Hypothalamic Slices Research Articles

Blast-Induced Mild Traumatic Brain Injury Alterations of Corticotropin-Releasing Factor Neuronal Activity in the Mouse Hypothalamic Paraventricular Nucleus.

Blast-induced mild traumatic brain injury (mbTBI) is the most common cause of TBI in US service members and veterans. Those exposed to TBI are at greater risk of developing neuropsychiatric disorders such as posttraumatic stress disorder, anxiety and depressive disorders, and substance use disorders following TBI. Previously, we have demonstrated that mbTBI increases anxiety-like behaviors in mice and dysregulates stress at the level of corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus (PVN). To expand on how mTBI may dysregulate the stress axis centrally, here PVN CRF neuronal activity was evaluated using whole cell-patch clamp recordings in hypothalamic slices from sham and mbTBI adult male CRF:tdTomato mice 7 days post-injury. We found that mbTBI generally did not affect the neuronal excitability and intrinsic membrane properties of PVN CRF neurons; this injury selectively increased the frequency of spontaneous neuronal firing of PVN CRF neurons localized to the dorsal PVN (dPVN) but not ventral PVN (vPVN). Consistently, mbTBI-induced dPVN CRF hyperactivity was associated with pre- and post-synaptic depression of spontaneous GABAergic transmission onto dPVN CRF neurons suggesting that mbTBI-induced GABAergic synaptic dysfunction may underlie dPVN CRF neuronal hyperactivity and increases in dPVN CRF signaling. The present results provide the first evidence for mbTBI-induced alterations in PVN CRF neuronal activity and GABAergic synaptic function that could mediate hypothalamic CRF dysregulation following mbTBI contributing to stress psychopathology associated with blast injury.

GKRP-dependent modulation of feeding behavior by tanycyte-released monocarboxylates.

Objectives: Glucokinase Regulatory Protein (GKRP) is the only known endogenous modulator of glucokinase (GK) localization and activity to date, and both proteins are localized in tanycytes, radial glia-like cells involved in metabolic and endocrine functions in the hypothalamus. However, the role of tanycytic GKRP and its impact on the regulation of feeding behavior has not been investigated. Here, we hypothesize that GKRP regulates feeding behavior by modulating tanycyte-neuron metabolic communication in the arcuate nucleus.Methods: We used primary cultures of tanycytes to evaluate the production of lactate and β-hydroxybutyrate (βHB). Similarly, we examined the electrophysiological responses to these metabolites in pro-opiomelanocortin (POMC) neurons in hypothalamic slices. To evaluate the role of GKRP in feeding behavior, we generated tanycyte-selective GKRP-overexpressing and GKRP-knock down mice (GKRPt-OE and GKRPt-KD respectively) using adenovirus-mediated transduction.Results: We demonstrated that lactate release induced by glucose uptake is favored in GKRP-KD tanycytes. Conversely, tanycytes overexpressing GKRP showed an increase in βHB efflux induced by low glucose concentration. In line with these findings, the excitability of POMC neurons was enhanced by lactate and decreased in the presence of βHB. In GKRPt-OE rats, we found an increase in post-fasting food avidity, whereas GKRPt-KD caused a significant decrease in feeding and body weight, which is reverted when MCT1 is silenced.Conclusion: Our study highlights the role of tanycytic GKRP in metabolic regulation and positions this regulator of GK as a therapeutic target for boosting satiety in patients with obesity problems.

Tachykinins amplify the action of capsaicin on central histaminergic neurons

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

L-type calcium channel subtypes mediating hypothalamic dysfunction caused by amyloid-beta.

