Differential effects of recombinant adeno-associated virus-mediated neuropeptide Y overexpression in the hypothalamic paraventricular nucleus and lateral hypothalamus on feeding behavior.

It is well known that neuropeptide Y (NPY) increases food intake. The hypothalamic paraventricular nucleus (PVN) and the lateral hypothalamus (LH) are both involved in the acute, hyperphagic effects of NPY. Although it is obvious that increased energy intake may lead to obesity, it is less understood which aspects of feeding behavior are affected and whether one or multiple neural sites mediate the effects of long-term increased NPY signaling. By long-term overexpressing NPY in either the PVN or the LH, we uncovered brain site-specific effects of NPY on meal frequency, meal size, and diurnal feeding patterns. In rats injected with adeno-associated virus-NPY in the PVN, increased food intake resulted from an increase in the amount of meals consumed, whereas in rats injected in the LH, increased food intake was attributable to increased meal size. Interestingly, food intake and body weight gain were only temporarily increased in PVN-injected rats, whereas in LH-injected rats hyperphagia and body weight gain remained for the entire 50 d. Moreover, in LH-NPY rats, but not in PVN-NPY rats, diurnal rhythmicity with regard to food intake and body core temperature was lost. These data clearly show that the NPY system differentially regulates energy intake and energy expenditure in the PVN and LH, which together adjust energy balance.

Viral mediated neuropeptide Y expression in the rat paraventricular nucleus results in obesity.

OBJECTIVE: Chronic central administration of neuropeptide Y (NPY) has dramatic effects on energy balance; however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. RESEARCH METHODS AND PROCEDURES: Recombinant adeno-associated viral particles containing NPY (rAAV-NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature, and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, agouti-related protein (AgRP), and pro-opiomelanocortin in the arcuate nucleus were studied. RESULTS: Food intake was increased specifically in the light period, and dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass; elevated plasma insulin, leptin, and adiponectin; decreased AgRP expression in the arcuate nucleus; and decreased insulin sensitivity; whereas plasma corticosterone was unaffected. DISCUSSION: These data suggest that increased NPY expression targeted at the PVN is sufficient to induce obesity. Interestingly, plasma concentrations of leptin and insulin were elevated before a rise in food intake, which suggests that NPY in the PVN influences leptin and insulin secretion independently from food intake. This strengthens the role of the PVN in regulation of energy balance by NPY.

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system.

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.

Anatomical demonstration of the suprachiasmatic nucleus-pineal pathway.

A polysynaptic pathway is proposed to transmit light information from the retina through the suprachiasmatic nucleus of the hypothalamus (SCN) to the pineal. In the present study, the powerful transneuronal tracer, pseudorabies virus (PRV), was used to provide a detailed description of this pathway. PRV injected into the pineal subsequently labeled the superior cervical ganglion, the intermediolateral column of the upper thoracic cord, the autonomic division of the paraventricular nucleus of the hypothalamus (PVN), and the SCN. Neurons in the autonomic division of the PVN were the only PRV-labeled neurons in the hypothalamus shown to receive input from the SCN as demonstrated by the presence of vasoactive intestinal polypeptide axonal contacts. This observation concurred with the presence of ventrally placed neurons in the SCN that could only be observed a day after the appearance of PVN-labeled neurons. Nevertheless the majority of the neurons were found in the dorsomedial position of the SCN, associated with the vasopressin-containing population of SCN neurons. Confocal laser scanning microscopy showed double-labeled neurons containing PRV and vasopressin or PRV and vasoactive intestinal polypeptide. Specificity of tracing was also established by prior removal of the superior cervical ganglion, resulting in a complete absence of the tracer but in the pineal. Thus, the present study provides the anatomical basis for circadian control of melatonin secretion.

Rescue of HSV-1 neurovirulence is associated with induction of brain interleukin-1 expression, prostaglandin synthesis and neuroendocrine responses.

Several HSV-1 neurovirulence genes have been mapped but the mechanisms by which they affect host-virus interactions are not known. We have previously mapped HSV-1 neurovirulence to the UL53 gene region of the viral DNA by transfer of this gene from the neurovirulent R-19 strain to the non-neurovirulent R-15 strain in the generation of the p-71 recombinant, in which neurovirulence was rescued. In the present study we inoculated these strains into the paraventricular nucleus (PVN) of the hypothalamus of rats. We examined: (1) Clinical course of encephalitis. (2) Hypothalamic-pituitary-adrenocortical (HPA) axis function. (3) Brain cytokine gene mRNA expression and prostaglandin E2 (PGE2) production. (4) The relation of these parameters to viral replication and to cellular inflammation. In R-15 infected rats no signs of disease were observed. There was a temporary inflammatory reaction and IL-1 beta transcription in the PVN area. The function of the HPA axis was similar to control rats. Only slight increase in brain PGE2 production was found. In R-19 and p-71 infected rats, overt clinical signs of encephalitis and cellular inflammation in the PVN area were observed within 3 days post-infection (p.i). All rats died between 4-7 days p.i. These strains induced IL-1 beta transcription in the hypothalamus as well as in extra-hypothalamic brain regions in which no cellular inflammation was found. Basal serum ACTH and' CS were markedly elevated and hypothalamic CRF-41 content was significantly reduced as compared to R-15 infected rats. Both strains markedly increased brain PGE2 production. HSV-1 brain titers at 3 days p.i. were 100-fold lower than the inoculum titer although clinical signs of encephalitis were prominent. The results suggest that rescue of HSV-1 neurovirulence by the UL53 gene region of the viral genome is associated with enhancement of viral-induced brain IL-1 beta gene expression, increased brain PGE2 synthesis and hypersecretion of HPA axis hormones. Viral-induced brain derived cytokines and prostaglandins may contribute to the clinical syndrome of acute herpetic encephalitis in particular at early stages of the disease when virus load in the brain is low and cellular infiltrates are not widespread.