Dopaminergic dominance in the ventral medial hypothalamus: A pivotal regulator for methamphetamine-induced pathological aggression.

Methamphetamine (METH) abuse is associated with a spectrum of behavioral consequences, among which heightened aggression presents a significant challenge. However, the causal role of METH's impact in aggression and its target circuit mechanisms remains largely unknown. We established an acute METH exposure-aggression mouse model to investigate the role of ventral tegmental area (VTA) dopaminergic neurons and ventral medial hypothalamus VMH glutamatergic neuron. Our findings revealed that METH-induced VTA dopamine excitability activates the ventromedial hypothalamus (VMH) glutamatergic neurons, contributing to pathological aggression. Notably, we uncovered a dopaminergic transmission within the VTA-VMH circuit that exclusively functioned under METH influence. This dopaminergic pathway emerged as a potential key player in enabling dopamine-related pathological aggression, with heightened dopaminergic excitability implicated in various psychiatric symptoms. Also, the modulatory function of this pathway opens new possibilities for targeted therapeutic strategies for intervention to improve treatment in METH abuse and may have broader implications for addressing pathological aggression syndromes.

Role of Dorsomedial Hypothalamus GABAergic Neurons in Sleep-Wake States in Response to Changes in Ambient Temperature in Mice.

Good sleep quality is essential for maintaining the body's attention during wakefulness, which is easily affected by external factors such as an ambient temperature. However, the mechanism by which an ambient temperature influences sleep-wake behaviors remains unclear. The dorsomedial hypothalamus (DMH) has been reported to be involved in thermoregulation. It also receives projection from the preoptic area, which is an important region for sleep and energy homeostasis and the suprachiasmatic nucleus-a main control area of the clock rhythm. Therefore, we hypothesized that the DMH plays an important role in the regulation of sleep related to ambient temperatures. In this study, we found that cold exposure (24/20/16/12 °C) increased wakefulness and decreased non-rapid eye movement (NREM) sleep, while warm exposure (32/36/40/44 °C) increased NREM sleep and decreased wakefulness compared to 28 °C conditions in wild-type mice. Then, using non-specific and specific apoptosis, we found that lesions of whole DMH neurons and DMH γ-aminobutyric acid (GABA)-ergic neurons induced by caspase-3 virus aggravated the fluctuation of core body temperature after warm exposure and attenuated the change in sleep-wake behaviors during cold and warm exposure. However, chemogenetic activation or inhibition of DMH GABAergic neurons did not affect the sleep-wake cycle. Collectively, our findings reveal an essential role of DMH GABAergic neurons in the regulation of sleep-wake behaviors elicited by a change in ambient temperature.

Electroconvulsive seizures lead to lipolytic-induced gene expression changes in mediobasal hypothalamus and decreased white adipose tissue mass.

AIMS: Electroconvulsive seizure (ECS) therapy is highly effective in the treatment of several psychiatric disorders, including depression. Past studies have shown that the rodent model of ECS reveals the activation of multiple brain regions including the hypothalamus, suggesting that this method of brain stimulation broadly regulates central neuronal function, which results in peripheral function. The ventromedial nucleus of the hypothalamus (VMH) plays an important role in feeding and energy homeostasis. Our previous study showed that ECS increases the expression of anorexigenic factors in the VMH and has an anorexigenic effect in a mouse model. Since the VMH is also suggested to play a critical role in the peripheral lipid metabolism of white adipose tissue (WAT), we hypothesized that ECS alters lipid metabolism via activation of the VMH. METHODS AND RESULTS: Here, we demonstrate that repeated ECS suppresses the fat mass of epididymal WAT and significantly increases the expression levels of lipolytic and brown adipose tissue markers such as Adrb3, Hsl/Lipe, and Ppargc1a. In the VMH, ECS increased the expression of multiple genes, notably Bdnf, Adcyap1, and Crhr2, which are not only anorexigenic factors but are also modulators of lipid metabolism. Furthermore, gold-thioglucose-induced hypothalamic lesions affecting the VMH abolished the effect of ECS on the WAT, indicating that hypothalamus activation is required for the phenotypic changes seen in the epididymal WAT. CONCLUSION: Our data demonstrates a new effect of ECS on the lipid metabolism of WAT via induction of hypothalamic activity involving the VMH.