In Alzheimer's disease (AD), non-cognitive manifestations attributable to hypothalamic dysfunction such as systemic metabolic deficits can occur prior to the cognitive decline (Ishii, M. and Iadecola C., Cell Metabolism 22:761,2015). We previously found in Tg2576 mice overexpressing the Swedish mutation of amyloid precursor protein that amyloid-beta pathology can cause dysfunction in hypothalamic arcuate neuropeptide Y (NPY) neurons, associated with low adiposity and low plasma leptin levels prior to memory decline or plaque formation (Ishii, M. et al., J Neuroscience 34:9096,2014). Furthermore, the hypothalamic arcuate NPY neuronal dysfunction was caused by amyloid-beta modulating low-threshold activated L-type calcium channels (Ishii, M. et al., J Neuroscience 39:8816,2019). However, which of the two major brain L-type calcium channel subtypes Cav 1.2 or Cav 1.3 is involved has not been established. To examine the role of Cav 1.2, we used the Cre-lox system in genetic mouse models to knock-out Cav 1.2 in hypothalamic arcuate NPY neurons (Cav 1.2 cKO). Cav 1.2 cKO mice were bred to NPY-GFP and Tg2576 mice. Two to 3-month-old male or female mice were used for all studies. Voltage-gated L-type calcium currents from hypothalamic slices containing arcuate NPY neurons were recorded using whole-cell voltage-clamp. Cytoplasmic Ca2+ levels were measured ratiometrically by loading Fura-2AM in dissociated arcuate NPY neurons and recording the emitted fluorescence using an inverted fluorescent microscope. Exogenous oligomeric amyloid-β42 caused a significant increase in cytoplasmic Ca2+ levels and L-type calcium currents in arcuate NPY neurons to a similar extent in both Cav 1.2 cKO and wild-type (WT) littermate mice. These effects of amyloid-β42 were reversed by the L-type calcium channel blocker nimodipine. In vivo, Cav 1.2 cKO mice bred to Tg2576 mice had similar body weight deficits compared to Tg2576 mice without deletion of Cav 1.2 in the arcuate NPY neurons. These results suggest that Cav 1.2 does not have a significant role in amyloid-beta mediated dysfunction of hypothalamic arcuate NPY neurons and that Cav 1.3 is likely the major target of amyloid-beta in these neurons. Future studies modulating Cav 1.3 in hypothalamic neurons will help elucidate the mechanisms underlying hypothalamic dysfunction caused by amyloid-beta pathology and its therapeutic potential in AD.

Glycolytic metabolism and activation of Na+ pumping contribute to extracellular acidification in the central clock of the suprachiasmatic nucleus: Differential glucose sensitivity and utilization between oxidative and non-oxidative glycolytic pathways

BackgroundThe central clock of the suprachiasmatic nucleus (SCN) controls the metabolism of glucose and is sensitive to glucose shortage. However, it is only beginning to be understood how metabolic signals such as glucose availability regulate the SCN physiology. We previously showed that the ATP-sensitive K+ channel plays a glucose-sensing role in regulating SCN neuronal firing at times of glucose shortage. Nevertheless, it is unknown whether the energy-demanding Na+/K+-ATPase (NKA) is also sensitive to glucose availability. Furthermore, we recently showed that the metabolically active SCN constantly extrudes H+ to acidify extracellular pH (pHe). This study investigated whether the standing acidification is associated with Na+ pumping activity, energy metabolism, and glucose utilization, and whether glycolysis- and mitochondria-fueled NKAs may be differentially sensitive to glucose shortage. MethodsDouble-barreled pH-selective microelectrodes were used to determine the pHe in the SCN in hypothalamic slices. ResultsNKA inhibition with K+-free (0-K+) solution rapidly and reversibly alkalinized the pHe, an effect abolished by ouabain. Mitochondrial inhibition with cyanide acidified the pHe but did not inhibit 0-K+-induced alkalinization. Glycolytic inhibition with iodoacetate alkalinized the pHe, completely blocked cyanide-induced acidification, and nearly completely blocked 0-K+-induced alkalinization. The results indicate that glycolytic metabolism and activation of Na+ pumping contribute to the standing acidification. Glucoprivation also alkalinized the pHe, nearly completely eliminated cyanide-induced acidification, but only partially reduced 0-K+-induced alkalinization. In contrast, hypoglycemia preferentially and partially blocked cyanide-induced acidification. The result indicates sensitivity to glucose shortage for the mitochondria-associated oxidative glycolytic pathway. ConclusionGlycolytic metabolism and activation of glycolysis-fueled NKA Na+ pumping activity contribute to the standing acidification in the SCN. Furthermore, the oxidative and non-oxidative glycolytic pathways differ in their glucose sensitivity and utilization, with the oxidative glycolytic pathway susceptible to glucose shortage, and the non-oxidative glycolytic pathway able to maintain Na+ pumping even in glucoprivation.

