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1.  Slc25a12 disruption alters myelination and neurofilaments: A model for a hypomyelination syndrome and childhood neurodevelopmental disorders 
Biological psychiatry  2009;67(9):887-894.
Background
SLC25A12, a susceptibility gene for autism spectrum disorders (ASDs) that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate/glutamate carrier (AGC1). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and ATP production.
Methods
We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies.
Results
Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In P13-14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cellautonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetyl aspartate (NAA) and/or alterations in the NADH/NAD+ ratio, resulting in myelin defects.
Conclusions
Our data implicate AGC1 activity in myelination and in neuronal structure, and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development contributing to increased autism susceptibility.
doi:10.1016/j.biopsych.2009.08.042
PMCID: PMC4067545  PMID: 20015484
Malate/aspartate shuttle; mitochondria; N-acetyl aspartate (NAA); neuron-oligodendrocyte interactions; pyruvate
2.  Schizophrenia research in 2013: are we making progress? 
Neurobiology of disease  2013;53:1-2.
The diagnostic category of schizophrenia encompasses a range of disabilities with distinct phenotypic features typically manifesting in young adulthood. Classically the purview of psychiatrists, it is now apparent that close cross disciplinary collaboration and iterative comparison of datasets is a must if we intend to make significant in-roads in preventing both the slide into full blown disease and ensuring that susceptible individuals maintain best quality of life as they age. For example, understanding whether there are specific development windows that permit environmental triggers acting on specific genetic backgrounds to unmask and ensconce the relevant phenotypes is one area where epidemiology, clinicians, and basic neuroscientists likely possess key pieces of the puzzle. A mind-set shift towards coherent collaborative strategies aimed at uncovering fundamental neurobiological principles that govern the dynamic way in which the brain continually moulds and reshapes structure and function could speed progress in the field.
doi:10.1016/j.nbd.2013.01.021
PMCID: PMC4049623  PMID: 23380149
3.  Coreleased Orexin and Glutamate Evoke Nonredundant Spike Outputs and Computations in Histamine Neurons 
Cell Reports  2014;7(3):697-704.
Summary
Stable wakefulness requires orexin/hypocretin neurons (OHNs) and OHR2 receptors. OHNs sense diverse environmental cues and control arousal accordingly. For unknown reasons, OHNs contain multiple excitatory transmitters, including OH peptides and glutamate. To analyze their cotransmission within computational frameworks for control, we optogenetically stimulated OHNs and examined resulting outputs (spike patterns) in a downstream arousal regulator, the histamine neurons (HANs). OHR2s were essential for sustained HAN outputs. OHR2-dependent HAN output increased linearly during constant OHN input, suggesting that the OHN→HANOHR2 module may function as an integral controller. OHN stimulation evoked OHR2-dependent slow postsynaptic currents, similar to midnanomolar OH concentrations. Conversely, glutamate-dependent output transiently communicated OHN input onset, peaking rapidly then decaying alongside OHN→HAN glutamate currents. Blocking glutamate-driven spiking did not affect OH-driven spiking and vice versa, suggesting isolation (low cross-modulation) of outputs. Therefore, in arousal regulators, cotransmitters may translate distinct features of OHN activity into parallel, nonredundant control signals for downstream effectors.
Graphical Abstract
Highlights
•Natural orexin release generates unique signatures of brain activity•Unlike classical transmitter glutamate, orexin release produces enduring communication•Orexin transmission requires a distinct neural firing code•Orexin transmission is necessary for brain histamine neurons to integrate inputs
Stable wakefulness requires brain orexin/hypocretin neurons, whose loss causes narcoleptic instability of consciousness. These neurons produce several neuroexcitatory transmitters, such as orexins and glutamate. The logic of this is unclear, because it seems energetically wasteful to make several transmitters serving the same function of excitation. Schöne et al. found, however, that when naturally coreleased, orexin and glutamate each create unique signatures of brain activity, which are mutually independent and useful for different but complementary purposes. This may offer insights into why orexins are indispensable for stable consciousness.
doi:10.1016/j.celrep.2014.03.055
PMCID: PMC4022832  PMID: 24767990
4.  Conditional Ablation of Orexin/Hypocretin Neurons: A New Mouse Model for the Study of Narcolepsy and Orexin System Function 
The Journal of Neuroscience  2014;34(19):6495-6509.