Morphological and functional evidence for sexual dimorphism in neurokinin B signalling in the retrochiasmatic area of sheep.

Neurokinin B (NKB) is critical for fertility in humans and stimulates gonadotrophin-releasing hormone/luteinising hormone (LH) secretion in several species, including sheep. There is increasing evidence that the actions of NKB in the retrochiasmatic area (RCh) contribute to the induction of the preovulatory LH surge in sheep. In the present study, we determined whether there are sex differences in the response to RCh administration of senktide, an agonist to the NKB receptor (neurokinin receptor-3 [NK3R]), and in NKB and NK3R expression in the RCh of sheep. To normalise endogenous hormone concentrations, animals were gonadectomised and given implants to mimic the pattern of ovarian steroids seen in the oestrous cycle. In females, senktide microimplants in the RCh produced an increase in LH concentrations that lasted for at least 8 hours after the start of treatment, whereas a much shorter increment (approximately 2 hours) was seen in males. We next collected tissue from gonadectomised lambs 18 hours after the insertion of oestradiol implants that produce an LH surge in female, but not male, sheep for immunohistochemical analysis of NKB and NK3R expression. As expected, there were more NKB-containing neurones in the arcuate nucleus of females than males. Interestingly, there was a similar sexual dimorphism in NK3R-containing neurones in the RCh, NKB-containing close contacts onto these RCh NK3R neurones, and overall NKB-positive fibres in this region. These data demonstrate that there are both functional and morphological sex differences in NKB-NK3R signalling in the RCh and raise the possibility that this dimorphism contributes to the sex-dependent ability of oestradiol to induce an LH surge in female sheep.

Neurog2 Acts as a Classical Proneural Gene in the Ventromedial Hypothalamus and Is Required for the Early Phase of Neurogenesis.

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.

Effect of central JAZF1 on glucose production is regulated by the PI3K-Akt-AMPK pathway.

The role of central juxtaposed with another zinc finger gene 1 (JAZF1) in glucose regulation remains unclear. Here, we activated mediobasal hypothalamus (MBH) JAZF1 in high-fat diet (HFD)-fed rats by an adenovirus expressing JAZF1 (Ad-JAZF1). We evaluated the changes in the hypothalamic insulin receptor (InsR)-PI3K-Akt-AMPK pathway and hepatic glucose production (HGP). To investigate the impact of MBH Ad-JAZF1 on HGP, we activated MBH JAZF1 in the presence or absence of ATP-dependent potassium (KATP ) channel inhibition, hepatic branch vagotomy (HVG), or an AMPK activator (AICAR). In HFD-fed rats, MBH Ad-JAZF1 decreased body weight and food intake, and inhibited HGP by increasing hepatic insulin signaling. Under insulin stimulation, MBH Ad-JAZF1 increased InsR and Akt phosphorylation, and phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) formation; however, AMPK phosphorylation was decreased in the hypothalamus. The positive effect of MBH JAZF1 on hepatic insulin signaling and HGP was prevented by treatment with a KATP channel inhibitor or HVG. The metabolic impact of hypothalamic JAZF1 was also blocked by MBH AICAR. Ad-JAZF1 treatment in SH-SY5Y cells resulted in an elevation of InsR and Akt phosphorylation following insulin stimulation. Our findings show that hypothalamic JAZF1 regulates HGP via the InsR-PI3K-Akt-AMPK pathway and KATP channels.

Regulation of prepubertal dynorphin secretion in the medial basal hypothalamus of the female rat.

The onset of puberty is the result of an increase in secretion of hypothalamic gonadotrophin-releasing hormone (GnRH). This action is a result of not only the development of stimulatory inputs to its release, but also the gradual decrease in inhibitory inputs that restrain release of the peptide prior to pubertal onset. Dynorphin (DYN) is one of the inhibitory inputs produced in the medial basal hypothalamus (MBH); however, little is known about what substance(s) control its prepubertal synthesis and release. Because neurokinin B (NKB) increases in the hypothalamus as puberty approaches, we considered it a candidate for such a role. An initial study investigated the acute effects of an NKB agonist, senktide, on the secretion of DYN from MBH tissues incubated in vitro. In other experiments, central injections of senktide were administered to animals for 4 days then MBHs were collected for assessment of DYN synthesis or for the in vitro secretion of both DYN and GnRH. Because insulin-like growth factor (IGF)-1 has been shown to play an important role at puberty, additional animals received central injections of this peptide for 4 days to assess NKB and DYN synthesis or the in vitro secretion of NKB. The results obtained show that senktide administration up-regulates the NKB receptor protein, at the same time as suppressing the DYN and its receptor. Senktide consistently suppressed DYN and elevated GnRH secretion in the same tissue incubates from both the acute and chronic studies. IGF-1 administration caused an increase in NKB protein, at the same time as decreasing DYN protein. Furthermore, the central administration of IGF-1 caused an increase in NKB release, an action blocked by the IGF-1 receptor blocker, JB-1. These results indicate that the IGF-1/NKB pathway contributes to suppressing the DYN inhibitory tone on prepubertal GnRH secretion and thus facilitates the puberty-related increase in the release of GnRH to accelerate the onset of puberty.

Hypothalamic concentrations of kisspeptin and gonadotropin-releasing hormone during the breeding season and non-breeding season in ewes.

PROBLEM: Current methods to quantify kisspeptin (KP) are limited. To this end, a radioimmunoassay (RIA) specific for KP was developed and validated. We hypothesized that use of a RIA would reveal multiple hypothalamic regions as targets of negative seasonal feedback of estradiol on KP production in sheep. METHOD OF STUDY: Ovariectomized (OVX) ewes bearing a subcutaneous implant of estradiol were euthanized during the breeding season (BS) (n = 4) and non-breeding season (NBS) (n = 3). Coronal sections of preoptic area (POA), anterior hypothalamic area (AHA), and mediobasal hypothalamus (MBH) were collected, as well as the median eminence (ME), cortex, brain stem, and cerebellum. Amounts of KP and gonadotropin-releasing hormone (GnRH) in individual hypothalamic nuclei were quantified by radioimmunoassay. RESULTS: Concentration and content of KP were lower during the NBS than the BS in the MBH (P < 0.01) and POA (P < 0.01). Levels of KP in tissue adjacent to the POA and MBH were much lower, and neither concentration nor content of KP differed between the BS and NBS. Kisspeptin was also detected in the cortex, brain stem, and cerebellum, but concentrations were not affected by season. In addition, concentration and content of GnRH in the POA, AHA, MBH, and ME were similar between seasons. CONCLUSION: Our RIA results indicate that in addition to the MBH, the POA and AHA appear to be involved in the seasonal negative feedback of estradiol on KP expression.

Recurrent glucose deprivation leads to the preferential use of lactate by neurons in the ventromedial hypothalamus.

Increased GABAergic output in the ventromedial hypothalamus (VMH) contributes to counterregulatory failure in recurrently hypoglycemic (RH) rats, and lactate, an alternate fuel source in the brain, contributes to this phenomenon. The current study assessed whether recurring bouts of glucose deprivation enhanced neuronal lactate uptake and, if so, whether this influenced γ-aminobutyric acid (GABA) output and the counterregulatory responses. Glucose deprivation was induced using 5-thioglucose (5TG). Control rats received an infusion of artificial extracellular fluid. These groups were compared with RH animals. Subsequently, the rats underwent a hypoglycemic clamp with microdialysis. To test whether 5TG affected neuronal lactate utilization, a subgroup of 5TG-treated rats was microinjected with a lactate transporter inhibitor [cyano-4-hydroxycinnamate (4CIN)] just before the start of the clamp. Both RH and 5TG raised VMH GABA levels, and this was associated with impaired counterregulatory responses. 4CIN reduced VMH GABA levels and restored the hormone responses in the 5TG group. We then evaluated [14C]lactate uptake in hypothalamic neuronal cultures. Recurring exposure to low glucose increased monocarboxylate transporter-2 mRNA expression and augmented lactate uptake. Taken together, our data suggest that glucose deprivation, per se, enhances lactate utilization in hypothalamic neurons, and this may contribute to suppression of the counterregulatory responses to hypoglycemia.