Evaluation of VGF peptides as potential anti-obesity candidates in pre-clinical animal models

VGF is a peptide precursor expressed in neuroendocrine cells that is suggested to play a role in the regulation of energy homeostasis. VGF is proteolytically cleaved to yield multiple bioactive peptides. However, the specific actions of VGF-derived peptides on energy homeostasis remain unclear. The aim of the present work was to investigate the role of VGF-derived peptides in energy homeostasis and explore the pharmacological actions of VGF-derived peptides on body weight in preclinical animal models. VGF-derived peptides (NERP-1, NERP-2, PGH-NH2, PGH−OH, NERP-4, TLQP-21, TLQP-30, TLQP-62, HHPD-41, AQEE-30, and LQEQ-19) were synthesized and screened for their ability to affect neuronal activity in vitro on hypothalamic brain slices and modulate food intake and energy expenditure after acute central administration in vivo. In addition, the effects of NERP-1, NERP-2, PGH-NH2, TLQP-21, TLQP-62, and HHPD-41 on energy homeostasis were studied after chronic central infusion. NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased the functional activity of hypothalamic neuronal networks. However, none of the peptides altered energy homeostasis after either acute or chronic ICV administration. The present data do not support the potential use of the tested VGF-derived peptides as novel anti-obesity drug candidates.

Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake.

The metabolic syndrome, which comprises obesity and diabetes, is a major public health problem and the awareness of energy homeostasis control remains an important worldwide issue. The energy balance is finely regulated by the central nervous system (CNS), notably through neuronal networks, located in the hypothalamus and the dorsal vagal complex (DVC), which integrate nutritional, humoral and nervous information from the periphery. The glial cells’ contribution to these processes emerged few year ago. However, its underlying mechanism remains unclear. Glial connexin 43 hemichannels (Cx43 HCs) enable direct exchange with the extracellular space and can regulate neuronal network activity. In the present study, we sought to determine the possible involvement of glial Cx43 HCs in energy balance regulation. We here show that Cx43 is strongly expressed in the hypothalamus and DVC and is associated with glial cells. Remarkably, we observed a close apposition of Cx43 with synaptic elements in both the hypothalamus and DVC. Moreover, the expression of hypothalamic Cx43 mRNA and protein is modulated in response to fasting and diet-induced obesity. Functionally, we found that Cx43 HCs are largely open in the arcuate nucleus (ARC) from acute mice hypothalamic slices under basal condition, and significantly inhibited by TAT-GAP19, a mimetic peptide that specifically blocks Cx43 HCs activity. Moreover, intracerebroventricular (i.c.v.) TAT-GAP19 injection strongly decreased food intake, without further alteration of glycaemia, energy expenditures or locomotor activity. Using the immediate early gene c-Fos expression, we found that i.c.v. TAT-GAP19 injection induced neuronal activation in hypothalamic and brainstem nuclei dedicated to food intake regulation. Altogether, these results suggest a tonic delivery of orexigenic molecules associated with glial Cx43 HCs activity and a possible modulation of this tonus during fasting and obesity.

Astrocytic Modulation of Supraoptic Oxytocin Neuronal Activity in Rat Dams with Pup-Deprivation at Different Stages of Lactation.

