MC3R links nutritional state to childhood growth and the timing of puberty.

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development1. The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure2. Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation.

Development of leptin-sensitive circuits.

Energy homeostasis is achieved by the integration of peripheral metabolic signals by neural circuits. The organisation and function of neural circuits regulating energy homeostasis has been the subject of intense investigation and has led to the definition of a core circuitry in the hypothalamus that interacts with key regions in the brain stem, which appear to mediate many of the effects of the adipocyte-derived hormone leptin on feeding and energy balance. Recent data on the ontogeny of these pathways indicate that, in rodents, these feeding circuits primarily form during neonatal life and remain structurally and functionally immature until 3 weeks of life. Our understanding of the mechanisms promoting the formation of these critical circuits has been advanced significantly by recent evidence showing that neonatal leptin acts as a neurotrophic factor promoting the development of projections from the arcuate nucleus of the hypothalamus. Together with an expanding literature on the role of nutritional factors to affect health, these discoveries may contribute to our understanding on perinatally acquired predisposition to later disease, such as obesity and diabetes.

Developmental programming of hypothalamic feeding circuits.

The hypothalamus plays a critical role in the regulation of food intake and body weight, and recent work has defined a core circuitry in the hypothalamus that appears to mediate many of the effects of the adipocyte-derived hormone leptin on feeding and glucose homeostasis. However, until recently, little was known about the development of these critical pathways. This review summarizes recent advances regarding the post-natal development of 'metabolic' projections from the arcuate nucleus of the hypothalamus. Evidence accumulated primarily in mice indicates that these circuits develop after birth and remain both structurally and functionally immature until the second week of life. Recent studies have begun to identify cues governing development of these pathways, and leptin appears to play a crucial neurotrophic role in the development of the hypothalamic circuits regulating food intake and adiposity. The neurodevelopmental actions of leptin appear specifically to be restricted to a neonatal critical period that coincides with the naturally occurring surge in leptin. In addition, the timing and amplitude of the post-natal leptin surge has important consequences for normal body weight regulation and glucose homeostasis later in life. Ultimately, these data promise to provide new insight into the mechanisms by which alteration of perinatal nutrition may have long-term consequences on body weight regulation and adiposity in the offspring.

Neurons in the principal nucleus of the bed nuclei of the stria terminalis provide a sexually dimorphic GABAergic input to the anteroventral periventricular nucleus of the hypothalamus.

Neurons of the principal nucleus of the bed nuclei of the stria terminalis (BSTp) process pheromonal and viscerosensory stimuli associated with reproduction and relay this information to preoptic and hypothalamic cell groups that regulate reproductive function. The anteroventral periventricular nucleus of the hypothalamus (AVPV), a nucleus involved in the regulation of gonadotropin secretory patterns, receives dense projections from BSTp neurons in males but not in females. By injecting the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL), into the BSTp of rats and immunohistochemically colocalizing the GABA synthetic enzyme, GAD65, to PHAL-immunoreactive fibers in the AVPV, we tested the hypothesis that these sex-specific projections arise from BSTp neurons that synthesize the inhibitory neurotransmitter GABA. Although dense GAD65-immunoreactive fiber terminals were observed in both the male and female AVPV, higher numbers of GAD65-labeled terminals were found in the male, and those localized to PHAL-immunoreactive fibers were seen almost exclusively in males. Treatment of newborn females with testosterone or neonatal orchidectomy of males reversed these sex differences, while GAD65-immunoreactivity in the AVPV was not altered in response to exogenous hormone treatments administered to peripubertal animals. Our results suggest that projections from BSTp neurons constitute a stable, sex-specific GABAergic input to the AVPV that is patterned permanently by perinatal hormone exposure.

Target-dependent sexual differentiation of a limbic-hypothalamic neural pathway.

Neural pathways between sexually dimorphic forebrain regions develop under the influence of sex steroid hormones during the perinatal period, but how these hormones specify precise sex-specific patterns of connectivity is unknown. A heterochronic coculture system was used to demonstrate that sex steroid hormones direct development of a sexually dimorphic limbic-hypothalamic neural pathway through a target-dependent mechanism. Explants of the principal nucleus of the bed nuclei of the stria terminalis (BSTp) extend neurites toward explants of the anteroventral periventricular nucleus (AVPV) derived from male but not female rats. Coculture of BSTp explants from male rats with AVPV explants derived from females treated in vivo with testosterone for 9 d resulted in a high density of neurites extending from the BSTp to the AVPV explant, as was the case when the BSTp explants were derived from females and the AVPV explants were derived from males or androgen-treated females. These in vitro findings suggest that during the postnatal period testosterone induces a target-derived, diffusible chemotropic activity that results in a sexually dimorphic pattern of connectivity.