Nitric oxide acutely modulates hypothalamic and neurohypophyseal carbon monoxide and hydrogen sulphide production to control vasopressin, oxytocin and atrial natriuretic peptide release in rats.

Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso- and hypertonicity, we determined the effects of NO (600 µmol L-1 sodium nitroprusside), CO (100 µmol L-1 tricarbonylchloro[glycinato]ruthenium [II]) and H2 S (10 mmol L-1 sodium sulphide) donors and nitric oxide synthase (NOS) (300 µmol L-1 Nω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200 µmol L-1 Zn(II) deuteroporphyrin IX 2,4-bis-ethylene glycol [ZnDPBG]) and cystathionine ß-synthase (CBS) (100 µmol L-1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system.

The glutamine-glutamate cycle regulates synaptic glutamate release in the ventrolateral ventromedial nucleus of the hypothalamus of perinatal female rats.

The astrocytic glutamine (Gln)-glutamate (Glu) cycle (GGC) supplies Gln for the regulation of glutamatergic synaptic transmission (GST) in the adult hippocampus. Increased synaptic Glu release in the perinatal ventrolateral ventromedial nucleus of the hypothalamus (vlVMH) modulates sexual differentiation, however, whether GGC regulates GST in the perinatal vlVMH has not been determined. Sex differences in oestradiol (E2 ) levels exist in the neonatal hypothalamus, and E2 increases levels of glutamine synthetase and glutaminase, two key enzymes involved in the GGC. Thus, it is hypothesised that sexually dimorphic phenotypes may exist in glutamatergic synapses associated with the GGC in the vlVMH in perinatal rats. Whole-cell voltage-clamp recordings in vlVMH neurones in brain slices from male and female pups revealed that pharmacological disruption of the GGC by α-(methylamino) isobutyric acid (5 mmol L-1 ), which blocks neuronal Gln uptake; or by l-methionine sulphoximine (1.5 mmol L-1 ), which inhibits astrocytic Gln synthesis, decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in female but not male pups. By contrast, GGC interruptions decreased evoked (e)EPSC amplitudes in both sexes following increased synaptic activity produced by a period of stimulation. In male pups, the decreased eEPSCs were attributable to reduced Glu release, as assessed by paired-pulse stimulations, whereas, in female pups, they were attributable to decreased Glu content in the synaptic vesicles, as measured by strontium-evoked mEPSCs. The l-methionine sulphoximine-mediated decrease in eEPSCs was rapidly rescued by exogenous Gln in female but not male pups. The reductions in mEPSCs and eEPSCs in female pups were accompanied by enhanced blocking effects of the low-affinity Glu AMPA receptor antagonist, γ-d-glutamylglycine, consistent with diminished Glu release. In conclusion, female, but not male pups, rely on constitutive astrocytic Gln for sustained synaptic Glu release in the vlVMH. This glutamatergic synaptic phenotype may be associated with brain and behaviour feminisation and/or defeminisation in rats.

Neuronal Dnmt1 Deficiency Attenuates Diet-Induced Obesity in Mice.

Aberrant neuronal DNA methylation patterns have been implicated in the promotion of obesity development; however, the role of neuronal DNA methyltransferases (Dnmts), enzymes that catalyze DNA methylation, in energy balance remains poorly understood. We investigated whether neuronal Dnmt1 regulates normal energy homeostasis and obesity development using a neuronal Dnmt1 knockout (ND1KO) mouse model, Dnmt1fl/fl Synapsin1Cre, which specifically deletes Dnmt1 in neurons. Neuronal Dnmt1 deficiency reduced adiposity in chow-fed mice and attenuated obesity in high-fat diet (HFD)-fed male mice. ND1KO male mice had reduced food intake and increased energy expenditure with the HFD. Furthermore, these mice had improved insulin sensitivity, as measured using an insulin tolerance test. The HFD-fed ND1KO mice had smaller fat pads and upregulation of thermogenic genes in brown adipose tissue. These data suggest that neuronal Dnmt1 plays an important role in regulating energy homeostasis. Notably, ND1KO male mice had elevated estrogen receptor-α (ERα) gene expression in the medial hypothalamus, which previously has been shown to control body weight. Immunohistochemistry experiments revealed that ERα protein expression was upregulated specifically in the dorsomedial region of the ventromedial hypothalamus, a region that might mediate the central effect of leptin. We conclude that neuronal Dnmt1 regulates energy homeostasis through pathways controlling food intake and energy expenditure. In addition, ERα expression in the dorsomedial region of the ventromedial hypothalamus might mediate these effects.

