Impact of Early Consumption of High-Fat Diet on the Mesolimbic Dopaminergic System.

Increasing evidence suggest that consumption of high-fat diet (HFD) can impact the maturation of brain circuits, such as during adolescence, which could account for behavioral alterations associated with obesity. In the present study, we used behavioral sensitization to amphetamine to investigate the effect of periadolescent HFD exposure (pHFD) in rats on the functionality of the dopamine (DA) system, a central actor in food reward processing. pHFD does not affect responding to an acute injection, however, a single exposure to amphetamine is sufficient to induce locomotor sensitization in pHFD rats. This is paralleled by rapid neurobiological adaptations within the DA system. In pHFD-exposed animals, a single amphetamine exposure induces an increase in bursting activity of DA cells in the ventral tegmental area (VTA) as well as higher DA release and greater expression of (tyrosine hydroxylase, TH) in the nucleus accumbens (NAc). Post-synaptically, pHFD animals display an increase in NAc D2 receptors and c-Fos expression after amphetamine injection. These findings highlight the vulnerability of DA system to the consumption of HFD during adolescence that may support deficits in reward-related processes observed in obesity.

Expression of GABA(A) receptor alpha3-, theta-, and epsilon-subunit mRNAs during rat CNS development and immunolocalization of the epsilon subunit in developing postnatal spinal cord.

Ionotropic GABA(A) receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA(A) receptors consist of alpha1-, beta2-, and gamma2-subunits that are co-expressed in numerous brain areas. alpha3-, theta-, And epsilon-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of epsilon and theta transcripts before birth, to a theta and alpha3 expression at birth, and finally to a grouped epsilon, theta and alpha3 expression postpartum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), epsilon and theta appeared to have a greater spatial distribution before the dominance of the alpha3 subunit transcript around birth. We also revealed expression of alpha3, theta, and epsilon in the developing spinal cord and identified neurons that express epsilon in the postnatal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of alpha3-, theta- and epsilon-subunits may be assembled at a regional and developmental level in the brain.

Immunocytochemical detection of cholecystokinin and corticotrophin-releasing hormone neuropeptides in the hypothalamic paraventricular nucleus of the jerboa (Jaculus orientalis): modulation by immobilisation stress.

The hypothalamic response to an environmental stress implicates the corticotrophin-releasing hormone (CRH) neuroendocrine system of the hypothalamic parvicellular paraventricular nucleus (PVN) in addition to other neuropeptides coexpressed within CRH neurones and controlling the hypothalamo-pituitary-adrenal (HPA) axis activity as well. Such neuropeptides are vasopressin, neurotensin and cholecystokinin (CCK). It has previously been demonstrated that the majority of the CRH neuronal population coexpresses CCK after a peripheral stress in rats. In the present study, we explored such neuroendocrine plasticity in the jerboa in captivity as another animal model. In particular, we studied CCK and CRH expression within the hypothalamic PVN by immunocytochemistry in control versus acute immobilisation stress-submitted jerboas. The results show that CCK- and CRH-immunoreactive neuronal systems are located in the hypothalamic parvicellular PVN. The number of CCK-immunoreactive neurones within the PVN was significantly increased (138% increase) in stressed animals compared to controls. Similarly, the number of CRH-containing neurones was higher in stressed jerboas (128%) compared to controls. These results suggest that the neurogenic stress caused by immobilisation stimulates CCK as well as CRH expression in jerboas, which correlates well with previous data obtained in rats using other stressors. The data obtained also suggest that, in addition to CRH, CCK is another neuropeptide involved in the response to stress in jerboa, acting by controlling HPA axis activity. Because CCK is involved in the phenotypical plasticity of CRH-containing neurones in response to an environmental stress, we also explored their coexpression by double immunocytochemistry within the PVN and the median eminence (i.e. the site of CRH and CCK corelease in the rat) following jerboa immobilisation. The results show that CCK is not coexpressed within CRH neurones in either control or stressed jerboa, suggesting differences between jerboas and rats in the neuroendocrine regulatory mechanisms of the stress response involving CRH and CCK. The adaptative physiological mechanisms to environmental conditions might vary from one mammal species to another.

Vasopressin-containing neurons of the hypothalamic parvocellular paraventricular nucleus of the jerboa: plasticity related to immobilization stress.

