Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca2+) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca2+ fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease.SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation.

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons.

Optogenetic activation of melanin-concentrating hormone neurons increases non-rapid eye movement and rapid eye movement sleep during the night in rats.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on - 4 min off) for 24 h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Reversed synaptic effects of hypocretin and NPY mediated by excitatory GABA-dependent synaptic activity in developing MCH neurons.

In mature neurons, GABA is the primary inhibitory neurotransmitter. In contrast, in developing neurons, GABA exerts excitatory actions, and in some neurons GABA-mediated excitatory synaptic activity is more prevalent than glutamate-mediated excitation. Hypothalamic neuropeptides that modulate cognitive arousal and energy homeostasis, hypocretin/orexin and neuropeptide Y (NPY), evoked reversed effects on synaptic actions that were dependent on presynaptic GABA release onto melanin-concentrating hormone (MCH) neurons. MCH neurons were identified by selective green fluorescent protein (GFP) expression in transgenic mice. In adults, hypocretin increased GABA release leading to reduced excitation. In contrast, in the developing brain as studied here with analysis of miniature excitatory postsynaptic currents, paired-pulse ratios, and evoked potentials, hypocretin acted presynaptically to enhance the excitatory actions of GABA. The ability of hypocretin to enhance GABA release increases inhibition in adult neurons but paradoxically enhances excitation in developing MCH neurons. In contrast, NPY attenuation of GABA release reduced inhibition in mature neurons but enhanced inhibition during development by attenuating GABA excitation. Both hypocretin and NPY also evoked direct actions on developing MCH neurons. Hypocretin excited MCH cells by activating a sodium-calcium exchanger and by reducing potassium currents; NPY reduced activity by increasing an inwardly rectifying potassium current. These data for the first time show that both hypocretin and NPY receptors are functional presynaptically during early postnatal hypothalamic development and that both neuropeptides modulate GABA actions during development with a valence of enhanced excitation or inhibition opposite to that of the adult state, potentially allowing neuropeptide modulation of use-dependent synapse stabilization.

Vasopressin and oxytocin excite MCH neurons, but not other lateral hypothalamic GABA neurons.

Neurons that synthesize melanin-concentrating hormone (MCH) colocalize GABA, regulate energy homeostasis, modulate water intake, and influence anxiety, stress, and social interaction. Similarly, vasopressin and oxytocin can influence the same behaviors and states, suggesting that these neuropeptides may exert part of their effect by modulating MCH neurons. Using whole cell recording in MCH-green fluorescent protein (GFP) transgenic mouse hypothalamic brain slices, we found that both vasopressin and oxytocin evoked a substantial excitatory effect. Both peptides reversibly increased spike frequency and depolarized the membrane potential in a concentration-dependent and tetrodotoxin-resistant manner, indicating a direct effect. Substitution of lithium for extracellular sodium, Na(+)/Ca(2+) exchanger blockers KB-R7943 and SN-6, and intracellular calcium chelator BAPTA, all substantially reduced the vasopressin-mediated depolarization, suggesting activation of the Na(+)/Ca(2+) exchanger. Vasopressin reduced input resistance, and the vasopressin-mediated depolarization was attenuated by SKF-96265, suggesting a second mechanism based on opening nonselective cation channels. Neither vasopressin nor oxytocin showed substantial excitatory actions on lateral hypothalamic inhibitory neurons identified in a glutamate decarboxylase 67 (GAD67)-GFP mouse. The primary vasopressin receptor was vasopressin receptor 1a (V1aR), as suggested by the excitation by V1aR agonist [Arg(8)]vasotocin, the selective V1aR agonist [Phe(2)]OVT and by the presence of V1aR mRNA in MCH cells, but not in other nearby GABA cells, as detected with single-cell RT-PCR. Oxytocin receptor mRNA was also detected in MCH neurons. Together, these data suggest that vasopressin or oxytocin exert a minimal effect on most GABA neurons in the lateral hypothalamus but exert a robust excitatory effect on presumptive GABA cells that contain MCH. Thus, some of the central actions of vasopressin and oxytocin may be mediated through MCH cells.

Excitatory neuromodulator reduces dopamine release, enhancing prolactin secretion.

Hypothalamic dopamine neurons inhibit pituitary prolactin secretion. In this issue of Neuron, Lyons et al. provide evidence for a novel model, whereby the excitatory neuropeptide TRH depolarizes gap-junction-coupled dopamine neurons, leading to a shift in the population pattern of action potentials from phasic burst firing to regular tonic firing, hypothetically reducing dopamine release while increasing total spike number.