TNFα drives mitochondrial stress in POMC neurons in obesity.

Consuming a calorically dense diet stimulates microglial reactivity in the mediobasal hypothalamus (MBH) in association with decreased number of appetite-curbing pro-opiomelanocortin (POMC) neurons; whether the reduction in POMC neuronal function is secondary to the microglial activation is unclear. Here we show that in hypercaloric diet-induced obese mice, persistently activated microglia in the MBH hypersecrete TNFα that in turn stimulate mitochondrial ATP production in POMC neurons, promoting mitochondrial fusion in their neurites, and increasing POMC neuronal firing rates and excitability. Specific disruption of the gene expressions of TNFα downstream signals TNFSF11A or NDUFAB1 in the MBH of diet-induced obese mice reverses mitochondrial elongation and reduces obesity. These data imply that in a hypercaloric environment, persistent elevation of microglial reactivity and consequent TNFα secretion induces mitochondrial stress in POMC neurons that contributes to the development of obesity.

The blockage of ventromedial hypothalamus CRF type 2 receptors impairs escape responses in the elevated T-maze.

In a previous study, the administration of corticotrophin-releasing factor (CRF) into the dorsomedial hypothalamus (DMH), a region that modulates defensive reactions, was shown to facilitate elevated T-maze (ETM) avoidance responses, an anxiogenic-like effect. Intra-DMH administration of the CRF type 1 receptor (CRFR1) antagonist antalarmin induced anxiolytic-like effects and counteracted the anxiogenic effects of CRF. The present study further investigates the role played by CRF receptors of the medial hypothalamus in anxiety. For that, male wistar rats were treated with CRFR1 and CRFR2-modulating drugs in the DMH or VMH, another hypothalamic nucleus implicated with defensive and emotional behavior, and tested in the ETM for inhibitory avoidance and escape measurements. In clinical terms, these responses have been respectively related to generalized anxiety and panic disorder. All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. The results showed that intra-VMH CRF or antalarmin did not alter ETM avoidance or escape performance. Intra-VMH injection of the CRFR2 preferential antagonist antisauvagine-30 or of the selective CRFR2 antagonist astressin 2-B inhibited escape performance, a panicolytic-like effect, without altering avoidance reactions. The CRFR2 agonist urocortin-2 intra-VMH was by itself without effect but blocked the effects of astressin 2-B. None of the drugs administered into the DMH altered ETM measurements. Additionally, none of the compounds altered locomotor activity measurements. These results suggest that VMH CRFR2 modulate a defensive response associated with panic disorder and are of relevance to the better understanding of the neural mechanisms underlying this pathological condition.

Cholinergic neurons in the dorsomedial hypothalamus regulate food intake.

