprotachykinin mRNA; however, there were no statistically significant changes in the number of cells that express detectable levels of beta-preprotachykinin mRNA in the ventrolateral portion of the ventromedial nucleus. Estrogen treatment produced two peaks of beta-preprotachykinin mRNA expression, the first at 2 h and the second at 48 h after the injection of estrogen. These data indicate that estrogen has both rapid and prolonged effects on beta-preprotachykinin mRNA levels, suggesting that estrogen may affect different cellular mechanisms relevant to the induction of beta-preprotachykinin mRNA expression.

Estrogen regulates preproenkephalin-A mRNA levels in the rat ventromedial nucleus: temporal and cellular aspects.

Numerous studies suggest that the ventromedial nucleus of the hypothalamus is a primary locus of control for reproductive behavior in the female rat. The display of lordosis behavior is estrogen dependent and its regulation appears to involve the activity of endogenous opioid circuits in the mediobasal hypothalamus and brainstem. Hypothalamic levels of preproenkephalin-A mRNA and the neuropeptide which it encodes, methionine-enkephalin, are dramatically up-regulated by estrogen. To characterize the temporal and cellular aspects of the effect of an acute exposure to estrogen on preproenkephalin-A mRNA levels in the ventromedial nucleus and arcuate nucleus of the hypothalamus, ovariectomized female rats were injected with 50 micrograms estradiol benzoate and used for quantitative in situ hybridization histochemistry of preproenkephalin-A mRNA. In the ventromedial nucleus, estrogen treatment caused a biphasic increase in the number of preproenkephalin-A mRNA cells. Maximal numbers of cells expressing preproenkephalin-A mRNA were observed at 1 and 48 h after estrogen injection. At 4 and 96 h after estrogen injection, the numbers of preproenkephalin-A mRNA cells were similar to that which was seen in the ovariectomized female. The induction of preproenkephalin-A mRNA expression in cells of the arcuate nucleus followed a similar pattern that peaked 48 h after exposure to estrogen. Levels of preproenkephalin-A mRNA per cell did not change in either nucleus as a function of estrogen treatment. Thus, acute estrogen treatment induces expression of preproenkephalin-A mRNA in populations of cells in the ventromedial hypothalamus and arcuate nucleus in which preproenkephalin-A mRNA expression is undetectable by in situ hybridization in the absence of circulating estrogen.

Estrogen and stress interact to regulate the hypothalamic expression of a human proenkephalin promoter-beta-galactosidase fusion gene in a site-specific and sex-specific manner.

Gonadal steroids and physiological stressors affect the regulation of proenkephalin (PPE) gene expression in the paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei. To examine the effects of these modulators at the cellular level, the current study utilized a transgenic mouse line that expresses a human proenkephalin promoter/bacterial beta-galactosidase fusion gene (ENK-1). Previous studies have demonstrated that the regulatory sequences included in this transgene are sufficient to support appropriate transcriptional regulation of the reported gene in the PVN of male ENK-1 mice in response to stress. The present experimental paradigm was designed to examine possible interactions of sex and circulating estrogen levels with the opioid responses to acute systemic stressors, an intraperitoneal injection of hypertonic (1.5 M) or isotonic (0.15 M) saline. Adult ENK-1 mice were gonadally intact, gonadectomized, or 21 days postpartum. Forty-eight hours before perfusion, castrated males and ovariectomized females received either 10 micrograms estradiol benzoate or oil vehicle and 4 animals per group received no further treatment. Six h before perfusion, remaining animals received a single intraperitoneal injection of either hypertonic or isotonic saline. Tissues were sectioned through the hypothalamus and processed for X-gal histochemistry. In the VMH of ovariectomized females that received isotonic saline, estrogen significantly elevated transgene expression. This effect was not seen in females that only received estrogen or in those that received the severe systemic stressor of a injection of hypertonic saline. Estrogen and stress did not interact to elevate transgene expression in the VMH of males. A different pattern of expression was observed in the PVN; injection of hypertonic saline induced transgene expression only in gonadally intact males and in castrated males given estrogen. These findings demonstrate that stress and estrogen have sex-specific and site-specific regulatory effects on the expression of a PPE promoter transgene in hypothalamic neurons.

Regulation of cholecystokinin receptors in the ventromedial nucleus of the hypothalamus: sex steroid hormone effects.