Appropriate interactions between astrocytes and oxytocin neurons in the hypothalamo- neurohypophysial system are essential for normal lactation. To further explore the mechanisms underlying astrocytic modulation of oxytocin neuronal activity, we observed astrocytic plasticity in the supraoptic nucleus of lactating rats with intermittent pup-deprivation (PD, 20h/day) at early (day 1-5) and middle (day 8-12) stages of lactation. PD at both stages decreased suckling duration and litter's body weight gain. They also significantly increased the expression of glial fibrillary acidic protein (GFAP) in Western blots while increased GFAP filaments and the colocalization of GFAP filaments with aquaporin 4 (AQP4) puncta in astrocyte processes surrounding oxytocin neuronal somata in immunohistochemistry in the supraoptic nucleus. Suckling between adjacent milk ejections but not shortly after them decreased molecular association between GFAP and AQP4. In hypothalamic slices from male rats, oxytocin treatment (0.1nmol/L, 10min) significantly reduced the length of GFAP filaments and AQP4 puncta in the processes but increased GFAP staining in the somata. These oxytocin effects were blocked by pretreatment of the slices with N-(1,3,4-Thiadiazolyl) nicotinamide (TGN-020, inhibitor of AQP4, 10µmol/L, 5min before oxytocin). In addition, inhibition of AQP4 with TGN-020 blocked excitation in oxytocin neurons evoked by prostaglandin E2, a downstream signal of oxytocin receptor and mediator of oxytocin-evoked burst firing, in whole-cell patch-clamp recordings. These results indicate that AQP4-associated astrocytic plasticity is essential for normal oxytocin neuronal activity during lactation and that PD-evoked hypogalactia is associated with astrocytic process expansion following increased GFAP and AQP4 expressions.

Morphological plasticity of the tuberoinfundibular dopaminergic neurones in the rat during the oestrous cycle and lactation

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones are critical with respect to regulating prolactin secretion from the anterior pituitary. Under most physiological conditions, they are stimulated by prolactin to release dopamine into the median eminence which subsequently suppresses further prolactin secretion from the lactotrophs. During lactation, the TIDA neurones are known to undergo both electrophysiological and neurochemical changes that alleviate this negative-feedback, thus allowing circulating prolactin levels to rise. The present study aimed to determine whether TIDA neurone morphology, most notably spine density, is also modified during lactation. This was achieved by stereotaxically injecting the arcuate nucleus of female, tyrosine hydroxylase-promoter driven Cre-recombinase transgenic rats with Cre-dependent adeno-associated virus-expressing Brainbow. This resulted in the highly specifici transfection of between 10% and 30% of the TIDA neurones, thus allowing the morphologies on multiple individual neurones to be examined in a single hypothalamic slice. The transfected neurones exhibited a range of complex forms, including a diversity of soma and location of axonal origin. Neuronal spine counting showed that the density of somatic, but not dendritic, spines was significantly higher during lactation than at any other reproductive stage. There was also a significant fall in somatic spine density across the oestrous cycle from dioestrus to oestrus. Although the functional characteristics of the additional somatic spines have not been determined, if, as might be expected, they represent an increased excitatory input to the TIDA neurones, this could have important physiological implications by perhaps supporting altered neurotransmitter release at their neuroendocrine terminals. Enhanced excitatory input may, for example, favour the release of the opioid peptide enkephalin rather than dopamine, which is potentially significant because the expression of the peptide is known to increase in the TIDA neurones during lactation and, in contrast to dopamine, it stimulates rather than inhibits prolactin secretion from the pituitary.