The corticotropin-releasing hormone (CRH) neurons of the hypothalamic parvocellular paraventricular nucleus (PVN) have a high potential for phenotypical plasticity, allowing them to rapidly modify their neuroendocrine output, depending upon the type of stressors. Indeed, these neurons coexpress other neuropeptides, such as cholecystokinin (CCK), vasopressin (VP), and neurotensin, subserving an eventual complementary function to CRH in the regulation of the pituitary. Unlike in rats, our previous data showed that in jerboas, CCK is not coexpressed within CRH neurons in control as well as stressed animals. The present study explored an eventual VP participation in the phenotypic plasticity of CRH neurons in the jerboa. We analyzed the VP expression within the PVN by immunocytochemistry in male jerboas submitted to acute stress. Our results showed that, contrary to CRH and CCK, no significant change concerned the number of VP-immunoreactive neurons following a 30-min immobilization. The VP/CRH coexpression within PVN and median eminence was investigated by double immunocytochemistry. In control as well as stressed animals, the CRH-immunopositive neurons coexpressed VP within cell bodies and terminals. No significant difference in the number of VP/CRH double-labeled cells was found between both groups. However, such coexpression was quantitatively more important into the posterior PVN as compared with the anterior PVN. This suggests an eventual autocrine/paracrine or endocrine role for jerboa parvocellular VP which is not correlated with acute immobilization stress. VP-immunoreactive neurons also coexpressed CCK within PVN and median eminence of control and stressed jerboas. Such coexpression was more important into the anterior PVN as compared with the posterior PVN. These results showed the occurrence of at least two VP neuronal populations within the jerboa PVN. In addition, the VP expression did not depend upon acute immobilization stress. These data highlight differences in the neuroendocrine regulatory mechanisms of the stress response involving CRH/CCK or VP. They also underline that adaptative physiological mechanisms to stress might vary from one mammal species to another.

Molecular and cellular properties of GnRH neurons revealed through transgenics in the mouse.

Recent advances in the use of gonadotropin-releasing hormone (GnRH) promoter-driven transgenics in the mouse are beginning to open up the once elusive GnRH neuronal phenotype to detailed molecular and cellular investigation. This review highlights progress in the development of GnRH promoter transgenic constructs and the understanding of murine gene sequences required for the correct temporal and spatial targeting of transgenes to the GnRH phenotype in vivo. Strategies enabling the identification of single, living GnRH neurons in the acute brain slice preparation are allowing gene profiling and electrophysiological experiments to be undertaken. Results so far indicate that, like other neurons, GnRH cells express a variety of sodium, potassium and calcium channels as well as GABAergic and glutamatergic receptors which are responsible for determining the membrane properties and firing characteristics of the GnRH neuron. Many of these receptors and channels appear to be expressed heterogeneously within the GnRH phenotype. Furthermore, several display distinct postnatal developmental expression profiles which are likely to be of consequence to the development of synchronized, pulsatile GnRH secretion in the adult animal.

Profiling gamma-aminobutyric acid (GABA(A)) receptor subunit mRNA expression in postnatal gonadotropin-releasing hormone (GnRH) neurons of the male mouse with single cell RT-PCR.

The present investigation has examined which subunits of the GABA(A) receptor are expressed by gonadotropin-releasing hormone (GnRH) neurons in the juvenile and adult male mouse. Cells of defined morphology, located in the medial septum (MS) and rostral preoptic area (POA), were patch-clamped in the acute brain slice preparation and their cell contents extracted. A reverse transcriptase polymerase chain reaction (RT-PCR) procedure using nested primers was used to establish individual GnRH mRNA-expressing cells which were then evaluated for eleven GABA(A) receptor (alpha1-5, beta1-3, gamma1-3) subunit transcripts. Single and multiple GABA(A) receptor subunit mRNAs were detected in approximately 70% of all GnRH neurons. A range of different subunit mRNAs (alpha1, alpha2, alpha5, beta1, beta2, beta3, gamma2) were found in juvenile GnRH neurons, with the alpha1gamma2 and alpha5gamma2 combinations encountered most frequently within individual cells. The expression profile in adult GnRH neurons was more extensive than that detected in juveniles with alpha1, alpha2, alpha3, alpha5, beta1, beta2, beta3, gamma1 and gamma2 subunits all being detected. The major difference in subunit profile between GnRH neurons located in the MS and POA involved the beta subunits. The principal postnatal developmental change was one of increasing overall subunit heterogeneity in maturing POA GnRH neurons. The profile of GABA(A) receptor subunit mRNAs detected in male GnRH neurons was quite different to that reported by us for female GnRH neurons in the mouse using the same RT-PCR approach. Together, these findings indicate that postnatal GnRH neurons are likely to express a range of GABA(A) receptor subunit mRNAs in a sexually dimorphic and developmentally-regulated manner.

