Reducing neuronal nitric oxide synthase (nNOS) expression in the ventromedial hypothalamus (VMH) increases body weight and blood glucose levels while decreasing physical activity in female mice.

Glucose-inhibited (GI) neurons of the ventromedial hypothalamus (VMH) depend on neuronal nitric oxide synthase (nNOS) and AMP-activated protein kinase (AMPK) for activation in low glucose. The Lopez laboratory has shown that the effects of estrogen on brown fat thermogenesis and white fat browning require inhibition of VMH AMPK. This effect of estrogen was mediated by downstream lateral hypothalamus (LH) orexin neurons 1,2 . We previously showed that estrogen inhibits activation of GI neurons in low glucose by inhibiting AMPK 3 . Thus, we hypothesized that VMH AMPK- and nNOS-dependent GI neurons project to and inhibit orexin neurons. Estrogen inhibition of AMPK in GI neurons would then disinhibit orexin neurons and stimulate brown fat thermogenesis and white fat browning, leading to decreased body weight. To test this hypothesis, we reduced VMH nNOS expression using nNOS shRNA in female mice and measured body weight, adiposity, body temperature, white and brown fat uncoupling protein (UCP1; an index of thermogenesis and browning), locomotor activity, and blood glucose levels. Surprisingly, we saw no effect of reduced VMH nNOS expression on body temperature or UCP1. Instead, body weight and adiposity increased by 30% over 2 weeks post injection of nNOS shRNA. This was associated with increased blood glucose levels and decreased locomotor activity. We also found that VMH nNOS-GI neurons project to the LH. However, stimulation of VMH-LH projections increased excitatory glutamate input onto orexin neurons. Thus, our data do not support our original hypothesis. Excitation of orexin neurons has previously been shown to increase physical activity, leading to decreased blood glucose and body weight 4 . We now hypothesize that VMH nNOS-GI neurons play a role in this latter function of orexin neurons.

First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.

Lateral hypothalamus hypocretin/orexin glucose-inhibited neurons promote food seeking after calorie restriction.

OBJECTIVE: The present study tests the hypothesis that changes in the glucose sensitivity of lateral hypothalamus (LH) hypocretin/orexin glucose-inhibited (GI) neurons following weight loss leads to glutamate plasticity on ventral tegmental area (VTA) dopamine neurons and drives food seeking behavior. METHODS: C57BL/6J mice were calorie restricted to a 15% body weight loss and maintained at that body weight for 1 week. The glucose sensitivity of LH hypocretin/orexin GI and VTA dopamine neurons was measured using whole cell patch clamp recordings in brain slices. Food seeking behavior was assessed using conditioned place preference (CPP). RESULTS: 1-week maintenance of calorie restricted 15% body weight loss reduced glucose inhibition of hypocretin/orexin GI neurons resulting in increased neuronal activation with reduced glycemia. The effect of decreased glucose on hypocretin/orexin GI neuronal activation was blocked by pertussis toxin (inhibitor of G-protein coupled receptor subunit Gαi/o) and Rp-cAMP (inhibitor of protein kinase A, PKA). This suggests that glucose sensitivity is mediated by the Gαi/o-adenylyl cyclase-cAMP-PKA signaling pathway. The excitatory effect of the hunger hormone, ghrelin, on hcrt/ox neurons was also blocked by Rp-cAMP suggesting that hormonal signals of metabolic status may converge on the glucose sensing pathway. Food restriction and weight loss increased glutamate synaptic strength (indexed by increased AMPA/NMDA receptor current ratio) on VTA dopamine neurons and the motivation to seek food (indexed by CPP). Chemogenetic inhibition of hypocretin/orexin neurons during caloric restriction and weight loss prevented these changes in glutamate plasticity and food seeking behavior. CONCLUSIONS: We hypothesize that this change in the glucose sensitivity of hypocretin/orexin GI neurons may drive, in part, food seeking behavior following caloric restriction.

The Antinarcolepsy Drug Modafinil Reverses Hypoglycemia Unawareness and Normalizes Glucose Sensing of Orexin Neurons in Male Mice.

Perifornical hypothalamus (PFH) orexin glucose-inhibited (GI) neurons that facilitate arousal have been implicated in hypoglycemia awareness. Mice lacking orexin exhibit narcolepsy, and orexin mediates the effect of the antinarcolepsy drug modafinil. Thus, hypoglycemia awareness may require a certain level of arousal for awareness of the sympathetic symptoms of hypoglycemia (e.g., tremors, anxiety). Recurrent hypoglycemia (RH) causes hypoglycemia unawareness. We hypothesize that RH impairs the glucose sensitivity of PFH orexin GI neurons and that modafinil normalizes glucose sensitivity of these neurons and restores hypoglycemia awareness after RH. Using patch-clamp recording, we found that RH enhanced glucose inhibition of PFH orexin GI neurons in male mice, thereby blunting activation of these neurons in low-glucose conditions. We then used a modified conditioned place preference behavioral test to demonstrate that modafinil reversed hypoglycemia unawareness in male mice after RH. Similarly, modafinil restored normal glucose sensitivity to PFH orexin GI neurons. We conclude that impaired glucose sensitivity of PFH orexin GI neurons plays a role in hypoglycemia unawareness and that normalizing their glucose sensitivity after RH is associated with restoration of hypoglycemia awareness. This suggests that the glucose sensitivity of PFH orexin GI neurons is a therapeutic target for preventing hypoglycemia unawareness.

Insulin actions on hypothalamic glucose-sensing neurones.