The sleep disorder narcolepsy results from loss of hypothalamic orexin/hypocretin neurons. Although narcolepsy onset is usually postpubertal, current mouse models involve loss of either orexin peptides or orexin neurons from birth. To create a model of orexin/hypocretin deficiency with closer fidelity to human narcolepsy, diphtheria toxin A (DTA) was expressed in orexin neurons under control of the Tet-off system. Upon doxycycline removal from the diet of postpubertal orexin-tTA;TetO DTA mice, orexin neurodegeneration was rapid, with 80% cell loss within 7 d, and resulted in disrupted sleep architecture. Cataplexy, the pathognomic symptom of narcolepsy, occurred by 14 d when ∼5% of the orexin neurons remained. Cataplexy frequency increased for at least 11 weeks after doxycycline. Temporary doxycycline removal followed by reintroduction after several days enabled partial lesion of orexin neurons. DTA-induced orexin neurodegeneration caused a body weight increase without a change in food consumption, mimicking metabolic aspects of human narcolepsy. Because the orexin/hypocretin system has been implicated in the control of metabolism and addiction as well as sleep/wake regulation, orexin-tTA; TetO DTA mice are a novel model in which to study these functions, for pharmacological studies of cataplexy, and to study network reorganization as orexin input is lost.
doi:10.1523/JNEUROSCI.0073-14.2014
PMCID: PMC4012309  PMID: 24806676
diphtheria toxin A fragment; hypocretin; model mice; narcolepsy; orexin; transgenic
5.  Molecular and neural mechanisms of sex pheromone reception and processing in the silkmoth Bombyx mori 
Male moths locate their mates using species-specific sex pheromones emitted by conspecific females. One striking feature of sex pheromone recognition in males is the high degree of specificity and sensitivity at all levels, from the primary sensory processes to behavior. The silkmoth Bombyx mori is an excellent model insect in which to decipher the underlying mechanisms of sex pheromone recognition due to its simple sex pheromone communication system, where a single pheromone component, bombykol, elicits the full sexual behavior of male moths. Various technical advancements that cover all levels of analysis from molecular to behavioral also allow the systematic analysis of pheromone recognition mechanisms. Sex pheromone signals are detected by pheromone receptors expressed in olfactory receptor neurons in the pheromone-sensitive sensilla trichodea on male antennae. The signals are transmitted to the first olfactory processing center, the antennal lobe (AL), and then are processed further in the higher centers (mushroom body and lateral protocerebrum) to elicit orientation behavior toward females. In recent years, significant progress has been made elucidating the molecular mechanisms underlying the detection of sex pheromones. In addition, extensive studies of the AL and higher centers have provided insights into the neural basis of pheromone processing in the silkmoth brain. This review describes these latest advances, and discusses what these advances have revealed about the mechanisms underlying the specific and sensitive recognition of sex pheromones in the silkmoth.
doi:10.3389/fphys.2014.00125
PMCID: PMC3978319  PMID: 24744736
insect; silkmoth; olfaction; sex pheromone; pheromone-source searching behavior
6.  Mitigation of murine focal cerebral ischemia by the hypocretin/orexin system is associated with reduced inflammation 
Background and Purpose
Brain ischemia causes immediate and delayed cell death that is exacerbated by inflammation. Recent studies show that hypocretin-1/orexin-A (Hcrt-1) reduces ischemic brain injury, and Hcrt-positive neurons modulate infection-induced inflammation. Here we tested the hypothesis that Hcrt plays a protective role against ischemia by modulating inflammation.
Methods
Orexin/ataxin-3 (AT) mice, a transgenic strain in which Hcrt-producing neurons degenerate in early adulthood, and wild type (WT) mice were subjected to transient middle cerebral artery occlusion (MCAO). Infarct volume, neurological score, and spontaneous home cage activity were assessed. Inflammation was measured using immunohistochemistry, ELISA and assessment of cytokine mRNA levels.
Results
Infarct volumes 24 and 48 hours after MCAO were significantly larger, neurological score was worse, and spontaneous activity decreased in AT compared to WT mice.
