Locus coeruleus mediates cold stress-induced polycystic ovary in rats

Previous reports about the rat ovary have shown that cold stress promotes ovarian morphological alterations related to a polycystic ovary (PCO) condition through activation of the ovarian sympathetic nerves. Because the noradrenergic nucleus locus coeruleus (LC) is activated by cold stress and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway, this study aimed to evaluate the LC`s role in cold stress-induced PCO in rats. Ovarian morphology and endocrine and sympathetic functions were evaluated after 8 wk of chronic intermittent cold stress (4 C, 3 h/d) in rats with or without LC lesion. The effect of acute and chronic cold stress upon the LC neuron activity was confirmed by Fos protein expression in tyrosine hydroxylase-immunoreactive neurons. Cold stress induced the formation of follicular cysts, type III follicles, and follicles with hyperthecosis alongside increased plasma estradiol and testosterone levels, irregular estrous cyclicity, and reduced ovulation. Considering estradiol release in vitro, cold stress potentiated the ovarian response to human chorionic gonadotropin. Ovarian norepinephrine (NE) was not altered after 8 wk of stress. However, LC lesion reduced NE activity in the ovary of cold-stressed rats, but not in controls, and prevented all the cold stress effects evaluated. Cold stress increased the number of Fos/tyrosine hydroxylase-immunoreactive neurons in the LC, but this effect was more pronounced for acute stress as compared with chronic stress. These results show that cold stress promotes PCO in rats, which apparently depends on ovarian NE activity that, under this condition, is regulated by the noradrenergic nucleus LC.

Ovarian-Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (W) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity. (C) 2009 Elsevier B.V. All rights reserved.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

Melanopsin and Clock Genes: Regulation by Light and Endothelin in the Zebrafish ZEM-2S Cell Line

It is well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In the teleost, Danio rerio, peripheral oscillators can be directly synchronized by light. Danio rerio ZEM-2S embryonic cells respond to light with differential growth: cells kept in constant light exhibited a strong inhibition of proliferation, whereas in cells kept in light:dark (LD) cycles (14L:10D and 10L:14D) or in constant darkness (DD), the doubling times were not statistically different. We demonstrated by RT-PCR followed by PCR that ZEM-2S cells express two melanopsins, Opn4x and Opn4m, and the six Cry genes. The presence of the protein OPN4x was demonstrated by immunocytochemistry. The pattern of temporal expression of the genes Opn4x, Per1, Cry1b, and Clock was studied in ZEM-2S cells kept for five days in 12L:12D or DD. In 12L:12D, the clock genes Per 1 and Cry1b exhibited robust circadian expression, while Opn4x and Clock expression seemed to vary in an ultradian pattern. Both Per1 and Cry1b genes had higher expression during the L phase; Clock gene had an increase in expression coincident with the D phase, and during the subjective night. In DD, the temporal variation of Per1 and Cry1b genes was greatly attenuated but not extinguished, and the higher expressions were shifted to the transition times between subjective day and night, demonstrating that Per and Cry1b were synchronized by the LD cycle. Clock and Opn4x kept the ultradian oscillation, but the rhythm was not statistically significant. As endothelins (ET) have been reported to be a potent stimulator of Per genes in rodents, we investigated the effect of endothelin on ZEM-2S cells, which express ETA receptors. Cells were kept in 12D:12L for five days, and then treated with 10-11 to 10-8M ET-1 for 24h. ET-1 exhibited a biphasic effect on Opn4x expression. At 10-11M, the hormone exerted a highly significant stimulation of Opn4x expression during the L phase and introduced a circadian oscillatory pattern. At 10-10M, a significant increase was seen at ZT21 and ZT0 (i.e., at the end of the D phase and beginning of the L phase), whereas 10-9 and 10-8M ET-1 inhibited the expression of Opn4x at most ZTs. Clock expression was unaffected by 10-8M ET-1; however, in the presence of lower concentrations, the expression was enhanced at some ZTs, strengthening the ultradian oscillation. ET-1 at 10-11 and 10-10M had no effect on Per1 circadian expression; however, 10-9 and 10-8M ET-1 reduced the amplitude of Per1 expression in the beginning of the L phase. ET-1 effects were less evident on Cry 1b. For both genes, the reduction in expression was not sufficient to abolish the circadian oscillatory pattern. Based on these results and data in the literature, a link between ET-1 stimulation of ETA receptors may be established by E4BP4 binding to the promoters and consequent inhibition of gene expression.

EXPRESSION OF COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT IN THE RAT FOREBRAIN DURING POSTNATAL DEVELOPMENT

Cocaine- and amphetamine-regulated transcript (CART) is widespread in the rodent brain. CART has been implicated in many different functions including reward, feeding, stress responses, sensory processing, learning and memory formation. Recent studies have suggested that CART may also play a role in neural development. Therefore, in the present study we compared the distribution pattern and levels of CART mRNA expression in the forebrain of male and female rats at different stages of postnatal development: P06, P26 and P66. At 6 days of age (P06), male and female rats showed increased CART expression in the somatosensory and piriform cortices, indusium griseum, dentate gyrus, nucleus accumbens, and ventral premammillary nucleus. Interestingly, we found a striking expression of CART mRNA in the ventral posteromedial and ventral posterolateral thalamic nuclei. This thalamic expression was absent at P26 and P66. Contrastingly, at P06 CART mRNA expression was decreased in the arcuate nucleus. Comparing sexes, we found increased CART mRNA expression in the anteroventral periventricular nucleus of adult females. In other regions including the CA1, the lateral hypothalamic area and the dorsomedial nucleus of the hypothalamus, CART expression was not different comparing postnatal ages and sexes. Our findings indicate that CART gene expression is induced in a distinct temporal and spatial manner in forebrain sites of male and female rats. They also suggest that CART peptide participate in the development of neural pathways related to selective functions including sensory processing, reward and memory formation. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

Forebrain projections to brainstem nuclei involved in the control of mandibular movements in rats

Mandibular movements occur through the triggering of trigeminal motoneurons. Aberrant movements by orofacial muscles are characteristic of orofacial motor disorders, such as nocturnal bruxism (clenching or grinding of the dentition during sleep). Previous studies have suggested that autonomic changes occur during bruxism episodes. Although it is known that emotional responses increase jaw movement, the brain pathways linking forebrain limbic nuclei and the trigeminal motor nucleus remain unclear. Here we show that neurons in the lateral hypothalamic area, in the central nucleus of the amygdala, and in the parasubthalamic nucleus, project to the trigeminal motor nucleus or to reticular regions around the motor nucleus (Regio h) and in the mesencephalic trigeminal nucleus. We observed orexin co-expression in neurons projecting from the lateral hypothalamic area to the trigeminal motor nucleus. In the central nucleus of the amygdala, neurons projecting to the trigeminal motor nucleus are innervated by corticotrophin-releasing factor immunoreactive fibers. We also observed that the mesencephalic trigeminal nucleus receives dense innervation from orexin and corticotrophin-releasing factor immunoreactive fibers. Therefore, forebrain nuclei related to autonomic control and stress responses might influence the activity of trigeminal motor neurons and consequently play a role in the physiopathology of nocturnal bruxism.

Expression Profile and Distribution of Efhc1 Gene Transcript During Rodent Brain Development

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Leptin Targets in the Mouse Brain

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin`s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRbI-RES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518-532, 2009. (C) 2009 Wiley-Liss, Inc.