Quantitative autoradiography was used to analyze cholecystokinin receptor (CCK-R) binding in the ventromedial hypothalamic nucleus (VMH) of gonadectomized rats treated with estrogen or testosterone. In the rostral VMH, sex steroids did not affect binding levels. In the caudal VMH, in both gonadectomized male and female rats, the levels of CCK-R binding were decreased 24 h after injection of 50 micrograms of estrogen benzoate but were not changed after injection of 300 micrograms of testosterone propionate. We hypothesize that these changes in CCK-R binding in the VMH reflect ligand-induced down-regulation that result from an estrogen-facilitation of stimulated CCK release in the VMH.

Microinjection of cholecystokinin into the medial preoptic nucleus facilitates lordosis behavior in the female rat.

We examined the effect of cholecystokinin (CCK) on lordosis behavior when administered into the medial preoptic area or the nucleus accumbens (NAcc) of ovariectomized estrogen-primed female rats. The frequency of lordotic responses was measured subsequent to unilateral microinjections of sulphated octapeptide CCK (sCCK-8) into the medial preoptic nucleus (MPN) or the NAcc. In the first experiment, three doses of sCCK-8 (1, 5, and 50 ng) microinjected into the MPN, and 50 ng injected into the NAcc produced a marked facilitation of lordosis. In a separate experiment, unilateral injections of an undiluted sCCK-8 antiserum into the MPN produced significant reduction in lordosis behavior in highly receptive females when compared with a normal rabbit serum injected control group. The results of the present study indicate that the CCK innervation of the MPN is involved in the neural regulation of lordosis behavior in the female rat.

Estrogen and tamoxifen differentially regulate beta-endorphin and cFos expression and neuronal colocalization in the arcuate nucleus of the rat.

Estrogen regulates hypothalamic gene expression, synthesis and release of the endogenous opioid peptide beta-endorphin (betaEND), although a consensus estrogen response element sequence has not been identified in the rat proopiomelanocortin (POMC) gene. POMC gene expression is also regulated by the activation of AP-1 promoter elements, which are known to be estrogen sensitive. The present studies examine whether estrogen modulates the hypothalamic POMC system through a non-classical mechanism involving AP-1 binding proteins such as cFos. Immunohistochemical double-labeling for betaEND and cFos was used and immunoreactive (-ir) populations were quantified in the arcuate nucleus and periarcuate area across time using unbiased stereological methods. Ovariectomized rats were injected with 50 microg estradiol (E2), 500 microg tamoxifen citrate (TAM) or both (E2+TAM) and were perfused 1, 2, 4 or 48 h later. E2 rapidly increased numbers of cFos-ir, betaEND-ir and doubly-labeled cells after 4 h, and the number of betaEND-ir cells remained high 48 h later, suggesting that the stimulatory effects of cFos on POMC in the hypothalamus persist after the cFos signal decays. Treatment with TAM alone did not affect the numbers of immunoreactive cells, although E2+TAM blocked the E2-mediated induction in all immunoreactive populations. Similar effects were seen at the transcriptional level. E2 increased hypothalamic POMC mRNA after 4 h, while TAM treatment or coadministration of E2+TAM did not significantly change the levels of POMC mRNA. Cellular colocalization of betaEND-ir and cFos-ir supports a possible intracellular co-regulation of these peptides by an estrogen-dependent mechanism within a subset of hypothalamic neurons. It does not, however, appear that E2 acts directly through an AP-1 site within the POMC gene.

Actions of sex steroids on behaviours beyond reproductive reflexes.

The actions of sex steroids in the brain have been shown, from molecular to systems levels, to control reproductive behaviour in a wide range of vertebrates. It has become increasingly clear that gonadal steroid hormones have regulatory functions which extend far beyond the direct coordination of an animal's physiological state and its display of sexual behaviour. While some of these actions may include changes in mood or other behavioural measures, such as exploration or excitability, sex steroid hormones also influence neural plasticity, neuronal activity and, possibly, learning and memory, as reflected by long-term potentiation or age-related deficits. Here we describe two systems that have been used to explore the non-reproductive roles of gonadal steroid hormones. The first of these is to examine the oestrogen-sensitive opioid peptide gene expression in the hypothalamus. Currently, we are attempting to identify the types of behaviour which may be altered consequent to the oestrogenic induction of the preproenkephalin gene. The second approach involves studying the effects of progesterone at the neuronal cell membrane and characterizing the metabolites of progesterone which have benzodiazepine-like actions in the brain. A number of studies suggest that this may provide an alternative mechanism through which progesterone can influence mood or behaviour.