Macrophage/microglial infiltration and myeloperoxidase-positive cells were higher in AT compared to WT mice. Pre-MCAO intracerebroventricular injection of Hcrt-1 significantly reduced infarct volume and macrophage/microglial infiltration in both genotypes, and improved neurological score in AT mice. Post-MCAO treatment decreased infarct size in both WT and AT mice, but had no effect on neurological score in either genotype. Microglia express the Hcrt-1 receptor following MCAO. TNFα production by LPS-stimulated microglial BV2 cells was significantly reduced by Hcrt-1 pretreatment. Sham AT mice exhibit increased brain TNFα and IL-6 mRNA, suggesting chronic inflammation.
Conclusion
Loss of Hcrt neurons in AT mice resulted in worsened stroke outcomes, which were reversed by administration of exogenous Hcrt-1. The mechanism underlying Hcrt-mediated neuroprotection includes attenuation of inflammatory responses following ischemic insult.
doi:10.1161/STROKEAHA.112.681700
PMCID: PMC3638929  PMID: 23349191
Hypocretin; orexin; brain ischemia; inflammation; neurobehavior
7.  NrCAM regulating neural systems and addiction related behaviors 
Addiction biology  2012;10.1111/j.1369-1600.2012.00469.x.
We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine, or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety-provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM. Glutaminase appears to be involved in NrCAM-related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, PLG, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety-like behavior. Thus, NrCAM could affect addiction-related behaviors via at least partial modulation of some glutamatargic pathways and neural function in brain.
doi:10.1111/j.1369-1600.2012.00469.x
PMCID: PMC3470748  PMID: 22780223
behavior; cell adhesion molecule; glutamate
8.  Orexin neurons suppress narcolepsy via 2 distinct efferent pathways 
The loss of orexin neurons in humans is associated with the sleep disorder narcolepsy, which is characterized by excessive daytime sleepiness and cataplexy. Mice lacking orexin peptides, orexin neurons, or orexin receptors recapitulate human narcolepsy phenotypes, further highlighting a critical role for orexin signaling in the maintenance of wakefulness. Despite the known role of orexin neurons in narcolepsy, the precise neural mechanisms downstream of these neurons remain unknown. We found that targeted restoration of orexin receptor expression in the dorsal raphe (DR) and in the locus coeruleus (LC) of mice lacking orexin receptors inhibited cataplexy-like episodes and pathological fragmentation of wakefulness (i.e., sleepiness), respectively. The suppression of cataplexy-like episodes correlated with the number of serotonergic neurons restored with orexin receptor expression in the DR, while the consolidation of fragmented wakefulness correlated with the number of noradrenergic neurons restored in the LC. Furthermore, pharmacogenetic activation of these neurons using designer receptor exclusively activated by designer drug (DREADD) technology ameliorated narcolepsy in mice lacking orexin neurons. These results suggest that DR serotonergic and LC noradrenergic neurons play differential roles in orexin neuron–dependent regulation of sleep/wakefulness and highlight a pharmacogenetic approach for the amelioration of narcolepsy.
doi:10.1172/JCI71017
PMCID: PMC3904620  PMID: 24382351
9.  Effects of a newly developed potent orexin-2 receptor-selective antagonist, compound 1 m, on sleep/wakefulness states in mice 
Orexins (also known as hypocretins) play critical roles in the regulation of sleep/wakefulness states by activating two G-protein coupled receptors (GPCRs), orexin 1 (OX1R) and orexin 2 receptors (OX2R). In order to understand the differential contribution of both receptors in regulating sleep/wakefulness states we compared the pharmacological effects of a newly developed OX2R antagonist (2-SORA), Compound 1 m (C1 m), with those of a dual orexin receptor antagonist (DORA), suvorexant, in C57BL/6J mice. After oral administration in the dark period, both C1m and suvorexant decreased wakefulness time with similar efficacy in a dose-dependent manner. While C1m primarily increased total non-rapid eye movement (NREM) sleep time without affecting episode durations and with minimal effects on REM sleep, suvorexant increased both total NREM and REM sleep time and episode durations with predominant effects on REM sleep. Fos-immunostaining showed that both compounds affected the activities of arousal-related neurons with different patterns. The number of Fos-IR noradrenergic neurons in the locus coeruleus was lower in the suvorexant group as compared with the control and C1m-treated groups. In contrast, the numbers of Fos-IR neurons in histaminergic neurons in the tuberomamillary nucleus and serotonergic neurons in the dorsal raphe were reduced to a similar extent in the suvorexant and C1m groups as compared with the vehicle-treated group. Together, these results suggest that an orexin-mediated suppression of REM sleep via potential activation of OX1Rs in the locus coeruleus may possibly contribute to the differential effects on sleep/wakefulness exerted by a DORA as compared to a 2-SORA.