Molecular cloning and characterization of two putative G protein-coupled receptors which are highly expressed in the central nervous system.

We have cloned from a rat hypothalamic cDNA library two closely related G protein-coupled receptors (GPCRs) which we have designated GPCR/CNS1 and GPCR/CNS2. The peptide sequences of these two G protein-coupled receptors shared 42% identity with each other and were next most closely related to the endothelin receptors and the bombesin-like peptide receptors (approximately 25% identity). Northern blot analysis showed that both GPCR/CNS1 and GPCR/CNS2 were very highly expressed in rat brain. In situ hybridization of rat brain demonstrated broad distribution of both receptors throughout the central nervous system. GPCR/CNS1 appeared to be expressed primarily in glial cells of the fiber tracts, while GPCR/CNS2 was expressed primarily in cells of the gray matter. The different distribution patterns of these two receptors in rat brain suggests distinct functional roles for each receptor in the central nervous system. Expression of these two receptors in Xenopus oocytes showed no response to any known endothelin and bombesin-like peptides. Therefore, the endogenous ligands and physiological significance of GPCR/CNS1 and GPCR/CNS2 remain to be elucidated, but may be related to the endothelins or bombesins. The very abundant expression in brain by these two receptors, however, suggests that they play important roles in the central nervous system.

Hormonal regulation of glutamate receptor gene expression in the anteroventral periventricular nucleus of the hypothalamus.

Glutamate plays an important role in mediating the positive feedback effects of ovarian steroids on gonadotropin secretion, and the preoptic region of the hypothalamus is a likely site of action of glutamate. The anteroventral periventricular nucleus (AVPV) of the preoptic region is an essential part of neural pathways mediating hormonal feedback on gonadotropin secretion, and it appears to provide direct inputs to gonadotropin releasing hormone (GnRH)-containing neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are abundant in the AVPV, as are cells that express AMPA receptor subtypes (GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals. However, AVPV neurons do not seem to express detectable levels of kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr after treatment. In contrast, estrogen appears to suppress levels of NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian steroids may provide positive feedback to GnRH neurons is by altering the sensitivity of AVPV neurons to glutamatergic activation.

Organization and regulation of sexually dimorphic neuroendocrine pathways.

Reproduction depends on the co-ordinated expression of stereotypical behaviors and precisely timed physiological events, yet the neurobiological mechanisms underlying the integration of sensory and hormonal information that is crucial to this process have remained difficult to define. A variety of experimental approaches has provided compelling evidence that the anteroventral periventricular nucleus (AVPV) of the preoptic region plays a particularly important role in the neural control of gonadotropin secretion. It is larger in female rats, contains high densities of neurons that express receptors for ovarian steroid hormones and appears to provide direct projections to gonadotropin releasing hormone neurons in the hypothalamus. Moreover, it receives inputs from a variety of distinct sensory systems known to influence secretion of luteinizing hormone from the anterior pituitary. Thus, the AVPV appears to represent an important nodal point in sexually dimorphic forebrain circuits for the integration of sensory and hormonal information that influence reproduction. Examples of neurohumoral integration at the level of functional neural systems, individual neurons in the AVPV, or at the molecular level have been identified which provide new insight into how the hypothalamus co-ordinates expression of sex specific reproductive behaviors with gonadotropin secretion.

Development of a sexually dimorphic projection from the bed nuclei of the stria terminalis to the anteroventral periventricular nucleus in the rat.

The principal nucleus of the bed nuclei of the stria terminalis (BSTp) is larger in male rats and conveys olfactory information relevant for reproduction to the hypothalamus. In males, the BSTp provides a massive projection to the anteroventral periventricular nucleus of the preoptic region (AVPV), which in contrast to most sexually dimorphic nuclei contains more neurons in female rats. Injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin into the BSTp of adult female rats failed to demonstrate the strong projection to the AVPV observed previously in males. The ontogeny of this robust sex difference was examined by using the axonal marker DiI. The projection from the BSTp to the AVPV is established between postnatal day 9 (P9) and P10 in male rats and seems to be maintained during the juvenile period. Although labeled fibers extended from the BSTp toward the preoptic region in both male and female neonates, a similar connection with the AVPV was not apparent in female rats at any of the ages studied, and the density of labeled axons in the AVPV of P10 males was 20-fold greater than that of P10 females. A projection from the BSTp to the medial preoptic nucleus was also weaker in females but was much more substantial than that to the AVPV. These findings suggest that a sex- and region-specific activity influences the development of the projection from the BSTp to the AVPV, producing a sexually dimorphic architecture in pathways that convey olfactory information to the hypothalamus.

Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rat.

The anteroventral periventricular nucleus of the hypothalamus (AVPV) is a sexually dimorphic nucleus in the preoptic region that appears to be a nodal point in forebrain circuits, mediating hormonal feedback on gonadotropin secretion. The results of anterograde transport experiments indicate that the AVPV sends ascending projections to the ventral part of the lateral septal nucleus, the parastrial nucleus, and the region adjacent to the vascular organ of the lamina terminalis (OVLT) that contains a subpopulation of gonadotropin releasing hormone (GnRH)-containing neurons. The majority of projections from the AVPV pass caudally through the periventricular zone of the hypothalamus and form dense terminal fields in the periventricular nuclei, parvicellular parts of the paraventricular nucleus, and in the arcuate nucleus. Inputs to medial zone nuclei are more limited, with substantial projections to only the medial preoptic and dorsomedial nuclei. The AVPV sends few projections to the caudal brainstem, but terminals were observed reliably in the periaqueductal gray and medial part of the nucleus of the solitary tract. Anterograde double-labeling experiments demonstrate terminals derived from neurons in the AVPV in close apposition to GnRH-containing neurons in the preoptic region, and to dopaminergic neurons in the arcuate nucleus. Thus, the organization of projections from the AVPV in female rats suggests that neurons in this nucleus may influence the secretion of luteinizing hormone and prolactin through direct projections to GnRH neurons and tuberoinfundibular dopaminergic neurons.

Induction of neuropeptide Y gene expression in the dorsal medial hypothalamic nucleus in two models of the agouti obesity syndrome.

Dominant mutations at the agouti locus induce several phenotypic changes in the mouse including yellow pigmentation (phaeomelanization) of the coat and adult-onset obesity. Nonpigmentary phenotypic changes associated with the agouti locus are due to ectopic expression of the agouti-signaling protein (ASP), and the pheomelanizing effects on coat color are due to ASP antagonism of alpha-MSH binding to the melanocyte MC1 receptor. Recently it has been demonstrated that pharmacological antagonism of hypothalamic melanocortin receptors or genetic deletion of the melanocortin 4 receptor (MC4-R) recapitulates aspects of the agouti obesity syndrome, thus establishing that chronic disruption of central melanocortinergic signaling is the cause of agouti-induced obesity. To learn more about potential downstream effectors involved in these melanocortinergic obesity syndromes, we have examined expression of the orexigenic peptides galanin and neuropeptide Y (NPY), as well as the anorexigenic POMC in lethal yellow (A(y)), MC4-R knockout (MC4-RKO), and leptin-deficient (ob/ob) mice. No significant changes in galanin or POMC gene expression were seen in any of the obese models. In situ hybridizations using an antisense NPY probe demonstrated that in obese A(y) mice, arcuate nucleus NPY mRNA levels were equivalent to that of their C57BL/6J littermates. However, NPY was expressed at high levels in a new site, the dorsal medial hypothalamic nucleus (DMH). Expression of NPY in the DMH was also seen in obese MC4-RKO homozygous (-/-) mice, but not in lean heterozygous (+/-) or wild type (+/+) control mice. This identifies the DMH as a brain region that is functionally altered by the disruption of melanocortinergic signaling and suggests that this nucleus, possibly via elevated NPY expression, may have an etiological role in the melanocortinergic obesity syndrome.

Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic region of the mouse.

Although it has been known for some time that estrogen exerts a profound influence on brain development a definitive demonstration of the role of the classical estrogen receptor (ERalpha) in sexual differentiation has remained elusive. In the present study we used a sexually dimorphic population of dopaminergic neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) to test the dependence of sexual differentiation on a functional ERalpha by comparing the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the AVPV of wild-type (WT) mice with that of mice in which the ERalpha had been disrupted by homologous recombination (ERKOalpha). Only a few ERalpha-immunoreactive neurons were detected in the AVPV of ERKOalpha mice, and the number of TH-immunoreactive neurons was three times that of WT mice, suggesting that disruption of the ERalpha gene feminized the number of TH-immunoreactive neurons. In contrast, the AVPV contains the same number of TH-immunoreactive neurons in testicular feminized male mice as in WT males, indicating that sexual differentiation of this population of neurons is not dependent on an intact androgen receptor. The number of TH-immunoreactive neurons in the AVPV of female ERKOalpha mice remained higher than that of WT males, but TH staining appeared to be lower than that of WT females. Thus, the sexual differentiation of dopamine neurons in the AVPV appears to be receptor specific and dependent on the perinatal steroid environment.

