Optimization of whole-brain rabies virus tracing technology for small cell populations.

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Hypothalamic dopamine signaling regulates brown fat thermogenesis.

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.

Hypothalamic kappa opioid receptor mediates both diet-induced and melanin concentrating hormone-induced liver damage through inflammation and endoplasmic reticulum stress.

UNLABELLED: The opioid system is widely known to modulate the brain reward system and thus affect the behavior of humans and other animals, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and adenoviral vectors overexpressing or silencing κOR were stereotaxically delivered in the lateral hypothalamic area (LHA) of rats. Vagal denervation was performed to assess its effect on liver metabolism. Endoplasmic reticulum (ER) stress was inhibited by pharmacological (tauroursodeoxycholic acid) and genetic (overexpression of the chaperone glucose-regulated protein 78 kDa) approaches. The peripheral effects on lipid metabolism were assessed by histological techniques and western blot. We show that in the LHA κOR directly controls hepatic lipid metabolism through the parasympathetic nervous system, independent of changes in food intake and body weight. κOR colocalizes with melanin concentrating hormone receptor 1 (MCH-R1) in the LHA, and genetic disruption of κOR reduced melanin concentrating hormone-induced liver steatosis. The functional relevance of these findings was given by the fact that silencing of κOR in the LHA attenuated both methionine choline-deficient, diet-induced and choline-deficient, high-fat diet-induced ER stress, inflammation, steatohepatitis, and fibrosis, whereas overexpression of κOR in this area promoted liver steatosis. Overexpression of glucose-regulated protein 78 kDa in the liver abolished hypothalamic κOR-induced steatosis by reducing hepatic ER stress. CONCLUSIONS: This study reveals a novel hypothalamic-parasympathetic circuit modulating hepatic function through inflammation and ER stress independent of changes in food intake or body weight; these findings might have implications for the clinical use of opioid receptor antagonists. (Hepatology 2016;64:1086-1104).

Diet-induced neuropeptide expression: feasibility of quantifying extended and highly charged endogenous peptide sequences by selected reaction monitoring.

Understanding regulation and action of endogenous peptides, especially neuropeptides, which serve as inter- and intracellular signal transmitters, is key in understanding a variety of functional processes, such as energy balance, memory, circadian rhythm, drug addiction, etc. Therefore, accurate and reproducible quantification of these bioactive endogenous compounds is highly relevant. The biosynthesis of endogenous peptides, involving multiple possible trimming and modification events, hinders the de novo prediction of the active peptide sequences, making MS-based measurements very valuable in determining the actual active compounds. Here, we report an extended selected reaction monitoring (SRM)-based strategy to reproducibly and quantitatively monitor the abundances of a set of 15 endogenously occurring peptides from Rattus norvegicus hypothalamus. We demonstrate that SRM can be extended toward reproducible detection and quantification of peptides, bearing characteristics very different from tryptic peptides. We show that long peptide sequences, producing precursors with up to five and MS2 fragment ions with up to three charges, can be targeted by SRM on a triple quadrupole instrument. Using this approach to quantify endogenous peptide levels in hypothalami of animals subjected to different diets revealed several significant changes, most notably the significant upregulation of VGF-derived signaling peptide AQEE-30 upon high caloric feeding.

Anticipation of meals during restricted feeding increases activity in the hypothalamus in rats.

Rats exposed to timed restricted meals develop anticipation of food. They increase their activity levels in the hours preceding food access; this has been described as food-anticipatory activity (FAA). In the present study, we show the involvement of regions of the hypothalamus [arcuate nucleus, dorsomedial hypothalamus (DMH) and lateral hypothalamus] in the early development of FAA in rats exposed to the activity-based anorexia (ABA) model. We thereby used two different paradigms, rats exposed to the ABA model (ABA-normal) and rats exposed to the same restraint in food access but on a random feeding schedule (ABA-random). The latter group of rats were not able to anticipate food. We found a strong correlation between the expression of food anticipation measured by running-wheel activity and Fos expression levels in the DMH of ABA-normal rats, whereas no correlation was found in ABA-random rats. In contrast, in the randomly fed ABA rats only, a strong negative correlation was found between the neuronal activity in the hypothalamic area and the percentage body weight loss. Interestingly, these results imply that anticipation of meals during food restriction more strongly affects activation in the hypothalamus than negative energy balance alone. We conclude that during the early stages of development of FAA, the DMH plays a role in anticipation of food during periods of negative energy balance.