Enhanced excitatory input to melanin concentrating hormone neurons during developmental period of high food intake is mediated by GABA.

In contrast to the local axons of GABA neurons of the cortex and hippocampus, lateral hypothalamic neurons containing melanin concentrating hormone (MCH) and GABA send long axons throughout the brain and play key roles in energy homeostasis and mental status. In adults, MCH neurons maintain a hyperpolarized membrane potential and most of the synaptic input is inhibitory. In contrast, we found that developing MCH neurons received substantially more excitatory synaptic input. Based on gramicidin-perforated patch recordings in hypothalamic slices from MCH-green fluorescent protein transgenic mice, we found that GABA was the primary excitatory synaptic transmitter in embryonic and neonatal ages up to postnatal day 10. Surprisingly, glutamate assumed only a minor excitatory role, if any. GABA plays a complex role in developing MCH neurons, with its actions conditionally dependent on a number of factors. GABA depolarization could lead to an increase in spikes either independently or in summation with other depolarizing stimuli, or alternately, depending on the relative timing of other depolarizing events, could lead to shunting inhibition. The developmental shift from depolarizing to hyperpolarizing occurred later in the dendrites than in the cell body. Early GABA depolarization was based on a Cl(-)-dependent inward current. An interesting secondary depolarization in mature neurons that followed an initial hyperpolarization was based on a bicarbonate mechanism. Thus during the early developmental period when food consumption is high, MCH neurons are more depolarized than in the adult, and an increased level of excitatory synaptic input to these orexigenic cells is mediated by GABA.

Mu-opioid receptor-mediated depression of the hypothalamic hypocretin/orexin arousal system.

Arousal and maintenance of a wake state is dependent on the hypothalamic hypocretin/orexin system. We found that hypocretin neurons are depressed by opiates, drugs of abuse that reduce cognitive alertness. Met-enkephalin (mENK), an endogenous opioid, and exogenous opiates such as morphine inhibited the hypocretin system by direct actions on the cell body that include reduced spike frequency, hyperpolarization, increased G-protein-coupled inwardly rectifying K(+) channel current, and attenuated calcium current, and indirectly through reducing excitatory synaptic tone by a presynaptic mechanism. CTAP (H-d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH(2)) and naloxone, antagonists of mu-opioid receptors, blocked mu agonist actions. In the absence of exogenous opioids, mu receptor antagonists enhanced activity of the hypocretin system, suggesting ongoing inhibition by endogenous receptors. Morphine pretreatment attenuated subsequent excitatory responses to hypocretin, suggesting a long-lasting depression caused by opiate exposure. Chronic exposure to morphine reduced subsequent responses to morphine and to mENK, but increased the response to opioid receptor antagonists. Together, these data are consistent with the view that the hypocretin system may be an important direct target for drugs of abuse, including opiates, that induce sedation and mental lethargy.

Rapid direct excitation and long-lasting enhancement of NMDA response by group I metabotropic glutamate receptor activation of hypothalamic melanin-concentrating hormone neurons.

The effect of group I metabotropic glutamate receptor (mGluR1 and mGluR5) activation on identified melanin-concentrating hormone (MCH) neurons was studied using patch-clamp recording in hypothalamic slices from green fluorescent protein-expressing transgenic mice. S-3,5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist, depolarized MCH cells and increased spike frequency. The mGluR-mediated depolarization was not blocked with tetrodotoxin but was significantly reduced by replacement of extracellular Na+ with Tris, by Ni2+ or the Na+/Ca2+ exchanger blocker KB-R7943, or with BAPTA in the pipette, consistent with a mechanism based on activation of the Na+/Ca2+ exchanger. DHPG also decreased potassium currents. DHPG-induced depolarization was reduced by either mGluR1 or mGluR5 antagonists, suggesting involvement of both receptor subtypes. DHPG-induced depolarization desensitized; blockade of mGluR1 prevented the desensitization. Group I mGluR activation enhanced NMDA-evoked currents; this enhancement was remarkably long lasting and could be blocked by protein kinase A or C blockers. DHPG potentiated electrically evoked NMDA receptor-mediated postsynaptic currents, and mGluR5 antagonists blocked this action. Group I mGluRs increased spontaneous EPSCs in MCH neurons, possibly by stimulation of nearby mGluR-expressing hypocretin neurons. We found no tonic activation of mGluRs. However, electrical stimulation produced a slow inward current, which could be blocked by group I mGluR antagonists, suggesting high, but not low, levels of synaptically released glutamate activated mGluRs. Together, group I mGluRs increase MCH neuron activity by multiple presynaptic and postsynaptic mechanisms, suggesting mGluRs may therefore play a role in hypothalamic signaling relating to MCH neuron modulation of food intake and energy metabolism.