Co-expression of steroid hormone receptors in opioid peptide-containing neurons correlates with patterns of gene expression during the estrous cycle.

The anteroventral periventricular nucleus (AVPV) of the preoptic region represents an essential component of neural pathways regulating gonadotropin secretion, and contains sexually dimorphic populations of neurons that express dynorphin or enkephalin. In the present study we used in situ hybridization to measure prodynorphin (PDYN) and proenkephalin (PENK) mRNA in the AVPV of intact animals killed on each day of the cycle. Levels of PDYN mRNA were lowest in animals killed on the afternoon of proestrus and then increased by over 60% by the morning of the following day. Expression of PENK mRNA was generally stable during the cycle, but a small yet significant reduction was detected on proestrus relative to levels of PENK mRNA in animals killed on the day of diestrus. In addition, we used double in situ hybridization to demonstrate that the majority of PDYN mRNA-containing neurons express both estrogen (50%) and progesterone receptor (85%) mRNAs. Only one quarter of the PENK-containing neurons also co-express estrogen receptor mRNA, and fewer than 10% of the PENK mRNA neurons express PR mRNA. Thus, the differential expression of PDYN and PENK during the cycle generally correlates with distinct differences in the degree of colocalization of ER and PR mRNA in PDYN and PENK mRNA-containing neurons in the AVPV.

Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus.

The anteroventral periventricular nucleus (AVPV) is a nodal point in neural circuits regulating secretion of gonadotropin and contains sexually dimorphic populations of hormonally regulated dopamine-, dynorphin-, and enkephalin-containing neurons. Because the tyrosine hydroxylase (TH), prodynorphin (PDYN), and proenkephalin (PENK) genes contain cAMP response elements that control their expression in their promoters, we used histochemical methods to determine whether ovarian steroids alter expression of the cAMP response element-binding protein (CREB) in the AVPV. Because the ability of CREB to activate transcription depends on phosphorylation at Ser133, we also evaluated the effects of acute steroid treatment on levels of phosphorylated CREB (pCREB) in AVPV neurons by using an antibody that differentiates between CREB and pCREB. Treatment of ovariectomized rats with estradiol treatments caused a significant induction in the number of pCREB-immunoreactive nuclei within 30 min that was maintained for at least 4 hr, but did not alter CREB immunostaining in the AVPV. Pretreatment with the estrogen antagonist Nafoxidine blocked this induction. In contrast, acute administration of progesterone to estrogen-primed animals suppressed and then increased pCREB staining in the ASVPV at 30 and 60 min, respectively; no significant differences between experimental and control animals were apparent by 2 hr after progesterone treatment. Double-labeling experiments showed that pCREB was colocalized with PDYN, PENK, or TH mRNA in the AVPV, suggesting that pCREB may mediate the effect of steroid hormones on gene expression in these neurons.

Ovarian steroid regulation of estrogen and progesterone receptor messenger ribonucleic acid in the anteroventral periventricular nucleus of the rat.

The anteroventral periventricular nucleus of the preoptic region (AVPV) represents a key site for hormonal feedback on gonadotropin secretion. It plays a critical role in the neural control of luteinizing hormone secretion and contains high densities of neurons that express receptors for estrogen and progesterone. In this study in situ hybridization was used to examine the expression of mRNAs encoding the estrogen (ER) and progesterone (PR) receptors in the AVPV during the estrous cycle. ER gene expression fluctuated during the cycle with the lowest levels of ER mRNA observed in animals killed on the afternoon of proestrus, and the highest levels present in animals killed during metestrus. This apparent inverse relationship between circulating levels of estradiol (E2) and ER mRNA levels in AVPV neurons was supported by the observation that treatment of ovariectomized rats with E2 suppressed expression of ER mRNA in the AVPV. The influence of progesterone (P4) on ER expression was less pronounced, but a significant increase in ER mRNA in the AVPV was detected 3 h after treatment with P4. In contrast, PR mRNA levels were highest in the AVPV during diestrus and lowest on the morning of proestrus suggesting that PR expression in the AVPV is regulated in a complex manner that may reflect the combined regulatory effects of E2 and P4. E2 treatment caused a dramatic induction of PR mRNA in the AVPV, but P4 did not affect PR mRNA expression acutely, although PR mRNA appears to be attenuated in the AVPV 27 h after P4 treatment. These findings suggest that ovarian steroid hormones regulate ER and PR gene expression in the AVPV during the estrous cycle, which may represent molecular events that contribute to cyclic changes in the responsiveness of AVPV neurons to steroid hormones.