New evidence for estrogen receptors in gonadotropin-releasing hormone neurons.

Estrogen exerts a critical regulatory influence upon the biosynthetic and secretory activity of the gonadotropin-releasing hormone (GnRH) neurons. It seems likely that estrogen regulates the behavior of the GnRH neuron through multiple transsynaptic, neuronal-glial, and direct membrane modes of action. Advances in our understanding of these mechanisms over the last 3 years are highlighted. In addition, very recent studies have begun to provide evidence for the expression of estrogen receptors (ERs) in GnRH neurons in the rodent. Although not yet firmly established, the current consensus supports the hypothesis that GnRH neurons express ERbeta. Evidence exists for ERbeta mRNA expression by GnRH neurons throughout development and ERbeta immunoreactivity has now also been detected in these cells. Murine GnRH neurons have further been shown to express estrogen receptor-related receptor-alpha, an orphan receptor thought to constitutively activate estrogen response elements. Together, these findings provide a cornerstone for the reassessment of the role of ERs and related receptors in the direct genomic and potential nontranscriptional actions of estrogen upon the GnRH neuron.

Late postnatal reorganization of GABA(A) receptor signalling in native GnRH neurons.

The molecular and cellular characteristics of the gonadotropin-releasing hormone (GnRH) neurons have been difficult to ascertain due to their scattered distribution within the basal forebrain. Using morphological criteria coupled with single cell RT-PCR postidentification, we have developed a method for investigating native GnRH neurons in the mouse brain and used it to examine the development of GABA(A) receptor signalling in this phenotype. Following the harvesting of the cytoplasmic contents of individual GnRH neurons, single cell multiplex RT-PCR experiments demonstrated that GABAA receptor alpha1-5, beta1-3 and gamma2 & 3 subunit transcripts were expressed by both neonatal (postnatal day 5) and juvenile (day 15-20) GnRH neurons in a heterogeneous manner. Following puberty, this profile was reduced to a predominant alpha1, alpha5, beta1, gamma2 subunit complement in rostral preoptic area GnRH neurons of the adult female. Whole-cell patch-clamp recordings revealed little difference between juvenile and adult GnRH neurons in their resting membrane potential and spontaneous firing rates. All GnRH neurons were found to be subjected to a tetrodotoxin-insensitive, tonic GABAergic barrage signalling through the GABA(A) receptor. However, marked heterogeneity in the sensitivity of individual juvenile GnRH neurons to GABA was revealed and, in parallel with the change in subunit mRNA profile, this was dramatically reduced in the reproductively competent adult GnRH neurons. These findings provide the first electrical and molecular characterization of the GnRH phenotype and demonstrate a novel pattern of late postnatal reorganization of native GABA(A) receptor gene expression and signalling in the GnRH neuronal population.

Transgenics identify distal 5'- and 3'-sequences specifying gonadotropin-releasing hormone expression in adult mice.

GnRH neurons play a critical role in regulating gonadotropin secretion, but their scattered distribution has prevented detailed understanding of their molecular and cellular properties in vivo. Using GnRH promoter-driven transgenics we have examined here the role of 5'- and 3'-murine GnRH sequences in specifying GnRH expression in the adult mouse. Transgenic mice bearing a lacZ construct incorporating 5.5 kb of 5'-, all the introns and exons, and 3.5 kb of 3'-murine GnRH sequence were found to express beta-galactosidase (betagal) immunoreactivity in approximately 85% of all GnRH neurons. Deletion of GnRH sequence 3' to exon II had no effect upon transgene expression in the GnRH population (89%) but resulted in the appearance of ectopic betagal immunoreactivity in several regions of the brain. The production of additional mice in which 5'-elements were deleted to leave only -2.1 kb of sequence resulted in an approximately 40% reduction in the number of GnRH neurons expressing betagal. Mice in which further deletion of 400 bp allowed only -1.7 kb of 5'-sequence to remain exhibited a complete absence of betagal immunoreactivity within GnRH and other neurons. These results suggest that elements 3' to exon II of the GnRH gene have little role in enabling GnRH expression within the GnRH phenotype but, instead, are particularly important in repressing the GnRH gene in non-GnRH neurons. In contrast, elements located between -2.1 and -1.7 kb of distal 5'-sequence appear to be critical for the in vivo activation of GnRH expression within GnRH neurons in the adult brain.

Suckling-induced changes in neuropeptide Y and proopiomelanocortin gene expression in the arcuate nucleus of the rat: evaluation of a putative intervention of prolactin.

Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC)-derived peptides located in the arcuate nucleus (ARC) have been postulated to be good candidates to play a modulatory role during lactation. In the present study, we first quantified, by in situ hybridization, lactation-induced changes in NPY and POMC gene expression throughout the ARC. In a second phase of the study, we attempted to determine whether any relationship exists between neuropeptide gene expression and the suckling stimulus itself. For this, we used experimental groups of animals submitted to suppression of the suckling stimulus by removal of pups and the subsequent restoration of the suckling stimulus by the return of the litter. Since lactation is characterized by an estrogen-deficient status [15], we attempted using ovariectomized (2 or 21 days) diestrous females to describe the changes in NPY gene expression observed during lactation. Since the suckling stimulus induces a strong prolactin (PRL) release, we completed this study by using an intravenous injection of PRL antiserum in order to discriminate the effects of PRL per se on the observed suckling-induced changes in neuropeptide gene expression. Freely nursing lactating females exhibited a large increase in NPY mRNA expression as compared to diestrous females (10.10 +/- 0.50 vs. 4.51 +/- 0.35). After suppression of the suckling stimulus by removal of pups, this increase intensified during short-term suppression of 16 h (15.37 +/- 0.67) and was reversed following long-term suppression of 36 h (12.35 +/- 0.61). Ovariectomized diestrous animals showed significant changes in NPY mRNA expression as compared to lactating females (5.25 +/- 0.42 vs. 10.10 +/- 0.50). Lactating females submitted to PRL immunoneutralization by PRL antiserum showed a slight increase in NPY mRNA expression as compared to non-injected lactating females (13.75 +/- 0.51 vs. 12.95 +/- 0.59). Freely nursing lactating females showed a decrease in POMC mRNA expression (8.27 +/- 0.33) whereas suppression of suckling by removal of the pups (9.52 +/- 0.45) resulted in a return to diestrous POMC mRNA levels (10.77 +/- 0.36). We showed that restoration of suckling by the return of the litter induced an increase in POMC gene expression (12.55 +/- 0.66). By lowering circulating levels of PRL with PRL antiserum after restoration of suckling, we observed a decrease in POMC mRNA expression (9.81 +/- 0.46). Results of this study showed that the increase in NPY mRNA in the medial ARC during lactation did not appear to be due either to gonadal steroid-deficient status or to the suckling-induced hyperprolactinemia. If freely nursing lactating females showed a moderate decrease in POMC gene expression, restoration of the suckling stimulus by return of the pups provoked an increase in POMC gene expression which seemed to depend on high endogenous levels of PRL.

Suckling-induced Fos-immunoreactivity in subgroups of hypothalamic POMC neurons of the lactating rat: investigation of a role for prolactin.

Attention has recently been focused on lactation-induced modifications of activity of neuronal populations in the arcuate nucleus (ARC) of the mediobasal hypothalamus. The ARC hosts the tubero-infundibular dopaminergic (TIDA system) responsible for the neuroendocrine control of prolactin (PRL), and other non-neuroendocrine neuronal populations, such as neuropeptide Y (NPY)- and proopiomelanocortin (POMC)-containing systems that are important modulators of hypothalamic gonadoliberin (GnRH) secretion. Our longstanding interest in the functional anatomy of the ARC led us to investigate whether the suckling stimulus would trigger an expression of Fos-ir in specific arcuate neuronal populations and to possibly characterize responsive neurons by using double-labeling immunohistochemistry. Freely nursing lactating females expressed strong Fos-ir in neurons of the ARC compared to diestrous females. Fos-ir was encountered in neurons not belonging to the TIDA system and that was for a large proportion identical to the POMCergic neurons. We showed that, in lactating females submitted to suppression of the suckling stimulus by removal of the pups, the pattern of expression of Fos-ir is similar to that seen in diestrous females and that, a pattern of expression of Fos-ir indistinguishable from that observed during free lactation is reinstated a short time after the return of the pups and restoration of the suckling stimulus, suggesting that this expression of Fos-ir strictly depends upon the presence of the newborns and the suckling stimulus. By lowering circulating levels of the PRL with bromocryptine-or PRL antiserum-treatment, we noticed a decrease in the number of (beta-endorphin + Fos)-ir neurons compared to non-injected freely nursing lactating females. By maintaining high levels of circulating PRL with haloperidol-treatment, we observed a number of colocalizations close to that observed in freely nursing lactating females. Our results suggest that during lactation a rostral subgroup of the arcuate POMCergic neuronal population is activated at least partially in response to the suckling-induced secretion of PRL and that this activation participates in maintaining the endocrine and/or metabolic demands of the lactational status.