Cannabinoids excite hypothalamic melanin-concentrating hormone but inhibit hypocretin/orexin neurons: implications for cannabinoid actions on food intake and cognitive arousal.

Cannabinoids modulate energy homeostasis and decrease cognitive arousal, possibly by acting on hypothalamic neurons including those that synthesize melanin-concentrating hormone (MCH) or hypocretin/orexin. Using patch-clamp recordings, we compared the actions of cannabinoid agonists and antagonists on identified MCH or hypocretin neurons in green fluorescent protein-expressing transgenic mice. The cannabinoid type-1 receptor (CB1R) agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpho linylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212,2) depolarized MCH cells and increased spike frequency; in contrast, WIN55,212,2 hyperpolarized and reduced spontaneous firing of the neighboring hypocretin cells, both results consistent with reduced activity seen with intracerebral cannabinoid infusions. These effects were prevented by AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1R antagonist, and by tetrodotoxin, suggesting no postsynaptic effect on either neuron type. In MCH cells, depolarizing WIN55,212,2 actions were abolished by the GABA(A) receptor antagonist bicuculline, suggesting that the CB1R-mediated depolarization was attributable to reduced synaptic GABA release. WIN55,212,2 decreased spontaneous IPSCs, reduced the frequency but not amplitude of miniature IPSCs, and reduced electrically evoked synaptic currents in MCH cells. Glutamate microdrop experiments suggest that WIN55,212,2 acted on axons arising from lateral hypothalamus local inhibitory cells that innervate MCH neurons. In hypocretin neurons, the reduced spike frequency induced by WIN55,212,2 was attributable to presynaptic attenuation of glutamate release; CB1R agonists depressed spontaneous and evoked glutamatergic currents and reduced the frequency of miniature EPSCs. Cannabinoid actions on hypocretin neurons were abolished by ionotropic glutamate receptor antagonists. Together, these results show that cannabinoids have opposite effects on MCH and hypocretin neurons. These opposing actions could help explain the increase in feeding and reduction in arousal induced by cannabinoids.

Differential target-dependent actions of coexpressed inhibitory dynorphin and excitatory hypocretin/orexin neuropeptides.

The hypocretin/orexin arousal system plays a key role in maintaining an alert wake state. The hypocretin peptide is colocalized with an opioid peptide, dynorphin. As dynorphin may be coreleased with hypocretin, we asked what action simultaneous stimulation with the excitatory neuropeptide hypocretin and the inhibitory peptide dynorphin might exert on cells postsynaptic to hypocretin axons, including hypocretin neurons. Hypocretin neurons received direct synaptic contact from other hypocretin neurons but showed little direct response to hypocretin. Here, we show that mouse hypocretin neurons are acutely sensitive to dynorphin. Dynorphin inhibits the hypocretin system by direct postsynaptic actions (hyperpolarization, decreased spike frequency, increased GIRK (G-protein-gated inwardly rectifying K+ channel) current, and attenuated calcium current, and indirectly by reducing excitatory synaptic tone. Interestingly, a selective antagonist of kappa-opioid receptors enhanced activity of the hypocretin system, suggesting ongoing depression by endogenous hypothalamic opioids. Electrical stimulation of hypothalamic microslices that contained hypocretin cells and their axons evoked dynorphin release. Costimulation with dynorphin and hypocretin had three different effects on neurons postsynaptic to hypocretin axons: direct response to only one or the other of the two peptides [hypocretin cells respond to dynorphin, arcuate neuropeptide Y (NPY) cells respond to hypocretin], differential desensitization causing shift from inhibitory current to excitatory current with repeated coexposure (melanin-concentrating hormone neurons), synergistic direct excitation by hypocretin and presynaptic attenuation of inhibition by dynorphin (arcuate NPY neurons). These results suggest that hypocretin neurons may be able to exercise a high degree of modulatory control over postsynaptic targets using multiple neuropeptides with target-dependent actions.