Mechanisms of Action of Methionine Restriction and Fibroblast Growth Factor 21 (FGF21)

Abstract Objectives Many of the metabolic benefits of methionine restriction (MR) are mediated by FGF21. MR activates protein kinase R-like ER kinase (PERK) and nuclear respiratory factor 2 (NRF2) signaling pathways, which have also been tied to FGF21 production. Aim 1 is to elucidate the mechanism by which MR increases hepatic FGF21 production. FGF21 signaling in the brain is required for its metabolic effects. However, the neuronal circuits and mechanisms mediating FGF21’s metabolic effects remain unclear. Aim 2 is to determine the effects of FGF21 on neurons in the paraventricular nucleus (PVN). Methods Aim 1: HepG2 cells were treated with vehicle or NRF2 inhibitor (brusatol; 80 nM) for 4 h or PERK inhibitor (GSK2606414; 1 µM) for 2 h. Cells were then incubated in control or MR media for 16 h. FGF21 mRNA expression was measured. Aim 2: WT and Fgf21−/− mice were fed high-fat control or MR diets for 14 wk. 4 mice per group were perfused with formalin for immunohistochemistry. Patch-clamp study: Sympathetic, PVN neurons projecting to the rostral ventrolateral medulla were retrogradely-labeled and subsequently patched in acute hypothalamic slices and treated with vehicle or the FGF21 analog, LY2405319 (LY; 500 nM). Firing activity of PVN-RVLM neurons and the activity of GABAergic inhibitory inputs to these neurons were measured. Results Aim 1: MR increased FGF21 mRNA in HepG2 cells by 18-fold on average (P = 0.02). The ability of MR to increase FGF21 is abolished when cells are treated with the NRF2 inhibitor but is not affected by PERK inhibition. Aim 2: MR increased ERK phosphorylation in the PVN of WT, but not Fgf21−/− mice. LY either increased (33% of recorded neurons) or decreased (67% of recorded neurons) the firing activity of PVN-RVLM neurons suggesting that FGF21 effects may depend on their projecting targets. LY inhibited the frequency of GABA inhibitory postsynaptic current (IPSC) (42.02% decrease, P < 0.05). Conclusions In vitro analysis suggests that the ability of MR to increase FGF21 expression is dependent upon NRF2 activity, but independent of PERK. Our data support the hypothesis that FGF21 signals through the PVN modulating sympathetic-related PVN neuronal activity to produce its metabolic benefits. Funding Sources GSU Research Initiation Grant; Center for Neuroinflammation and Cardiometabolic Diseases (CNCD), GSU.

SAT-595 Early Weight Gain Impact onto the Arcuate Leptin Receptor Expressing Neurons Activity During Development

The arcuate nucleus (ARH) is considered the main mediator of the effects of leptin on energy homeostasis, as well, part of the hypothalamic circuitry through which the sex steroids mediate the ovulatory cycle and therefore reproduction. While it is known that aging influences synaptic plasticity of ARH neurons, the effects of increased weight gain early in life onto in the activity of ARH neurons are still poorly understood. In order to demonstrate whether prepubertal adiposity gain is able to modulate the synaptic transmission in leptin receptor (LepR)-expressing cells of the ARH, we employed a postnatal over-nutrition model by raising mice in small litters (SL), while control mice were maintained in regular litters (6-8 pups per litter). The spontaneous currents of LepR-expressing neurons in the ARH were measured by whole-cell patch-clamp recordings in hypothalamic slices obtained from prepubertal, pubertal and adult female LepR-reporter mice. As expected, mice raised in SL exhibited increased body weight compared to control mice at prepubertal and pubertal stage (prepubertal, t(59) = 6.9, P < 0.001; pubertal, t(59) = 5.3, P < 0.001), despite no difference at adult age (t(59) = 1.5, P = 0.4). By evaluating ARH neuronal activity we observed an increased average of excitatory and inhibitory currents frequency during development, in both control females (sEPSC: F(2, 46)= 17.76, P< 0.0001, sIPSC: F(2, 21)= 4.064, P= 0.0322) and in mice raised in SL (sEPSC: F(2, 49)= 27.76, P< 0.0001, sIPSC: F(2, 19)= 6.714, P= 0.0062). However, SL and control mice exhibited similar sEPSC amplitudes at all stages of development. A significant interaction between litter size and excitatory transmission frequency onto LepR-expressing cells were noted at the pubertal and adult stages (F(2, 95)= 3.164, P= 0.046), despite no changes in the amplitudes of these signals (interaction: F(2, 95) = 0.1516, P= 0.8596; age: F(2, 95) = 0.9961, P= 0.3731; litter size: F(1, 95) = 0.076, P= 0.7832). By evaluating the inhibitory transmission to ARH LepR-expressing neurons, no significant interaction between litter size and inhibitory transmission frequency were observed (F(2, 40) = 0.09271, P= 0.9117). However, the average sIPSC amplitude were significantly reduced in the ARH cells recorded from mice raised in SL, when compared to the control group (F(2, 42) = 22.86, P< 0.0001), despite no identifiable interaction between litter size and inhibitory transmission (F(2, 42) = 0.6619, P= 0.5212). Taken together our results suggest that early weight gain influences the excitatory transmission pattern in LepR-expressing neurons by increasing presynaptic excitatory inputs and suppressing postsynaptically transmission to ARH neurons.

Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

Regulation of feeding by somatostatin neurons in the tuberal nucleus

The tuberal nucleus (TN) is a hypothalamic region which is well described in humans but remains poorly defined in rodents. In this paper, the authors demonstrated by specific and sophisticated techniques the role of somatostatin expressing TN (TNSST) neurons in the control of food intake in mice. Interestingly, their results showed the specific role of TNSTT neurons in food intake as compared to SST neurons located in the arcuate nucleus. Furthermore, the authors showed that the appetite-promoting hormone, ghrelin, induced a robust activation of TNSST neurons in both in-vivo and in hypothalamic slices. They then confirmed that specific activation of those neurons by chemogenetic or optogenetic effectors indeed promoted a dramatic food consumption. TNSST neurons project to regions in the hypothalamus which are known to be involved in the control of feeding such as the paraventicular nucleus (PVN). These TNSST neuron promoted feeding by inhibiting downstream neurons.

NMDA receptor-dependent signalling pathways regulate arginine vasopressin expression in the paraventricular nucleus of the rat

The antidiuretic hormone arginine vasopressin (AVP) regulates water homeostasis, blood pressure and a range of stress responses. It is synthesized in the hypothalamus and released from the posterior pituitary into the general circulation upon a range of stimuli. While the mechanisms leading to AVP secretion have been widely investigated, the molecular mechanisms regulating AVP gene expression are mostly unclear. Here we investigated the neurotransmitters and signal transduction pathways that activate AVP gene expression in the paraventricular nucleus (PVN) of the rat using acute brain slices and quantitative real-time PCR. We show that stimulation with l-glutamate robustly induced AVP gene expression in acute hypothalamic brain slices containing the PVN. More specifically, we show that AVP transcription was stimulated by NMDA. Using pharmacological treatments, our data further reveal that the activation of ERK1/2 (PD184352), CaMKII (KN-62) and PI3K (LY294002; 740 Y-P) is involved in the NMDA-induced AVP gene expression in the PVN. Together, this study identifies NMDA-mediated cell signalling pathways that regulate AVP gene expression in the rat PVN.

Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension.

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K+ channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca2+-dependent K+ channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K+ channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension.NEW & NOTEWORTHY Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.