doi:10.3389/fnins.2014.00008
PMCID: PMC3907770  PMID: 24550770
orexin receptor antagonists; sleep; wakefulness; REM sleep; orexin
10.  OX1 and OX2 orexin/hypocretin receptor pharmacogenetics 
Orexin/hypocretin peptide mutations are rare in humans. Even though human narcolepsy is associated with orexin deficiency, this is only extremely rarely due to mutations in the gene coding prepro-orexin, the precursor for both orexin peptides. In contrast, coding and non-coding variants of the OX1 and OX2 orexin receptors have been identified in many human populations; sometimes, these have been associated with disease phenotype, although most confer a relatively low risk. In most cases, these studies have been based on a candidate gene hypothesis that predicts the involvement of orexins in the relevant pathophysiological processes. In the current review, the known human OX1/HCRTR1 and OX2/HCRTR2 genetic variants/polymorphisms as well as studies concerning their involvement in disorders such as narcolepsy, excessive daytime sleepiness, cluster headache, polydipsia-hyponatremia in schizophrenia, and affective disorders are discussed. In most cases, the functional cellular or pharmacological correlates of orexin variants have not been investigated—with the exception of the possible impact of an amino acid 10 Pro/Ser variant of OX2 on orexin potency—leaving conclusions on the nature of the receptor variant effects speculative. Nevertheless, we present perspectives that could shape the basis for further studies. The pharmacology and other properties of the orexin receptor variants are discussed in the context of GPCR signaling. Since orexinergic therapeutics are emerging, the impact of receptor variants on the affinity or potency of ligands deserves consideration. This perspective (pharmacogenetics) is also discussed in the review.
doi:10.3389/fnins.2014.00057
PMCID: PMC4018553  PMID: 24834023
orexin; hypocretin; G protein-coupled receptor; polymorphism; pharmacogenetics
11.  Hypocretin/orexin neurons contribute to hippocampus-dependent social memory and synaptic plasticity in mice 
Hypocretin/orexin (Hcrt) neurons in the lateral hypothalamus project throughout the brain, including to the hippocampus, where Hcrt receptors are widely expressed. Hcrt neurons activate these targets to orchestrate global arousal state, wake-sleep architecture, energy homeostasis, stress adaptation, and reward behaviors. Hcrt has recently been implicated in cognitive functions and social interaction. Here, we tested the hypothesis that Hcrt neurons are critical to social interaction, particularly social memory, using neurobehavioral assessment and electrophysiological approaches. The validated “two-enclosure homecage test” devices and procedure were used to test sociability, preference for social novelty (social novelty), and recognition memory. A conventional direct contact social test was conducted to corroborate the findings. We found that adult orexin/ataxin-3 transgenic mice (AT, in which Hcrt neurons degenerate by 3 months of age) displayed normal sociability and social novelty with respect to wildtype (WT) littermates. However, AT mice displayed deficits in long-term social memory. Nasal administration of exogenous Hcrt-1 restored social memory to an extent in AT mice.