Peptide YY(3-36) inhibits both anorexigenic proopiomelanocortin and orexigenic neuropeptide Y neurons: implications for hypothalamic regulation of energy homeostasis.

Peptide YY(3-36) (PYY(3-36)) is released by endocrine cells of the gut and may serve as an important long-distance neuropeptide signal relating energy balance information to the brain to depress food intake. The postulated mechanism is the activation of anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. In striking contrast, using voltage and current-clamp recording, we found that PYY(3-36) consistently, dose dependently, and reversibly inhibited POMC cells by reducing action potentials, hyperpolarizing the membrane potential, decreasing input resistance and inward calcium currents, increasing G-protein-gated inwardly rectifying K+ channel currents, and presynaptically inhibiting release of excitatory glutamate. Importantly, we found PYY(3-36) had similar inhibitory effects on identified orexigenic neuropeptide Y (NPY) neurons. In both cell types, these effects were blocked by BIIE0246, a Y2 receptor antagonist. Together, these data argue that anorexigenic actions of PYY(3-36) are mediated more likely by inhibition of NPY neurons. Dual PYY(3-36) inhibition of both NPY and POMC cells may temporarily reduce the contribution of arcuate cells to feeding circuits, enhancing the role of other CNS loci.

Direct excitation of hypocretin/orexin cells by extracellular ATP at P2X receptors.

Hypocretin/orexin (hcrt) neurons play an important role in hypothalamic arousal and energy homeostasis. ATP may be released by neurons or glia or by pathological conditions. Here we studied the effect of extracellular ATP on hypocretin cells using whole cell patch-clamp recording in hypothalamic slices of transgenic mice expressing green fluorescent protein (GFP) exclusively in hcrt-producing cells. Local application of ATP induced a dose-dependent increase in spike frequency. In the presence of TTX, ATP (100 microM) depolarized the cells by 7.8 +/- 1.2 mV. In voltage clamp under blockade of synaptic activity with the GABA(A) receptor antagonist bicuculline, and ionotropic glutamate receptor antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), ATP (100 microM) evoked an 18 pA inward current. The inward current was blocked by extracellular choline substitution for Na+, had a reversal potential of -27 mV, and was not affected by nominally Ca2+-free external buffer, suggesting that ATP activated a nonselective cation current. All excitatory effects of ATP showed rapid attenuation. ATP-induced excitatory actions were mimicked by nonhydrolyzable ATP-gamma-S but not by alpha,beta-MeATP and inhibited by the purinoceptor antagonists suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS). The current was potentiated by a decrease in bath pH, suggesting P2X2 subunit involvement. Frequency and amplitude of spontaneous and miniature synaptic events were not altered by ATP. Suramin, but not PPADS, caused a small suppression of evoked excitatory synaptic potentials. Together, these results show a depolarizing response to extracellular ATP that would lead to an increased activity of the hypocretin arousal system.

Direct and indirect inhibition by catecholamines of hypocretin/orexin neurons.

Hypothalamic hypocretin enhances arousal, similar to the actions of norepinephrine (NE). The physiological actions of NE were examined in hypocretin neurons identified by selective green fluorescent protein expression in transgenic mouse hypothalamic slices using whole-cell recording. NE induced an outward current, inhibited spike frequency, and hyperpolarized hypocretin neurons dose dependently. Similar actions were evoked by the selective alpha2 adrenergic agonist clonidine. The alpha2 antagonist idazoxan increased spike frequency, suggesting tonic NE-mediated inhibition. The NE-induced current was inwardly rectified, and the reversal potential was dependent on external potassium concentration; it was blocked by barium in the bath and by GTP-gamma-S in the pipette, suggesting activation of a G-protein inward rectifying K+ (GIRK) current. NE and clonidine decreased calcium currents evoked by depolarizing voltage steps. The selective alpha1 adrenergic agonist phenylephrine had no effect on membrane potential but did increase IPSC frequency; miniature IPSC frequency was also increased, in some cells without any effect on amplitude, suggesting a facilitative presynaptic action at alpha1 receptors on GABAergic axons that innervate hypocretin neurons. NE therefore inhibits hypocretin neurons directly through two mechanisms: activation of a GIRK current, depression of calcium currents, and indirectly through increased inhibitory GABA input. Similar to NE, dopamine and epinephrine reduced or blocked spikes and, in the presence of TTX, showed direct hyperpolarizing actions. The action of dopamine was blocked by the D2 receptor antagonist eticlopride, whereas a D1/5 antagonist had no effect. These data suggest that catecholamines evoke strong inhibitory actions on hypocretin neurons and suggest negative feedback from catecholamine cells that may be excited by hypocretin.

