Opposing actions of co-released GABA and neurotensin on the activity of preoptic neurons and on body temperature.

Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (PACAP) neurons firing activity. GABA-A receptor antagonist or genetic deletion of VGAT in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking VGAT resulted in excitation of PACAP neurons and hypothermia. Mice lacking VGAT expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5 °C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic PACAP neurons. Taken together our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic PACAP neurons is the cellular mechanism that triggers this response.

Activation of Preoptic Arginine Vasopressin Neurons Induces Hyperthermia in Male Mice.

Arginine vasopressin (AVP) is a neuropeptide acting as a neuromodulator in the brain and plays multiple roles, including a thermoregulatory one. However, the cellular mechanisms of action are not fully understood. Carried out are patch clamp recordings and calcium imaging combined with pharmacological tools and single-cell RT-PCR to dissect the signaling mechanisms activated by AVP. Optogenetics combined with patch-clamp recordings were used to determine the neurochemical nature of these neurons. Also used is telemetry combined with chemogenetics to study the effect of activation of AVP neurons in thermoregulatory mechanisms. This article reports that AVP neurons in the medial preoptic (MPO) area release GABA and display thermosensitive firing activity. Their optogenetic stimulation results in a decrease of the firing rates of MPO pituitary adenylate cyclase-activating polypeptide (PACAP) neurons. Local application of AVP potently modulates the synaptic inputs of PACAP neurons, by activating neuronal AVPr1a receptors and astrocytic AVPr1b receptors. Chemogenetic activation of MPO AVP neurons induces hyperthermia. Chemogenetic activation of all AVP neurons in the brain similarly induces hyperthermia and, in addition, decreases the endotoxin activated fever as well as the stress-induced hyperthermia.

Neurotensin induces hypothermia by activating both neuronal neurotensin receptor 1 and astrocytic neurotensin receptor 2 in the median preoptic nucleus.

Neurotensin (NTS) is a neuropeptide acting as a neuromodulator in the brain and is a very potent hypothermic agent. However, the cellular mechanisms of actions are not fully understood. Here we report that NTS increases the firing rate of preoptic GABAergic neurons by activating both neurotensin receptor 1 (NTSR1) and neurotensin receptor 2 (NTSR2), expressed by neurons and astrocytes, respectively. Downstream of NTSR1 the neuropeptide activated an inward current, calcium release from intracellular stores and, postsynaptically, increased frequency and amplitude of inhibitory synaptic events. NTSR2 activation in astrocytes resulted in increased excitatory input in preoptic GABAergic neurons, an effect which was dependent upon the activation of P2X4 receptors. We also found that neuromedin N acted as a selective agonist at the NTSR1. Surprisingly, activation of both NTSR1 and NTSR2 in the median preoptic nucleus was required for activating a full hypothermic response.

Central thermoreceptors.

Homeotherms maintain their core body temperature within a narrow range by employing multiple redundant mechanisms to control heat production and dissipation. Preoptic area/anterior hypothalamic (PO/AH) neurons receive thermal signals from peripheral and deep-body thermoreceptors as well as hormonal and metabolic signals. A population of PO/AH neurons termed warm-sensitive increase their firing temperature with warming and are considered central thermoreceptors. Electrophysiologic and pharmacologic experiments have provided descriptions of their characteristics and signaling mechanisms. These studies have also allowed insights into the mechanisms by which neurochemicals important in thermoregulation exert their influence. Finally, the cellular mechanism involved in the interactions between thermoregulation and other aspects of homeostasis, such as energy metabolism and osmoregulation, have started to be unraveled.

Gastrin-releasing peptide receptor mediates the excitation of preoptic GABAergic neurons by bombesin.

Bombesin, a pan agonist of the bombesin-like peptide receptor family, elicits potent hypothermia when applied centrally. The signaling mechanisms involved are not known. Here we report that GABAergic preoptic neurons express gastrin-releasing peptide (GRP) receptors and are directly excited by GRP or bombesin. This effect was abolished by a GRP receptor antagonist. A partially overlapping group of preoptic GABAergic neurons express bombesin-like receptor 3 (BRS3), however their activation results in a decrease in firing rate. The excitatory effects of bombesin or GRP were not affected by BRS3 antagonist. GRP activated a Ca2+-dependent inward nonselective cationic current and Ca2+ release from intracellular stores. Our data indicate that GRP receptors mediate the excitatory effects of bombesin in preoptic neurons.

Histamine receptor signaling in energy homeostasis.

Histamine modulates several aspects of energy homeostasis. By activating histamine receptors in the hypothalamus the bioamine influences thermoregulation, its circadian rhythm, energy expenditure and feeding. These actions are brought about by activation of different histamine receptors and/or the recruitment of distinct neural pathways. In this review we describe the signaling mechanisms activated by histamine in the hypothalamus, the evidence for its role in modulating energy homeostasis as well as recent advances in the understanding of the cellular and neural network mechanisms involved. This article is part of the Special Issue entitled 'Histamine Receptors'.

Electrical remodeling of preoptic GABAergic neurons involves the Kv1.5 subunit.

The electrogenic machinery of an excitable cell can adapt in response to changes in input, genetic deficit or in pathological conditions, however the underlying molecular mechanisms are not understood. In cases of genetic deletion it is commonly observed that a channel subunit from the same family replaces the missing one. We have previously reported that Kv4.2-/- preoptic GABAergic neurons display identical firing characteristics to those of wild-type neurons despite having reduced A-type currents, and that, surprisingly, they present a robust upregulation of a delayed rectifier current, the nature of which is unknown. Here, using pharmacology, qPCR and Western blots we report that, although the wild-type neurons express several Kv subunits, the upregulated current is conducted by the Kv1.5 subunit exclusively. Thus, this study reveals the molecular nature of a novel mechanism of electrical remodeling in central neurons.

Persistent histamine excitation of glutamatergic preoptic neurons.

Thermoregulatory neurons of the median preoptic nucleus (MnPO) represent a target at which histamine modulates body temperature. The mechanism by which histamine excites a population of MnPO neurons is not known. In this study it was found that histamine activated a cationic inward current and increased the intracellular Ca(2+) concentration, actions that had a transient component as well as a sustained one that lasted for tens of minutes after removal of the agonist. The sustained component was blocked by TRPC channel blockers. Single-cell reverse transcription-PCR analysis revealed expression of TRPC1, TRPC5 and TRPC7 subunits in neurons excited by histamine. These studies also established the presence of transcripts for the glutamatergic marker Vglut2 and for the H1 histamine receptor in neurons excited by histamine. Intracellular application of antibodies directed against cytoplasmic sites of the TRPC1 or TRPC5 channel subunits decreased the histamine-induced inward current. The persistent inward current and elevation in intracellular Ca(2+) concentration could be reversed by activating the PKA pathway. This data reveal a novel mechanism by which histamine induces persistent excitation and sustained intracellular Ca(2+) elevation in glutamatergic MnPO neurons.

Mechanism of H2 histamine receptor dependent modulation of body temperature and neuronal activity in the medial preoptic nucleus.

Histamine is involved in the central control of arousal, circadian rhythms and metabolism. The preoptic area, a region that contains thermoregulatory neurons is the main locus of histamine modulation of body temperature. Here we report that in mice, histamine activates H(2) subtype receptors in the medial preoptic nucleus (MPON) and induces hyperthermia. We also found that a population of glutamatergic MPON neurons express H(2) receptors and are excited by histamine or H(2) specific agonists. The agonists decreased the input resistance of the neuron and increased the depolarizing "sag" observed during hyperpolarizing current injections. Furthermore, at -60 mV holding potential, activation of H(2) receptors induced an inward current that was blocked by ZD7288, a specific blocker of the hyperpolarization activated cationic current (I(h)). Indeed, activation of H(2) receptors resulted in increased I(h) amplitude in response to hyperpolarizing voltage steps and a depolarizing shift in its voltage-dependent activation. The neurons excited by H(2) specific agonism expressed the HCN1 and HCN2 channel subunits. Our data indicate that at the level of the MPON histamine influences thermoregulation by increasing the firing rate of glutamatergic neurons that express H(2) receptors.

AdipoR1 and 2 are expressed on warm sensitive neurons of the hypothalamic preoptic area and contribute to central hyperthermic effects of adiponectin.