Organization of projections from the medial nucleus of the amygdala: a PHAL study in the rat.

The organization of axonal projections from the four recognized parts of the medial amygdalar nucleus (MEA) were characterized with the Phaesolus vulgaris leucoagglutinin (PHAL) method in male rats. The results indicate that the MEA consists of two major divisions, ventral and dorsal, and that the former may also consist of rostral and caudal regions. As a whole, the MEA generates centrifugal projections to several parts of the accessory and main olfactory sensory pathways, and projections to a) several parts of the intrahippocampal circuit (ventrally); b) the ventral striatum, ventral pallidum, and bed nuclei of the stria terminalis (BST) in the basal telencephaon; c) many parts of the hypothalamus; d) midline and medial parts of the thalamus; and e) the periaqueductal gray, ventral tegmental area, and midbrain raphé. The dorsal division of the MEA (the posterodorsal part) is characterized by projections to the principal nucleus of the BST, and to the anteroventral periventricular, medial, and central parts of the medial preoptic, and ventral premammillary hypothalamic nuclei. These hypothalamic nuclei project heavily to neuroendocrine and autonomic-related parts of the hypothalamic periventricular zone. The ventral division of the MEA (the anterodorsal, anteroventral, and posteroventral parts) is characterized by dense projections to the transverse and interfascicular nuclei of the BST, and to the lateral part of the medial preoptic, anterior hypothalamic, and ventromedial hypothalamic nuclei. However, dorsal regions of the ventral division provide rather dense inputs to the medial preoptic region and capsule of the ventromedial nucleus, whereas ventral regions of the ventral division preferentially innervate the anterior hypothalamic, dorsomedial, and ventral parts of the ventromedial nuclei. Functional evidence suggests that circuits associated with dorsal regions of the ventral division may deal with reproductive behavior, whereas circuits associated with ventral regions of the ventral division may deal preferentially with agonistic behavior.

Hormonal regulation of opioid peptide neurons in the anteroventral periventricular nucleus.

Steroid hormones provide a means of coordinating the activity of widespread neural systems that mediate endocrine, autonomic, and somatomotor aspects of reproductive processes that are essential for the propagation of mammalian species. Because these processes are quite different in each sex, the neural pathways that control them are also sexually differentiated. The anteroventral periventricular nucleus (AVPV) of the preoptic region occupies a nodal point in sexually dimorphic forebrain circuits and appears to play a critical role in regulating gonadotropin secretion. The AVPV contains sexually dimorphic populations of opioid peptide containing neurons that display different patterns of development and are differentially regulated in adult animals by gonadal steroids. Moreover, estrogen (ER) and progesterone (PR) receptors are expressed in AVPV neurons in a transmitter-specific way, and the expression of these nuclear transacting factors is differentially regulated by sex steroids. Thus, neurons in the AVPV show distinct patterns of hormonal regulation of gene expression, and distinct hormone receptor profiles.

Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat.