Neuropeptide Y inhibits hypocretin/orexin neurons by multiple presynaptic and postsynaptic mechanisms: tonic depression of the hypothalamic arousal system.

Neurons that release neuropeptide Y (NPY) have important effects on hypothalamic homeostatic regulation, including energy homeostasis, and innervate hypocretin neurons. Using whole-cell patch-clamp recording, we explored NPY actions on hypocretin cells identified by selective green fluorescent protein expression in mouse hypothalamic slices. NPY reduced spike frequency and hyperpolarized the membrane potential of hypocretin neurons. The NPY hyperpolarizing action persisted in tetrodotoxin (TTX), was mimicked by Y1 receptor-selective agonists [Pro34]-NPY and [D-Arg25]-NPY, and was abolished by the Y1-specific antagonist BIBP3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-amide], consistent with a direct activation of postsynaptic Y1 receptors. NPY induced a current that was dependent on extracellular potassium, reversed near the potassium equilibrium potential, showed inward rectification, was blocked by extracellular barium, and was abolished by GDP-betaS in the recording pipette, consistent with a G-protein-activated inwardly rectifying K+ (GIRK) current. [Pro34]-NPY evoked, and BIBP3226 blocked, the activation of the GIRK-type current, indicating mediation by a Y1 receptor. NPY attenuated voltage-dependent calcium currents mainly via a Y1 receptor subtype. BIBP3226 increased spontaneous spike frequency, suggesting an ongoing Y1 receptor-mediated NPY inhibition. In TTX, miniature EPSCs were reduced in frequency but not amplitude by NPY, NPY13-36, and [D-Trp32]-NPY, but not by [Pro34]-NPY, suggesting the presynaptic inhibition was mediated by a Y2/Y5 receptor. NPY had little effect on GABA-mediated miniature IPSCs but depressed spontaneous IPSCs. Together, these data support the view that NPY reduces the activity of hypocretin neurons by multiple presynaptic and postsynaptic mechanisms and suggest NPY axons innervating hypocretin neurons may tonically attenuate hypocretin-regulated arousal.

Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection.

Neurons that synthesize melanin-concentrating hormone (MCH) may modulate arousal and energy homeostasis. The scattered MCH neurons have been difficult to study, as they have no defining morphological characteristics. We have developed a viral approach with AAV for selective long-term reporter gene (GFP) expression in MCH neurons, allowing the study of their cellular physiology in hypothalamic slices. MCH neurons showed distinct membrane properties compared to other neurons infected with the same virus with a cytomegalovirus promoter. Transmitters of extrahypothalamic arousal systems, including norepinephrine, serotonin, and the acetylcholine agonist muscarine, evoked direct inhibitory actions. Orexigenic neuropeptide Y was inhibitory by pre- and postsynaptic mechanisms; an anorexigenic melanocortin agonist had no effect. In contrast, the hypothalamic arousal peptide hypocretin/orexin evoked a direct inward current and increased excitatory synaptic activity and spike frequency in the normally silent MCH neurons. Together, these data support the view that MCH neurons may integrate information within the arousal system in favor of energy conservation.

Group III metabotropic glutamate receptors maintain tonic inhibition of excitatory synaptic input to hypocretin/orexin neurons.

Hypocretin/orexin neurons play an important role in hypothalamic arousal. Synaptic glutamate input to hypocretin neurons regulates cell firing. We studied the actions of group III metabotropic glutamate receptors (mGluRs) in modulating the activity of hypocretin neurons using whole-cell voltage- and current-clamp recording in mouse whole hypothalamic slices or minislices consisting only of the lateral hypothalamus. Selective green fluorescent protein expression was used to detect live hypocretin neurons. The mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP-4) inhibited synaptic input to hypocretin neurons in a dose-dependent manner; both spontaneous glutamate and GABA-mediated synaptic currents were reduced in frequency. l-AP-4 also reduced the amplitude of postsynaptic potentials evoked by a stimulating electrode placed medial or lateral to the recorded cell. No postsynaptic effect of l-AP-4 was found relative to membrane potential, input resistance, or AMPA-evoked currents. l-AP-4 appeared to act by a presynaptic mechanism and reduced the frequency of both glutamate- and GABA-mediated miniature events recorded in the presence of tetrodotoxin, with no change in amplitude. (RS)-phosphonopentanoic acid (CPPG), a group III mGluR antagonist, suppressed the actions of l-AP-4. Of substantial interest, CPPG by itself increased synaptic activity recorded in hypocretin neurons, suggesting an ongoing inhibitory tone attributable to activation of group III mGluRs. Glutamatergic interneurons have been suggested to play a role in a positive feedback recruitment of hypocretin on hypocretin neurons. l-AP-4 blocked hypocretin-mediated increases in EPSCs and attenuated the hypocretin-mediated increase in spike frequency. Together, these data suggest that tonically active inhibitory mGluRs are expressed on local hypocretin-sensitive glutamate neurons within the lateral hypothalamus that modulate the output of the hypocretin arousal system.

