Adenoviral vector-mediated rescue of the OMP-null phenotype in vivo.

The use of gene deletion by homologous recombination to determine gene or protein function has wide application in vertebrate neurobiology. An ideal complement to gene deletion would be subsequent gene replacement to demonstrate re-acquisition of function. Here we used an adenoviral vector to replace the olfactory marker protein (OMP) gene in olfactory receptor neurons of adult OMP-null mice and demonstrated the subsequent re-acquisition of function. Our results show that short-term expression of OMP restores the kinetics of electrophysiological responses of OMP-null mice to those of the control phenotype. This adenoviral-mediated rescue of the OMP-null phenotype is consistent with involvement of OMP in olfactory transduction.

Leptin sensitivity in the developing rat hypothalamus.

In adults, the adipocyte-derived hormone, leptin, regulates food intake and body weight principally via the hypothalamic arcuate nucleus (ARC). During early postnatal development, leptin functions to promote the outgrowth of neuronal projections from the ARC, whereas a selective insensitivity to the effects of leptin on food intake appears to exist. To investigate the mechanisms underlying the inability of leptin to regulate food intake during early development, leptin signaling was analyzed both in vitro using primary cultures of rat embryonic ARC neurones and in vivo by challenging early postnatal rats with leptin. In neuronal cultures, despite the presence of key components of the leptin signaling pathway, no detectable activation of either signal transducer and activator of transcription 3 or the MAPK pathways by leptin was detected. However, leptin down-regulated mRNA levels of proopiomelanocortin and neuropeptide Y and decreased somatostatin secretion. Leptin challenge in vivo at postnatal d (P) 7, P14, P21, and P28 revealed that, in contrast to adult and P28 rats, mRNA levels of neuropeptide Y, proopiomelanocortin, agouti-related peptide and cocaine- and amphetamine-regulated transcript were largely unaffected at P7, P14, and P21. Furthermore, leptin stimulation increased the suppressor of cytokine signaling-3 mRNA levels at P14, P21, and P28 in several hypothalamic nuclei but not at P7, indicating that selective leptin insensitivity in the hypothalamus is coupled to developmental shifts in leptin receptor signaling. Thus, the present study defines the onset of leptin sensitivity in the regulation of energy homeostasis in the developing hypothalamus.

The OMP-lacZ transgene mimics the unusual expression pattern of OR-Z6, a new odorant receptor gene on mouse chromosome 6: implication for locus-dependent gene expression.

Reporter gene expression in the olfactory epithelium of H-lacZ6 transgenic mice mimics the cell-selective expression pattern known for some odorant receptor genes. The transgene construct in these mice consists of the lacZ coding region, driven by the proximal olfactory marker protein (OMP) gene promoter, and shows expression in a zonally confined subpopulation of olfactory neurons. To address mechanisms underlying the odorant receptor-like expression pattern of the lacZ construct, we analyzed the transgene-flanking region and identified OR-Z6, the first cloned odorant receptor gene that maps to mouse chromosome 6. OR-Z6 bears the highest sequence similarity (85%) to a human odorant receptor gene at the syntenic location on human chromosome 7. We analyzed the expression pattern of OR-Z6 in olfactory tissues of H-lacZ6 mice and show that it bears strong similarities to that mapped for beta-galactosidase. Expression of both genes in olfactory neurons is primarily restricted to the same medial subregion of the olfactory epithelium. Axons from both neuronal subpopulations project to the same ventromedial aspect of the anterior olfactory bulbs. Furthermore, colocalization analyses in H-lacZ6 mice demonstrate that OR-Z6-reactive glomeruli receive axonal input from lacZ-positive neurons as well. These results suggest that the expression of both genes is coordinated and that transgene expression in H-lacZ6 mice is regulated by locus-dependent mechanisms.

Leptin-target neurones of the rat hypothalamus express somatostatin receptors.

Hypothalamic leptinoceptive neurones can be visualized by histochemical demonstration of leptin-induced nuclear translocation of the signalling molecule STAT3. We investigated the relationship of the leptinoceptive neurones to the somatostatin signalling system. With double-labelling immunohistochemistry, we studied the colocalization of leptin-activated transcription factor, STAT3, with somatostatin receptor subtypes, sst1, sst2A, sst2B, sst3 and sst4, or the neuropeptide itself, in the rat hypothalamus. Immunoreactivity for all the entities was widely distributed throughout the entire hypothalamus. Despite the wide distribution, only few cases of colocalization of somatostatin with leptin-activated STAT3 were detected in the paraventricular, arcuate and dorsomedial nuclei. A moderate to high degree of colocalization of nuclear STAT3 and all investigated subtypes of somatostatin receptors was found in the lateral and dorsal hypothalamic areas and in the dorsomedial hypothalamic nucleus. Immunoreactivity for sst1, sst2B and sst4 was present in STAT3-containing nuclei of the paraventricular, periventricular, arcuate and ventromedial hypothalamic neurones, as well as in the retrochiasmatic and posterior hypothalamic areas. Despite the wide distribution of sst2A in the rat hypothalamus, few events of colocalization with leptin-activated STAT3 were observed in the dorsomedial nucleus and in the lateral and dorsal hypothalamic areas only. Many leptin-responsive neurones of the dorsal, lateral, periarcuate, perifornical and posterior hypothalamic areas, as well as in the ventromedial and dorsomedial hypothalamic nuclei, displayed sst3 immunoreactivity at their neuronal cilia. These results provide strong anatomical evidence for the direct interaction of leptin and the somatostatin systems in neuroendocrine control loops such as the energy homeostasis, growth or stress response.

Somatostatin, a negative-regulator of central leptin action in the rat hypothalamus.

Leptin-responsive neurons of the hypothalamus constitute a heterogeneous population expressing a vast array of different neuropeptides and neurotransmitters, some of which participate in the regulation of hunger and satiety. Here we report that somatostatin modulates the efficacy of leptin-signalling in the rat hypothalamus. Using a two-pulse paradigm at 30-min intervals, we delivered somatostatin or somatostatin receptor subtype-selective agonists in combination with leptin into the lateral cerebral ventricle of stereotaxically cannulated rats. To monitor the effect of somatostatin on the leptin-signalling pathway, we quantified changes in the leptin-mediated activation of STAT3, the signal transducer and activator of transcription 3. Successive administration of somatostatin and leptin diminished the level of STAT3-phosphorylation and nuclear STAT3 translocation in the ventromedial and dorsomedial hypothalamic nuclei, the lateral hypothalamic area, and the arcuate nucleus by about 40% compared to leptin administration alone. Furthermore, application of subtype-selective somatostatin receptor agonists suggests that the observed reduction in leptin-responsiveness is predominantly mediated by the sst3 receptor-subtype, followed by sst1 and sst2. In addition, the intensity of the negative-regulatory effect of somatostatin on leptin-signalling displayed regional differences for the three receptor-subtypes involved. Addressing the functional consequences of the diminished leptin-signalling, behavioural analyses showed that centrally applied somatostatin counteracts the leptin-mediated suppression of food intake. These results suggest that the pleiotropic effector somatostatin also plays a role in the central regulation of energy homeostasis.