Gap junction proteins in inhibitory neurons of the adult barrel neocortex.

Recent studies indicate that electrical coupling among cortical neurons may persist throughout development; electrophysiological recordings made in cortical slices from young rats reveal that numerous GABAergic neurons are electrically coupled. To determine whether these in vitro findings reflect an inhibitory neural circuit that could be functionally relevant in vivo in adult rodents, we sought to identify whether inhibitory, parvalbumin-containing neurons of the mature cortex express gap junction proteins. Immunohistochemistry was used to examine the laminar distribution of the gap junction-forming proteins connexin 32 (Cx32), connexin 36 (Cx36) and connexin 43 (Cx43) in the somatosensory cortex of the adult mouse. Double labeling immunofluorescence identified Cx32, Cx36 and Cx43 in cortical neurons that were immunoreactive (-ir) for the neuronal markers neurofilament 145 kDa and neuronal nuclei (NeuN). Parvalbumin-ir neurons throughout the cortical laminae were labeled with Cx32-ir, Cx36-ir and Cx43-ir. Stereological methods were used to quantify the extent of parvalbumin colocalization with connexins. Analysis indicated that approximately 40% of parvalbumin-ir neurons were double labeled with either Cx32-ir or Cx43-ir, and approximately 50% of parvalbumin-ir neurons were double labeled with Cx36. These findings establish an anatomical substrate for widespread electrical coupling of neurons in somatosensory cortex and suggest that gap junctions among inhibitory interneurons may persist into adulthood, providing an important mechanism for neuronal communication.

Tonic and synaptically evoked presynaptic inhibition of sensory input to the rat olfactory bulb via GABA(B) heteroreceptors.

Olfactory receptor neurons of the nasal epithelium send their axons, via the olfactory nerve (ON), to the glomeruli of the olfactory bulb (OB), where the axon terminals form glutamatergic synapses with the apical dendrites of mitral and tufted cells, the output cells of the OB, and with juxtaglomerular (JG) interneurons. Many JG cells are GABAergic. Here we show that, despite the absence of conventional synapses, GABA released from JG cells activates GABA(B) receptors on ON terminals and inhibits glutamate release both tonically and in response to ON stimulation. Field potential recordings and current-source density analysis, as well as intracellular and whole cell recording techniques were used in rat OB slices. Baclofen (2-5 microM), a GABA(B) agonist, completely suppressed ON-evoked synaptic responses of both mitral/tufted cells and JG cells, with no evidence for postsynaptic effects. Baclofen (0.5-1 microM) also reversed paired-pulse depression (PPD) of mitral/tufted cell responses to paired-pulse facilitation (PPF), and reduced depression of JG cell excitatory postsynaptic currents (EPSCs) during repetitive ON stimulation. These results suggest that baclofen reduced the probability of glutamate release from ON terminals. The GABA(B) antagonists CGP35348 or CGP55845A increased mitral/tufted cell responses evoked by single-pulse ON stimulation, suggesting that glutamate release from ON terminals is tonically suppressed via GABA(B) receptors. The same antagonists reduced PPD of ON-evoked mitral/tufted cell responses at interstimulus intervals 50-400 ms. This finding suggests that a single ON impulse evokes sufficient GABA release, presumably from JG cells, to activate GABA(B) receptors on ON terminals. Thus GABA(B) heteroreceptors on ON terminals are activated by ambient levels of extrasynaptic GABA, and by ON input to the OB. The time course of ON-evoked, GABA(B) presynaptic inhibition suggests that neurotransmission to M/T cells and JG cells will be significantly suppressed when ON impulses arrive in glomeruli at 2.5-20 Hz. GABA(B) receptor-mediated presynaptic inhibition of sensory input to the OB may play an important role in shaping the activation pattern of the OB glomeruli during olfactory coding.

Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic-hypothalamic circuit: comparison of adult with neonatal steroid treatments.

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system-hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK-expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK-expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression.