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.

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.

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.

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.

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'.

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.

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.

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.

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.

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.

Ceramide mediates the rapid phase of febrile response to IL-1beta.

IL-1beta was identified after a long search for the endogenous pyrogen. It acts by inducing synthesis of prostaglandin E2, which mediates the late phase of IL-1beta-induced fever. Here we show by radiotelemetry that the early phase of the fever response to IL-1beta is mediated by ceramide. Hypothalamic application of the cell-penetrating C2-ceramide mimics the rapid phase of the IL-1beta-induced fever. Inhibition of ceramide synthesis blocks the rapid phase of fever but does not affect the slower prostaglandin E2-dependent phase, which is blocked by indomethacin or by null mutation of the EP3 prostanoid receptor. Electrophysiological experiments on preoptic area/anterior hypothalamic neurons show that C2-ceramide, but not dihydroceramide, mimics the rapid hyperpolarizing effects of IL-1beta on the activity of warm-sensitive hypothalamic neurons. IL-1beta-mediated hyperpolarization is blocked by PP2, the selective inhibitor of the protein tyrosine kinase Src, which is known to be activated by ceramide. These in vivo and in vitro data suggest that ceramide fulfills the criteria for an endogenous pyrogen.

Membrane stretch accelerates activation and slow inactivation in Shaker channels with S3-S4 linker deletions.

At low P(open)(V) Shaker exhibits pronounced stretch-activation. Possible explanations for Shaker's sensitivity to tension include 1) Shaker channels are sufficiently distensible that stretch produces novel channel states and 2) Shaker channels expand in the plane of the membrane during voltage gating. For channels expressed in oocytes, we compared effects of patch stretch on Shaker and mutants that retain their voltage-gating ability but activate sluggishly because all or most of the S3-S4 linker has been deleted. Deletants had 10, 5, or 0 amino acid (aa) linkers, whereas wild-type is 31 aa. In deletants, though activation is exceptionally slow, slow inactivation is exceptionally quick; the resulting kinetic match was a bonus that allowed effects of stretch to be followed simultaneously in both processes. With the intact linker, an approximately 3 orders of magnitude mismatch in the two processes makes this impracticable. Standard stretch stimuli increased the rates and extent of activation by about the same degree in wild type and deletants, with effects especially pronounced near the foot of G(V). In deletants (where slow inactivation is strongly coupled to activation) stretch also accelerated slow inactivation. Maximum conductances were unaffected by stretch in all variants. In ramp clamp dose experiments, near-lytic patch stretch acted, for all variants, like a approximately 10 mV hyperpolarizing shift. These results suggested that, whether basal rates were high (wild type) or low (deletants), stretch acted by facilitating voltage-dependent activation. Channel activity was therefore simulated with/without "tension," tension being simulated via rate changes at voltage-dependent closed-closed transitions that might involve in-plane expansion (explanation 2). Simulated Delta P(open) arising from approximately 2 kT of "mechanical gating energy" mimicked experimental effects seen with comfortably sub-lytic stretch.

Prostaglandin E2-increased thermosensitivity of anterior hypothalamic neurons is associated with depressed inhibition.

Temperature responses of anterior hypothalamic neurons are considered key elements in the regulation of the temperature setpoint of homeotherms. We have investigated the sensitivity to warming of cultured neurons of the AH from mice with electrophysiological and immunocytochemical techniques. In control experiments, only approximately 9% of the 3- to 5-week-old cells exhibited changes of their basic firing rate when the temperature was raised from 37 degrees C to 40 degrees C. This ratio was increased to 27% after the cultures were "primed" by adding prostaglandin E2 (PGE2), an endogenous pyrogen, in the extracellular medium. In these neurons the firing rate was significantly increased, and the frequency of the gamma gamma-aminobutyric acid (GABA) inhibitory postsynaptic potentials was markedly decreased. In contrast, the resting potential and membrane resistance of the recorded cells remained unchanged. PGE2 was found to decrease the level of phosphorylation of the extracellular signal-regulated kinases 1 and 2 in a subset of GABAergic neurons that express the E-prostanoid receptor type 3. Inhibition of ERK1/2 by U0126 mimicked the effects of PGE2. These data indicate that PGE2 acts primarily on the excitability of GABAergic presynaptic cells, most likely via alterations of voltage-gated K+ channels. Our results also suggest that far from being an inherent property of a specialized class of neurons, the degree of thermosensitivity can be strongly modulated by synaptic activity and is a more adaptive property of hypothalamic neurons than previously thought.

Mechanosensitivity of N-type calcium channel currents.

Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.