Adiponectin can act in the brain to increase energy expenditure and reduce body weight by mechanisms not entirely understood. We found that adiponectin type 1 and type 2 receptors (AdipoR1 and AdipoR2) are expressed in warm sensitive neurons of the hypothalamic preoptic area (POA) which play a critical role in the regulation of core body temperature (CBT) and energy balance. Thus, we tested the ability of adiponectin to influence CBT in wild-type mice and in mice deficient for AdipoR1 or AdipoR2. Local injection of adiponectin into the POA induced prolonged elevation of core body temperature and decreased respiratory exchange ratio (RER) indicating that increased energy expenditure is associated with increased oxidation of fat over carbohydrates. In AdipoR1 deficient mice, the ability of adiponectin to raise CBT was significantly blunted and its ability to decrease RER was completely lost. In AdipoR2 deficient mice, adiponectin had only diminished hyperthermic effects but reduced RER similarly to wild type mice. These results indicate that adiponectin can contribute to energy homeostasis by regulating CBT by direct actions on AdipoR1 and R2 in the POA.

Insulin-like growth factor 1-mediated hyperthermia involves anterior hypothalamic insulin receptors.

The objective is to investigate the role of insulin-like growth factor 1 (IGF-1) in the regulation of core body temperature. Sequencing cDNA libraries from individual warm-sensitive neurons from the preoptic area (POA) of the hypothalamus, a region involved in the central control of thermoregulation, identified neurons that express both IGF-1 receptor (IGF-1R) and insulin receptor transcripts. The effects of administration of IGF-1 into the POA was measured by radiotelemetry monitoring of core temperature, brown adipose tissue (BAT) temperature, metabolic assessment, and imaging of BAT by positron emission tomography of 2-[(18)F]fluoro-2-deoxyglucose uptake combined with computed tomography. IGF-1 injection into the POA caused dose-dependent hyperthermia that could be blocked by pretreatment with the IGF-1R tyrosine kinase inhibitor, PQ401. The IGF-1-evoked hyperthermia involved activation of brown adipose tissue and was accompanied by a switch from glycolysis to fatty acid oxidation as a source of energy as shown by lowered respiratory exchange ratio. Transgenic mice that lack neuronal insulin receptor expression in the brain (NIRKO mice) were unable to mount the full hyperthermic response to IGF-1, suggesting that the IGF-1 mediated hyperthermia is partly dependent on expression of functional neuronal insulin receptors. These data indicate a novel thermoregulatory role for both IGF-1R and neuronal insulin receptors in IGF-1 activation of BAT and hyperthermia. These central effects of IGF-1 signaling may play a role in regulation of metabolic rate, aging, and the risk of developing type 2 diabetes.

Kv4.2 mediates histamine modulation of preoptic neuron activity and body temperature.

Histamine regulates arousal, circadian rhythms, and thermoregulation. Activation of H3 histamine receptors expressed by preoptic GABAergic neurons results in a decrease of their firing rate and hyperthermia. Here we report that an increase in the A-type K⁺ current in preoptic GABAergic neurons in response to activation of H3 histamine receptors results in decreased firing rate and hyperthermia in mice. The Kv4.2 subunit is required for these actions in spite of the fact that Kv4.2⁻/⁻ preoptic GABAergic neurons display A-type currents and firing characteristics similar to those of wild-type neurons. This electrical remodeling is achieved by robust upregulation of the expression of the Kv4.1 subunit and of a delayed rectifier current. Dynamic clamp experiments indicate that enhancement of the A-type current by a similar amount to that induced by histamine is sufficient to mimic its robust effect on firing rates. These data indicate a central role played by the Kv4.2 subunit in histamine regulation of body temperature and its interaction with pERK1/2 downstream of the H3 receptor. We also reveal that this pathway provides a mechanism for selective modulation of body temperature at the beginning of the active phase of the circadian cycle.

A pathologic cascade leading to synaptic dysfunction in alpha-synuclein-induced neurodegeneration.

Several neurodegenerative diseases are typified by intraneuronal alpha-synuclein deposits, synaptic dysfunction, and dementia. While even modest alpha-synuclein elevations can be pathologic, the precise cascade of events induced by excessive alpha-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this, we developed a quantitative model system to evaluate evolving alpha-synuclein-induced pathologic events with high spatial and temporal resolution, using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-alpha-synuclein. Transgenic alpha-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed, several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons ("vacant synapses") with diminished levels in the remainder, suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains, highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic alpha-synuclein leads to a loss of a number of critical presynaptic proteins, thereby inducing functional synaptic deficits.

Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons.

The preoptic area/anterior hypothalamus, a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase and was not dependent on synaptic activity. Furthermore, a population of non-GABAergic neurons was depolarized, and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca(2+) release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked, indicating that it represents a postsynaptic effect. Single-cell reverse transcription-PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons, while H1 receptors were expressed in non-GABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long-lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype-specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons that express H1 and H3 receptor subtypes, respectively.

Insulin causes hyperthermia by direct inhibition of warm-sensitive neurons.

OBJECTIVE: Temperature and nutrient homeostasis are two interdependent components of energy balance regulated by distinct sets of hypothalamic neurons. The objective is to examine the role of the metabolic signal insulin in the control of core body temperature (CBT). RESEARCH DESIGN AND METHODS: The effect of preoptic area administration of insulin on CBT in mice was measured by radiotelemetry and respiratory exchange ratio. In vivo 2-[(18)F]fluoro-2-deoxyglucose uptake into brown adipose tissue (BAT) was measured in rats after insulin treatment by positron emission tomography combined with X-ray computed tomography imaging. Insulin receptor-positive neurons were identified by retrograde tracing from the raphe pallidus. Insulin was locally applied on hypothalamic slices to determine the direct effects of insulin on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. RESULTS: Injection of insulin into the preoptic area of the hypothalamus induced a specific and dose-dependent elevation of CBT mediated by stimulation of BAT thermogenesis as shown by imaging and respiratory ratio measurements. Retrograde tracing indicates that insulin receptor-expressing warm-sensitive neurons activate BAT through projection via the raphe pallidus. Insulin applied on hypothalamic slices acted directly on intrinsically warm-sensitive neurons by inducing hyperpolarization and reducing firing rates. The hyperthermic effects of insulin were blocked by pretreatment with antibodies to insulin or with a phosphatidylinositol 3-kinase inhibitor. CONCLUSIONS: Our findings demonstrate that insulin can directly modulate hypothalamic neurons that regulate thermogenesis and CBT and indicate that insulin plays an important role in coupling metabolism and thermoregulation at the level of anterior hypothalamus.

Hypothalamic and dietary control of temperature-mediated longevity.

Temperature is an important modulator of longevity and aging in both poikilotherms and homeotherm animals. In homeotherms, temperature homeostasis is regulated primarily in the preoptic area (POA) of the hypothalamus. This region receives and integrates peripheral, central and environmental signals and maintains a nearly constant core body temperature (T(core)) by regulating the autonomic and hormonal control of heat production and heat dissipation. Temperature sensitive neurons found in the POA are considered key elements of the neuronal circuitry modulating these effects. Nutrient homeostasis is also a hypothalamically regulated modulator of aging as well as one of the signals that can influence T(core) in homeotherms. Investigating the mechanisms of the regulation of nutrient and temperature homeostasis in the hypothalamus is important to understanding how these two elements of energy homeostasis influence longevity and aging as well as how aging can affect hypothalamic homeostatic mechanisms.

Rapid, concurrent alterations in pre- and postsynaptic structure induced by naturally-secreted amyloid-beta protein.

In Alzheimer's disease increasing evidence attributes synaptic and cognitive deficits to soluble oligomers of amyloid beta protein (Abeta), even prior to the accumulation of amyloid plaques, neurofibrillary tangles, and neuronal cell death. Here we show that within 1-2 h picomolar concentrations of cell-derived, soluble Abeta induce specific alterations in pre- and postsynaptic morphology and connectivity in cultured hippocampal neurons. Clusters of presynaptic vesicle markers decreased in size and number at glutamatergic but not GABAergic terminals. Dendritic spines also decreased in number and became dysmorphic, as spine heads collapsed and/or extended long protrusions. Simultaneous time-lapse imaging of axon-dendrite pairs revealed that shrinking spines sometimes became disconnected from their presynaptic varicosity. Concomitantly, miniature synaptic potentials decreased in amplitude and frequency. Spine changes were prevented by blockers of nAChRs and NMDARs. Washout of Abeta within the first day reversed these spine changes. Further, spine changes reversed spontaneously by 2 days, because neurons acutely developed resistance to continuous Abeta exposure. Thus, rapid Abeta-induced synapse destabilization may underlie transient behavioral impairments in animal models, and early cognitive deficits in Alzheimer's patients.