The organization of projections from the four parts of the ventromedial nucleus (VMH) and a ventrolaterally adjacent region tentatively identified as the tuberal nucleus (TU) have been analyzed with small injections of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Extrinsic and intranuclear projections of each part of the VMH display clear quantitative differences, whereas the overall patterns of outputs are qualitatively similar. Overall, the VMH establishes massive intrahypothalamic terminal fields in other parts of the medial zone, tending to avoid the periventricular and lateral zones. The ventrolateral VMH is more closely related to other parts of the hypothalamus that also express gonadal steroid hormone receptors, including the medial preoptic, tuberal, and ventral premammillary nuclei, whereas other parts of the VMH are more closely related to the anterior hypothalamic and dorsal premammillary nuclei. All parts of the VMH project to the zona incerta (including the A13 region) and parts of the midline thalamus, including the paraventricular and parataenial nuclei and nucleus reuniens. The densest inputs to the septum are to the bed nuclei of the stria terminalis, where the ventrolateral and central VMH innervate the anteroventral and anterodorsal areas and transverse and interfascicular nuclei, whereas the anterior and dorsomedial VMH innervate the latter two. The central, lateral, and medial amygdalar nuclei receive substantial inputs from various parts of the VMH. Other regions of the telencephalon, including the nucleus accumbens and the piriform-amygdaloid, infralimbic, prelimbic, anterior cingulate, agranular insular, piriform, perirhinal, entorhinal, and postpiriform transition areas, also receive sparse inputs. All parts of the VMH send a massive, topographically organized projection to the periaqueductal gray. Other brainstem terminal fields include the superior colliculus, peripeduncular area, locus coeruleus, Barrington's nucleus, parabrachial nucleus, nucleus of the solitary tract, and the mesencephalic, pontine, gigantocellular, paragigantocellular, and parvicellular reticular nuclei. The projections of the TU are similar to, and a subset of, those from the VMH and are thus not nearly as widespread as those from adjacent parts of the lateral hypothalamic area. Because of these similarities, the TU may eventually come to be viewed most appropriately as the lateral component of the VMH itself. The functional implications of the present findings are discussed in view of evidence that the VMH plays a role in the expression of ingestive, affective, and copulatory behaviors.

Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain.

POMC, the precursor of ACTH, MSH, and beta-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. We report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacologically distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting a role in the central control of pituitary function. MC4-R is also unique in its expression in numerous cortical and brainstem nuclei. Together, MC3-R and/or MC-4R mRNA are found in every nucleus reported to bind MSH in the adult rat brain and define neuronal circuitry known to be involved in the control of diverse neuroendocrine and autonomic functions. The high degree of conservation, distinct pharmacology, and unique neuronal distribution of the MC4 receptor suggest specific and complex roles for the melanocortin peptides in neuroendocrine and autonomic control.

Cell-type-specific expression of catecholamine transporters in the rat brain.

The dopamine transporter (DAT) and norepinephrine transporter (NET) terminate catecholaminergic neurotransmission at synapses by high-affinity sodium-dependent reuptake into presynaptic terminals, and are the initial sites of action for drugs of abuse and antidepressants. In the present study, we used in situ hybridization combined with immunohistochemistry to study the distribution of DAT and NET mRNA in the adult rat brain. Cells were first immunolabeled with antisera directed against one of the catecholamine-synthetic enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), or phenylethanolamine-N-methyltransferase (PNMT), in order to identify dopaminergic, noradrenergic, or epinephrine-containing cells. The immunolabeled cells were subsequently assayed for their ability to express catecholamine transporter mRNAs by in situ hybridization using either a rat DAT or NET cRNA probe. All dopaminergic cell groups of the mesencephalon contained high levels of DAT mRNA but only the A12 and A13 dopaminergic cell groups of the diencephalon appear to express detectable levels of DAT. All norepinephrine-containing cell bodies in the brainstem (locus coeruleus and lateral tegmentum) appear to express NET mRNA. In contrast, epinephrine-containing cell bodies of the brainstem do not appear to express NET mRNA, which raises the possibility that epinephrine may utilize a transporter that is distinct from the other bioactive amines, or may act as an endocrine regulator that does not require rapid reuptake mechanisms. Moreover, the cell-type-specific expression of catecholamine transporters suggests that DAT and NET gene expression may be closely linked to cellular mechanisms that specify transmitter phenotype. The termination of neurotransmission is a critical component of neural signaling and depends on the rapid removal of neurotransmitters from the synaptic cleft. Pharmacological evidence indicates that the action of monoamines at the synapse is terminated predominantly by rapid reuptake into presynaptic nerve endings via neurotransmitter-specific, high-affinity, Na(+)-dependent membrane transporter proteins. The cDNAs encoding distinct transporter proteins for the monoamines dopamine, norepinephrine, and serotonin have been cloned, expressed, and characterized in a variety of heterologous systems (Blakely et al., 1991; Giros et al., 1991; Hoffman et al., 1991; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991; Usdin et al., 1991). Although the monoamine transporters share a high degree of sequence homology, they are distinguished by their monoamine substrate specificities and by their differential sensitivities to a wide spectrum of transport antagonists. For example, pharmacological agents that potently inhibit norepinephrine and serotonin transport, such as desmethylimipramine and citalopram, have little effect on the activity of the dopamine transporter (Javitch et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)