OBJECTIVE: Central cholinergic neural circuits play a role in the regulation of feeding behavior. The dorsomedial hypothalamus (DMH) is considered the appetite-stimulating center and contains cholinergic neurons. Here, we study the role of DMH cholinergic neurons in the control of food intake. METHODS: To selectively stimulate DMH cholinergic neurons, we expressed stimulatory designer receptors exclusively activated by designer drugs (DREADDs) and channelrhodopsins in DMH cholinergic neurons by injection of adeno-associated virus (AAV) vectors into the DMH of choline acetyltransferase (ChAT)-IRES-Cre mice. We also generated transgenic mice expressing channelrhodopsins in cholinergic neurons with the Cre-LoxP technique. To delete the Chat gene exclusively in the DMH, we injected an AAV carrying a Cre recombinase transgene into the DMH of floxed ChAT mice. Food intake was measured with and without selective stimulation of DMH cholinergic neurons. RESULTS: Mice lacking the Chat gene in the DMH show reduced body weight as compared to control. Chemogenetic activation of DMH cholinergic neurons promotes food intake. This orexigenic effect is further supported by experiments of optogenetic stimulation of DMH cholinergic neurons. DMH cholinergic neurons innervate pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus (ARC). Treatment with acetylcholine (ACh) enhances GABAergic inhibitory transmission to ARC POMC neurons that is blocked by the muscarinic receptor antagonist. Direct activation of cholinergic fibers in the ARC readily stimulates food intake that is also abolished by the muscarinic receptor antagonist. CONCLUSION: ACh released from DMH cholinergic neurons regulates food intake and body weight. This effect is mediated in part through regulation of ARC POMC neurons. Activation of muscarinic receptors on GABAergic axon terminals enhances inhibitory tone to ARC POMC neurons. Hence, this novel DMHACh â†’ ARCPOMC pathway plays an important role in the control of food intake and body weight.

Activation of orexin/hypocretin neurons is associated with individual differences in cued fear extinction.

Identifying the neurobiological mechanisms that underlie differential sensitivity to stress is critical for understanding the development and expression of stress-induced disorders, such as post-traumatic stress disorder (PTSD). Preclinical studies have suggested that rodents display different phenotypes associated with extinction of Pavlovian conditioned fear responses, with some rodent populations being resistant to extinction. An emerging literature also suggests a role for orexins in the consolidation processes associated with fear learning and extinction. To examine the possibility that the orexin system might be involved in individual differences in fear extinction, we used a Pavlovian conditioning paradigm in outbred Long-Evans rats. Rats showed significant variability in the extinction of cue-conditioned freezing and extinction recall, and animals were divided into groups based on their extinction profiles based on a median split of percent freezing behavior during repeated exposure to the conditioned cue. Animals resistant to extinction (high freezers) showed more freezing during repeated cue presentations during the within trial and between trial extinction sessions compared with the group showing significant extinction (low freezers), although there were no differences between these groups in freezing upon return to the conditioned context or during the conditioning session. Following the extinction recall session, activation of orexin neurons was determined using dual label immunohistochemistry for cFos in orexin positive neurons in the hypothalamus. Individual differences in the extinction of cue conditioned fear were associated with differential activation of hypothalamic orexin neurons. Animals showing poor extinction of cue-induced freezing (high freezers) had significantly greater percentage of orexin neurons with Fos in the medial hypothalamus than animals displaying significant extinction and good extinction recall (low freezers). Further, the freezing during extinction learning was positively correlated with the percentage of activated orexin neurons in both the lateral and medial hypothalamic regions. No differences in the overall density of orexin neurons or Fos activation were seen between extinction phenotypes. Although correlative, our results support other studies implicating a role of the orexinergic system in regulating extinction of conditioned responses to threat.

Role of Estradiol in the Regulation of Prolactin Secretion During Late Pregnancy.

Estrogen action is necessary for evidencing the stimulatory action of mifepristone and naloxone on prolactin (PRL) secretion during late pregnancy. Our aim is to determine the mechanism mediating this facilitator action of estrogens. To investigate the hypothalamic mechanisms involved in estrogen actions in PRL secretion at the end of pregnancy, we measured the effect of pretreatment with the estrogen antagonist tamoxifen on the expression of tyrosine hydroxylase (TH), hormone receptors (ERα and ß, PRs, PRLR(long)), and µ- and κ- opioid receptors (ORs) at mRNA (by semiquantitative RT-PCR) and protein (by western blot for TH, PRLR(long), ERα, PRs, µ- and ORs) levels in extracts of medial basal hypothalamus (MBH) and serum PRL, E2 and P4 levels (by RIA) in mifepristone- and naloxone-treated rats. Tamoxifen administration partially prevented PRL release induced by the combined treatment. TH expression diminished and ERα expression increased in mifepristone-treated rats at mRNA and protein levels and tamoxifen partially prevented these changes with no effect on PRs expression. Mifepristone increased PRLR(long) mRNA levels; this increase was blocked by tamoxifen. Combined tamoxifen and mifepristone treatment decreased µ- and k-ORs mRNA but not protein levels. In conclusion, E2 induces neuroadaptive mechanisms necessary to facilitate PRL release preceding delivery. Acting through ERα, E2 modulates hypothalamic dopaminergic neurons activity, regulating TH, µ- and κ-ORs and PRLR(long) expression, and is necessary for evidencing the effects of P4 withdrawal. Its presence on days 14 and 15 of pregnancy is crucial to facilitate the opioid system modulation of PRL secretion at the end of pregnancy in the rat.