Estradiol Protects Neuropeptide Y/Agouti-Related Peptide Neurons against Insulin Resistance in Females

When it comes to obesity, men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage wanes in postmenopausal women. A key diagnostic of the metabolic syndrome is insulin resistance in both peripheral tissues and brain, especially in the hypothalamus. Since the anorexigenic hormone 17β-estradiol (E₂) regulates food intake in part by inhibiting the excitability of the hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons, we hypothesized that E₂ would protect against insulin resistance in NPY/AgRP neurons with diet-induced obesity (DIO). Therefore, we did whole-cell recordings and single cell quantitative polymerase chain reaction in arcuate NPY^GFP neurons from both female and male mice to test the efficacy of insulin with DIO. The resting membrane potential and input resistance of NPY/AgRP neurons were significantly increased in DIO versus control-diet fed males. Most notably, the efficacy of insulin to activate K_ATP channels in NPY/AgRP neurons was significantly attenuated, although the K_ATP channel opener diazoxide was fully effective in NPY/AgRP neurons from DIO males, indicating that the K_ATP channels were expressed and functional. In contrast, insulin was fully efficacious to activate K_ATP channels in DIO females, and the response was reversed by the K_ATP channel blocker tolbutamide. However, the ability of insulin to activate K_ATP channels was abrogated with ovariectomy but fully restored with E₂ replacement. Insulin resistance in obese males was likely mediated by an increase in suppressor of cytokine signaling-3 (SOCS-3), protein tyrosine phosphatase B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) activity, since the expression of all 3 mRNAs were upregulated in the obese males but not in females. As proof of principle, pre-incubation of hypothalamic slices from DIO males with the PTP1B/TCPTP inhibitor CX08005 completely rescued the effects of insulin. Therefore, E₂ protects NPY/AgRP neurons in females against insulin resistance through, at least in part, attenuating phosphatase activity. The neuroprotective effects of E₂ may explain sex differences in the expression of metabolic syndrome that disappears with the loss of E₂ in aging.

The Na+/H+-Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca2+]i in the Suprachiasmatic Nucleus

The central clock in the suprachiasmatic nucleus (SCN) has higher metabolic activity than extra-SCN areas in the anterior hypothalamus. Here we investigated whether the Na+/H+ exchanger (NHE) may regulate extracellular pH (pHe), intracellular pH (pHi) and [Ca2+]i in the SCN. In hypothalamic slices bathed in HEPES-buffered solution a standing acidification of ~0.3 pH units was recorded with pH-sensitive microelectrodes in the SCN but not extra-SCN areas. The NHE blocker amiloride alkalinised the pHe. RT-PCR revealed mRNA for plasmalemmal-type NHE1, NHE4, and NHE5 isoforms, whereas the NHE1-specific antagonist cariporide alkalinised the pHe. Real-time PCR and western blotting failed to detect day-night variation in NHE1 mRNA and protein levels. Cariporide induced intracellular acidosis, increased basal [Ca2+]i, and decreased depolarisation-induced Ca2+ rise, with the latter two effects being abolished with nimodipine blocking the L-type Ca2+ channels. Immunofluorescent staining revealed high levels of punctate colocalisation of NHE1 with serotonin transporter (SERT) or CaV1.2, as well as triple staining of NHE1, CaV1.2, and SERT or the presynaptic marker Bassoon. Our results indicate that NHE1 actively extrudes H+ to regulate pHi and nimodipine-sensitive [Ca2+]i in the soma, and along with CaV1.2 may also regulate presynaptic Ca2+ levels and, perhaps at least serotonergic, neurotransmission in the SCN.

Aerobic exercise regulates synaptic transmission and reactive oxygen species production in the paraventricular nucleus of spontaneously hypertensive rats