An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector.

BACKGROUND: This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 x 108 or 1 x 109 genomic copies of AAV1-GFP and 1 x 105 transducing units of LV-dsRed. RESULTS: Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. CONCLUSIONS: This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

Optimization of adeno-associated viral vector-mediated gene delivery to the hypothalamus.

To efficiently deliver genes and short hairpin RNAs to the hypothalamus we aimed to optimize the transduction efficiency of adeno-associated virus (AAV) in the rat hypothalamus. We compared the transduction efficiencies of AAV2 vectors pseudotyped with AAV1, AAV8, and mosaic AAV1/2 and AAV2/8 coats with that of an AAV2 coated vector after injection into the lateral hypothalamus of rats. In addition, we determined the transduction areas and the percentage of neurons infected after injection of various titers and volumes of two AAV1-pseudotyped vectors in the paraventricular hypothalamus (PVN). Successful gene delivery to the hypothalamus was achieved with AAV1-pseudotyped AAV vectors. The optimal approach to transduce an area, with the size of the PVN, was to inject 1 x 10(9) genomic copies of an AAV1-pseudotyped vector in a volume of 1 microl. At a radius of 0.05 mm from the injection site almost all neurons were transduced. In addition, overexpression of AgRP with the optimal approach resulted in an increase in food intake and body weight when compared with AAV-GFP.

Hypothalamic neuropeptide expression of juvenile and middle-aged rats after early postnatal food restriction.

Rats subjected to early postnatal food restriction (FR) show persistent changes in energy balance. The hypothalamus plays a major role in the regulation of energy balance. Therefore, we hypothesized that early postnatal food restriction induces developmental programming of hypothalamic gene expression of neuropeptides involved in this regulation. In the hypothalamus of juvenile and middle-aged rats that were raised in control (10 pups) or FR litters (20 pups), gene expression was investigated for neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC); CRH and TRH in the paraventricular nucleus; and melanin-concentrating hormone (MCH) and orexin in the lateral hypothalamic area. Early postnatal FR acutely and persistently reduced body size. Juvenile FR rats had significantly reduced CART gene expression and increased MCH expression. In middle-aged FR rats, POMC and CART mRNA levels were significantly reduced. The ratio between expression of the ARC orexigenic peptides (NPY and AgRP) and anorexigenic peptides (POMC and CART) was increased in juvenile, but not in middle-aged, FR rats. These results suggest that in neonatal rats, FR already triggers the ARC, and to a lesser extent the lateral hypothalamic area, but not the paraventricular nucleus, to increase expression of orexigenic relative to anorexigenic peptides. In addition, with enduring small body size and normalized hypothalamic gene expression, the adult FR rats appeared to have accepted this smaller body size as normal. This suggests that the body weight set-point was differently programmed in animals with early postnatal FR.

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.

Induction of brain-region-specific forms of obesity by agouti.

Disruption of melanocortin (MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic region with a high density of MC receptors, induced acute onset hyperphagia and rapid weight gain that persisted for at least 6 weeks. In contrast, obesity and hyperphagia developed with a 3 week delay when Agouti was expressed in the dorsal medial hypothalamus. Agouti expression in the lateral hypothalamus (LH) did not affect food intake and body weight during regular diet, despite the presence of MC receptors in this region. However, during exposure to a high-fat diet, animals with Agouti expression in the LH exhibited a marked increase in body weight. Here we show that the LH is important for the protection against diet-induced obesity by controlling caloric intake during consumption of a high-fat diet. Together, this study provides evidence that different aspects of the Agouti-induced obesity syndrome, such as hyperphagia and diet responsiveness, are mediated by distinct brain regions and opens challenging opportunities for further understanding of pathophysiological processes in the development of the obesity syndrome.