DEPTOR in POMC neurons affects liver metabolism but is dispensable for the regulation of energy balance.

We have recently demonstrated that specific overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in the mediobasal hypothalamus (MBH) protects mice against high-fat diet-induced obesity, revealing DEPTOR as a significant contributor to energy balance regulation. On the basis of evidence that DEPTOR is expressed in the proopiomelanocortin (POMC) neurons of the MBH, the present study aimed to investigate whether these neurons mediate the metabolic effects of DEPTOR. Here, we report that specific DEPTOR overexpression in POMC neurons does not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Unlike the previous model, mice overexpressing DEPTOR only in POMC neurons 1) did not show differences in feeding behavior, 2) did not exhibit changes in locomotion activity and oxygen consumption, 3) did not show an improvement in systemic glucose metabolism, and 4) were not resistant to high-fat diet-induced obesity. These results support the idea that other neuronal populations are responsible for these phenotypes. Nonetheless, we observed a mild elevation in fasting blood glucose, insulin resistance, and alterations in liver glucose and lipid homeostasis in mice overexpressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.

Adropin acts in brain to inhibit water drinking: potential interaction with the orphan G protein-coupled receptor, GPR19.

Adropin, a recently described peptide hormone produced in the brain and liver, has been reported to have physiologically relevant actions on glucose homeostasis and lipogenesis, and to exert significant effect on endothelial function. We describe a central nervous system action of adropin to inhibit water drinking and identify a potential adropin receptor, the orphan G protein-coupled receptor, GPR19. Reduction in GPR19 mRNA levels in medial basal hypothalamus of male rats resulted in the loss of the inhibitory effect of adropin on water deprivation-induced thirst. The identification of a novel brain action of adropin and a candidate receptor for the peptide should extend and accelerate the study of the potential therapeutic value of adropin or its mimetics for the treatment of metabolic disorders.

Knockdown of neuropeptide Y in the dorsomedial hypothalamus reverses high-fat diet-induced obesity and impaired glucose tolerance in rats.

Neuropeptide Y (NPY) in the dorsomedial hypothalamus (DMH) plays an important role in the regulation of energy balance. While DMH NPY overexpression causes hyperphagia and obesity in rats, knockdown of NPY in the DMH via adeno-associated virus (AAV)-mediated RNAi (AAVshNPY) ameliorates these alterations. Whether this knockdown has a therapeutic effect on obesity and glycemic disorder has yet to be determined. The present study sought to test this potential using a rat model of high-fat diet (HFD)-induced obesity and insulin resistance, mimicking human obesity with impaired glucose homeostasis. Rats had ad libitum access to rodent regular chow (RC) or HFD. Six weeks later, an oral glucose tolerance test (OGTT) was performed for verifying HFD-induced glucose intolerance. After verification, obese rats received bilateral DMH injections of AAVshNPY or the control vector AAVshCTL, and OGTT and insulin tolerance test (ITT) were performed at 16 and 18 wk after viral injection (23 and 25 wk on HFD), respectively. Rats were killed at 26 wk on HFD. We found that AAVshCTL rats on HFD remained hyperphagic, obese, glucose intolerant, and insulin resistant relative to lean control RC-fed rats receiving DMH injection of AAVshCTL, whereas these alterations were reversed in NPY knockdown rats fed a HFD. NPY knockdown rats exhibited normal food intake, body weight, glucose tolerance, and insulin sensitivity, as seen in lean control rats. Together, these results demonstrate a therapeutic action of DMH NPY knockdown against obesity and impaired glucose homeostasis in rats, providing a potential target for the treatment of obesity and diabetes.