Subsequent to the discovery of insulin 100 years ago, great strides have been made in understanding its function, especially in the brain. It is now clear that insulin is a critical regulator of the neuronal circuitry controlling energy balance and glucose homeostasis. This review focuses on the effects of insulin and diabetes on the activity and glucose sensitivity of hypothalamic glucose-sensing neurones. We highlight the role of electrophysiological data in understanding how insulin regulates glucose-sensing neurones. A brief introduction describing the benefits and limitations of the major electrophysiological techniques used to investigate glucose-sensing neurones is provided. The mechanisms by which hypothalamic neurones sense glucose are discussed with an emphasis on those glucose-sensing neurones already shown to be modulated by insulin. Next, the literature pertaining to how insulin alters the activity and glucose sensitivity of these hypothalamic glucose-sensing neurones is described. In addition, the effects of impaired insulin signalling during diabetes and the ramifications of insulin-induced hypoglycaemia on hypothalamic glucose-sensing neurones are covered. To the extent that it is known, we present hypotheses concerning the mechanisms underlying the effects of these insulin-related pathologies. To conclude, electrophysiological data from the hippocampus are evaluated aiming to provide clues regarding how insulin might influence neuronal plasticity in glucose-sensing neurones. Although much has been accomplished subsequent to the discovery of insulin, the work described in our review suggests that the regulation of central glucose sensing by this hormone is both important and understudied.

Ventromedial hypothalamus glucose-inhibited neurones: A role in glucose and energy homeostasis?

The ventromedial hypothalamus (VMH) plays a complex role in glucose and energy homeostasis. The VMH is necessary for the counter-regulatory response to hypoglycaemia (CRR) that increases hepatic gluconeogenesis to restore euglycaemia. On the other hand, the VMH also restrains hepatic glucose production during euglycaemia and stimulates peripheral glucose uptake. The VMH is also important for the ability of oestrogen to increase energy expenditure. This latter function is mediated by VMH modulation of the lateral/perifornical hypothalamic area (lateral/perifornical hypothalamus) orexin neurones. Activation of VMH AMP-activated protein kinase (AMPK) is necessary for the CRR. By contrast, VMH AMPK inhibition favours decreased basal glucose levels and is required for oestrogen to increase energy expenditure. Specialised VMH glucose-sensing neurones confer the ability to sense and respond to changes in blood glucose levels. Glucose-excited (GE) neurones increase and glucose-inhibited (GI) neurones decrease their activity as glucose levels rise. VMH GI neurones, in particular, appear to be important in the CRR, although a role for GE neurones cannot be discounted. AMPK mediates glucose sensing in VMH GI neurones suggesting that, although activation of these neurones is important for the CRR, it is necessary to silence them to lower basal glucose levels and enable oestrogen to increase energy expenditure. In support of this, we found that oestrogen reduces activation of VMH GI neurones in low glucose by inhibiting AMPK. In this review, we present the evidence underlying the role of the VMH in glucose and energy homeostasis. We then discuss the role of VMH glucose-sensing neurones in mediating these effects, with a strong emphasis on oestrogenic regulation of glucose sensing and how this may affect glucose and energy homeostasis.

Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo.

Autophagy-lysosome pathway (ALP) disruption is considered pathogenic in multiple neurodegenerative diseases; however, current methods are inadequate to investigate macroautophagy/autophagy flux in brain in vivo and its therapeutic modulation. Here, we describe a novel autophagy reporter mouse (TRGL6) stably expressing a dual-fluorescence-tagged LC3 (tfLC3, mRFP-eGFP-LC3) by transgenesis selectively in neurons. The tfLC3 probe distributes widely in the central nervous system, including spinal cord. Expression levels were similar to endogenous LC3 and induced no detectable ALP changes. This ratiometric reporter registers differential pH-dependent changes in color as autophagosomes form, fuse with lysosomes, acidify, and degrade substrates within autolysosomes. We confirmed predicted changes in neuronal autophagy flux following specific experimental ALP perturbations. Furthermore, using a third fluorescence label in TRGL6 brains to identify lysosomes by immunocytochemistry, we validated a novel procedure to detect defective autolysosomal acidification in vivo. Thus, TRGL6 mice represent a unique tool to investigate in vivo ALP dynamics in specific neuron populations in relation to neurological diseases, aging, and disease modifying agents. Abbreviations: ACTB: actin, beta; AD: Alzheimer disease; AL: autolysosomes; ALP: autophagy-lysosome pathway; AP: autophagosome; APP: amyloid beta (Abeta) precursor protein; ATG5: autophagy related 5; ATG7: autophagy related 7; AV: autophagic vacuoles; CNS: central nervous system; CTSD: cathepsin D; CQ: chloroquine; DMEM: Dulbecco's modified Eagle's medium; GFP: green fluorescent protein; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2/GATE16: gamma-aminobutyric acid (GABA) receptor-associated protein-like 2; ICC: immunocytochemistry; ICV: intra-cerebroventricular; LAMP2: lysosomal-associated membrane protein 2; Leup: leupeptin; LY: lysosomes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RFP: red fluorescent protein; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; tfLC3: mRFP-eGFP-LC3; TRGL6: Thy1 mRFP eGFP LC3-line 6; PCR: polymerase chain reaction; PD: Parkinson disease.

Epoxyeicosatrienoic acids pretreatment improves amyloid ß-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes.

Amyloid-ß (Aß) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aß accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aß-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aß (1, 10 µM) exposure versus absence of Aß. MS-PPOH preincubation also aggravated Aß-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aß. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aß-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aß toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aß reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aß inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca(2+) influx in the subfornical organ.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.