Aerobic exercise lowers blood pressure in patients with hypertension, but the underlying mechanisms remain incompletely understood. The hypothalamic paraventricular nucleus (PVN) plays a key role in the control of sympathetic outflow and cardiovascular tone. We examined whether chronic aerobic exercise altered synaptic transmission and reactive oxygen species (ROS) production in the PVN. In the present study, spontaneously hypertensive rats (SHRs) were subjected to exercise training for 8 weeks, five times per week, with Wistar Kyoto (WKY) rats as the cohort control. Miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from the PVN in ex vivo hypothalamic slice preparations obtained after the last training, and biomarkers of oxidative stress and physical indexes were observed. The mean frequency and amplitude, as well as the rise time and the decay time constant of mIPSCs, significantly decreased in 20-wk-old SHRs compared to WKY 20-wk-old controls. In contrast to mIPSCs, only the mean mEPSC frequency was higher, and there were no other changes in mEPSCs in comparison to the control group. SHRs exhibited higher ROS, 8-OHdG, and MDA; and lower SOD1, SOD2, CAT, Ogg1, and SOD and CAT activity in the PVN. These SHRs also had a significant increase in heart rate, blood pressure and sympathetic nerve activity, and higher levels of norepinephrine (NE). Exercise training ameliorated all these abnormalities, resulting in an increase in the mean frequency, amplitude and kinetics of mIPSCs, accompanied by a decrease in the mean frequency of mEPSCs in the PVN. This study demonstrates that moderate intensity, high frequency exercise training induces a selective enhancement of inhibitory synaptic transmission in the PVN, which may dampen sympathetic activity and reduce blood pressure in hypertension. These changes may be due to antioxidant-related adaptations in the PVNs of SHRs.

Insulin receptor localization in the embryonic avian hypothalamus

The hypothalamus is a brain region critical for the homeostatic regulation of appetite and energy expenditure. Hypothalamic neuronal activity that is altered during development can produce permanent physiological changes later in life. For example, circulating hormones such as insulin have been shown to influence hypothalamic neuronal projections, leading to altered metabolism in adult rodents. While insulin signaling in the post-hatch chicken has been shown to mirror that of mammals, the developmental role of insulin in the avian embryonic hypothalamus remains largely unexplored. Here we present the earliest known evidence for insulin receptor (InsR) expression in embryonic avian hypothalamic nuclei governing energy homeostasis. RT-PCR analysis reveals InsR mRNA in E8, E10, and E12 neurons while western blot data demonstrate protein expression in E12 avian whole brain and hypothalamic lysates. Immunohistochemical analysis of avian hypothalamic brain slices demonstrates a shift in InsR localization from paraventricular expression in E8 to a more defined concentration of InsR in developmental regions resembling the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) in E12 time points. In addition, InsR expression appears in a heterogeneous pattern, suggesting receptor localization to subpopulations of avian hypothalamic neurons as early as E8. With increasing evidence suggesting energy homeostasis pathways may be altered via the gestational environment, it is important to understand how insulin signaling may affect embryogenesis. Our research provides evidence for the earliest known embryonic expression of InsR protein in the avian hypothalamus and may suggest a developmental role for insulin signaling in the early patterning of metabolic pathways in the central nervous system.

Late onset obesity in mice with targeted deletion of potassium inward rectifier Kir7.1 from cells expressing the melanocortin-4 receptor.

Energy stores in fat tissue are determined in part by the activity of hypothalamic neurones expressing the melanocortin-4 receptor (MC4R). Even a partial reduction in MC4R expression levels in mice, rats or humans produces hyperphagia and morbid obesity. Thus, it is of great interest to understand the molecular basis of neuromodulation by the MC4R. The MC4R is a G protein-coupled receptor that signals efficiently through GαS , and this signalling pathway is essential for normal MC4R function in vivo. However, previous data from hypothalamic slice preparations indicated that activation of the MC4R depolarised neurones via G protein-independent regulation of the ion channel Kir7.1. In the present study, we show that deletion of Kcnj13 (ie, the gene encoding Kir7.1) specifically from MC4R neurones produced resistance to melanocortin peptide-induced depolarisation of MC4R paraventricular nucleus neurones in brain slices, resistance to the sustained anorexic effect of exogenously administered melanocortin peptides, late onset obesity, increased linear growth and glucose intolerance. Some MC4R-mediated phenotypes appeared intact, including Agouti-related peptide-induced stimulation of food intake and MC4R-mediated induction of peptide YY release from intestinal L cells. Thus, a subset of the consequences of MC4R signalling in vivo appears to be dependent on expression of the Kir7.1 channel in MC4R cells.