Night eating and obesity in the EP3R-deficient mouse.

Adult mice carrying a null mutation of the prostanoid receptor EP3R (EP3R(-/-) mice) exhibit increased frequency of feeding during the light cycle of the day and develop an obese phenotype under a normal fat diet fed ad libitum. EP3R(-/-) mice show increased motor activity, which is not sufficient to offset the increased feeding leading to increased body weight. Altered "nocturnal" activity and feeding behavior is present from a very early age and does not seem to require age-dependent factors for the development of obesity. Obesity in EP3R(-/-) mice is characterized by elevated leptin and insulin levels and >20% higher body weight compared with WT littermates. Abdominal and subcutaneous fat and increased liver weight account for the weight increase in EP3R(-/-) mice. These observations expand the roles of prostaglandin E(2) signaling in metabolic regulation beyond the reported stimulation of leptin release from adipose tissue to involve actions mediated by EP3R in the regulation of sleep architecture and feeding behavior. The findings add to the growing literature on links between inflammatory signaling and obesity.

IL-1beta induces a MyD88-dependent and ceramide-mediated activation of Src in anterior hypothalamic neurons.

The proinflammatory cytokine interleukin 1beta (IL-1beta), acting at IL-1R1 receptors, affects neuronal signaling under both physiological and pathophysiological conditions. The molecular mechanism of the rapid synaptic actions of IL-1beta in neurons is not known. We show here that within minutes of IL-1beta exposure, the firing rate of anterior hypothalamic (AH) neurons in culture was inhibited. This effect was prevented by pre-exposure of the cells to the Src family inhibitor, PP2, suggesting the involvement of Src in the hyperpolarizing effects of IL-1beta. The IL-1beta stimulation of neurons induced a rapid increase in the phosphorylation of the tyrosine kinase Src and kinase suppressor of Ras (ceramide activated protein kinase (CAPK)/KSR) in neurons grown on glia from IL-1RI(-/-) mice. These effects of IL-1beta were dependent on the association of the cytosolic adaptor protein, MyD88, to the IL-1 receptor, and on the activation of the neutral sphingomyelinase, leading to production of ceramide. A cell-permeable analog of ceramide mimicked the effects of IL-1beta on the cultured AH neurons. These results suggest that ceramide may be the second messenger of the fast IL-1beta actions in AH neurons, and that this IL-1beta/ceramide pathway may underlie the fast non-transcription-dependent, electrophysiological effects of IL-1beta observed in AH neurons in vivo.

A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.

Several lines of evidence suggest that a hypoglutamatergic condition may induce a phenotypic loss of cortical parvalbumin (PV)-positive GABAergic interneurons, such as that observed in brain tissue of schizophrenic subjects. However, it is not known whether the loss of PV interneurons is a consequence of the hypoglutamatergic condition or a secondary aspect of the disease. We characterized the signaling and subunit expression of NMDA receptors in cultured cortical PV interneurons and determined whether a hypoglutamatergic condition, created by direct application of sublethal concentrations of ketamine or subunit-selective NMDA receptor antagonists, can affect the expression of the GABAergic markers as observed in vivo. Real-time PCR performed on mRNA isolated from single neurons showed that PV interneurons present a fivefold higher NR2A/NR2B ratio than pyramidal neurons. Brief, nontoxic, exposure to NMDA led to an increase in ERK1/2 (extracellular signal-regulated kinase 1/2) and cAMP response element-binding protein phosphorylation in PV interneurons, and this increase was blocked by the NR2A-selective antagonist NVP-AAM077. Application of the nonselective NMDA receptor antagonist ketamine, at sublethal concentrations, induced a time and dose-dependent decrease in parvalbumin and GAD67 immunoreactivity specifically in PV interneurons. These effects were reversible and were also observed with the NR2A-selective antagonist, whereas the NR2B-selective antagonist Ro-25-6981 only partially reduced GAD67 immunoreactivity. Coexposure to the calcium channel opener BayK, or the group I metabotropic glutamate receptor agonist DHPG [(RS)-3,5-dihydroxyphenylglycine] attenuated the decrease in GAD67 and parvalbumin induced by the NMDA receptor antagonists. These results suggest that the activity of NR2A-containing NMDA receptors play a pivotal role in the maintenance of the GABAergic function of PV interneurons.