Hypocretin/Orexin excites hypocretin neurons via a local glutamate neuron-A potential mechanism for orchestrating the hypothalamic arousal system.

Neurons that release hypocretin/orexin modulate sleep, arousal, and energy homeostasis; the absence of hypocretin results in narcolepsy. Here we present data on the physiological characteristics of these cells, identified with GFP in transgenic mouse brain slices. Hypocretin-1 and -2 depolarized hypocretin neurons by 15mV and evoked an increase in spike frequency (+366% from a 1-3 Hz baseline). The mechanism for this appears to be hypocretin-mediated excitation of local glutamatergic neurons that regulate hypocretin neuron activity, in part by presynaptic facilitation of glutamate release. This represents a possible mechanism for orchestrating the output of the diffuse hypothalamic arousal system. No direct effect of hypocretin on membrane properties of hypocretin cells was detected. Norepinephrine and serotonin, transmitters of other arousal systems, decreased spike frequency and evoked outward currents, whereas acetylcholine and histamine had little effect.

Excitatory actions of GABA increase BDNF expression via a MAPK-CREB-dependent mechanism--a positive feedback circuit in developing neurons.

During early neuronal development, GABA functions as an excitatory neurotransmitter, triggering membrane depolarization, action potentials, and the opening of plasma membrane Ca(2+) channels. These excitatory actions of GABA lead to a number of changes in neuronal structure and function. Although the effects of GABA on membrane biophysics during early development have been well documented, little work has been done to examine the possible mechanisms underlying GABA-regulated plastic changes in the developing brain. This study focuses on GABA-regulated kinase activity and transcriptional control. We utilized a combination of Western blotting and immunocytochemical techniques to examine two potential downstream pathways regulated by GABA excitation: the p42/44 mitogen-activated protein kinase (MAPK) cascade and the transcription factor cyclic AMP response element binding protein (CREB). During early development of cultured hypothalamic neurons (5 days in vitro), stimulation with GABA triggered activation of the MAPK cascade and phosphorylation of CREB at Ser 133. These effects were mediated by the GABA(A) receptor, since administration of the GABA(A) receptor-specific agonist muscimol (50 microM) triggered pathway activation, and pretreatment with the GABA(A)-receptor specific antagonist bicuculline (20 microM) blocked pathway activation. Immunocytochemistry revealed a spatial and temporal correlation between activation of the MAPK cascade and CREB phosphorylation. Pretreatment with the MAPK/ERK kinase (MEK) inhibitor U0126 (10 microM) attenuated CREB phosphorylation, indicating that the MAPK pathway regulates that activation state of CREB. In contrast to the excitatory effects observed during early development, in more mature neurons, GABA functions as an inhibitory transmitter. Consistent with this observation, GABA(A) receptor activation did not stimulate MAPK cascade activation or CREB phosphorylation in mature cultures (18 days in vitro). To determine whether GABA(A) receptor activation during early development stimulates gene expression, we examined the inducible expression of the neurotrophin brain-derived neurotrophic factor (BDNF). Both GABA and muscimol stimulated BDNF expression, and pretreatment with U0126 attenuated GABA-induced BDNF expression. Whole cell electrophysiological recording was used to assess the effects of BDNF on GABA release. BDNF (100 ng/ml) dramatically increased the frequency of excitatory GABAergic spontaneous postsynaptic currents. Together, these data suggest a positive excitatory feedback loop between GABA and BDNF expression during early development, where GABA facilitates BDNF expression, and BDNF facilitates the synaptic release of GABA. Signaling via the MAPK cascade and the transcription factor CREB appear to play a substantial role in this process.