Hippocampal slices taken from AT mice exhibited decreases in degree of paired-pulse facilitation (PPF) and magnitude of long-term potentiation (LTP), despite displaying normal basal synaptic neurotransmission in the CA1 area compared to WT hippocampal slices. AT hippocampi had lower levels of phosphorylated cyclic AMP-response element binding protein (pCREB), an activity-dependent transcription factor important for synaptic plasticity and long-term memory storage. Our studies demonstrate that Hcrt neurons play an important role in the consolidation of social recognition memory, at least in part through enhancements of hippocampal synaptic plasticity and CREB phosphorylation.
doi:10.1523/JNEUROSCI.3200-12.2013
PMCID: PMC3640412  PMID: 23516292
Hypocretins/orexins; Learning; Memory; Neuropeptides; Neuromodulation; Social interaction; Social recognition
12.  Differential Roles of Orexin Receptor-1 and -2 in the Regulation of Non-REM and REM Sleep 
Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX1R−/−, and OX2R−/− mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knock-out mice as compared with wild-type mice, with substantially larger attenuation in OX2R−/− mice than in OX1R−/− mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX1R−/− and OX2R−/− mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX1R mRNA, but OX2R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep through distinct neuronal pathways.
doi:10.1523/JNEUROSCI.6506-10.2011
PMCID: PMC3732784  PMID: 21525292
13.  The juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity 
Despite considerable effort, the identification of genes that regulate complex multigenic traits such as obesity has proven difficult with conventional methodologies. The use of a chromosome substitution strain-based mapping strategy based on deep congenic analysis over-came many of the difficulties associated with gene discovery and led to the finding that the juxtaparanodal proteins CNTNAP2 and TAG1 regulate diet-induced obesity. The effects of a mild Cntnap2 mutation on body weight were highly dependent on genetic background, as both obesity-promoting and obesity-resistant effects of Cntnap2 were observed on different genetic backgrounds. The more severe effect of complete TAG1 deficiency, by decreasing food intake, completely prevented the weight gain normally associated with high-fat-diet feeding. Together, these studies implicate two novel proteins in the regulation of diet-induced obesity. Moreover, as juxtaparanodal proteins have previously been implicated in various neurological disorders, our results suggest a potential genetic and molecular link between obesity and diseases such as autism and epilepsy.
doi:10.1007/s00335-012-9400-8
PMCID: PMC3650838  PMID: 22752552
14.  Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice 
PLoS ONE  2013;8(7):e70012.
Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.
doi:10.1371/journal.pone.0070012
PMCID: PMC3726545  PMID: 23922890
15.  Optic chiasm presentation of Semaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing 
Neuron  2012;74(4):676-690.
Summary
At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here we identify a tripartite molecular system for contralateral RGC projections: Semaphorin 6D and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth-promotion. Nr-CAM functions as a novel receptor for Sema6D. Sema6D, Plexin-A1 and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a novel mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.
doi:10.1016/j.neuron.2012.03.025
PMCID: PMC3361695  PMID: 22632726
16.  Orexin Gene Transfer into Zona Incerta Neurons Suppresses Muscle Paralysis in Narcoleptic Mice 
Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.
doi:10.1523/JNEUROSCI.6069-10.2011
PMCID: PMC3634582  PMID: 21508228
17.  Neurotensin Co-Expressed in Orexin-Producing Neurons in the Lateral Hypothalamus Plays an Important Role in Regulation of Sleep/Wakefulness States 
PLoS ONE  2013;8(4):e62391.
Both orexin and neurotensin are expressed in the lateral hypothalamic area (LHA) and have been implicated in the regulation of feeding, motor activity and the reward system. A double label immunofluorescence and in situ hybridization studies showed that neurotensin colocalizes with orexin in neurons of the LHA. Pharmacological studies suggested that neurotensin excites orexin-producing neurons (orexin neurons) through activation of neurotensin receptor-2 (NTSR-2) and non-selective cation channels. In situ hybridization study showed that most orexin neurons express neurotensin receptor-2 mRNA but not neurotensin receptor-1 (Ntsr-1) mRNA. Immunohistochemical studies showed that neurotensin-immunoreactive fibers make appositions to orexin neurons. A neurotensin receptor antagonist decreased Fos expression in orexin neurons and wakefulness time in wild type mice when administered intraperitoneally. However, the antagonist did not evoke any effect on these parameters in orexin neuron-ablated mice. These observations suggest the importance of neurotensin in maintaining activity of orexin neurons. The evidence presented here expands our understanding of the regulatory mechanism of orexin neurons.
doi:10.1371/journal.pone.0062391
PMCID: PMC3631195  PMID: 23620827
18.  Linking oligodendrocyte and myelin dysfunction to neurocircuitry abnormalities in schizophrenia 
Progress in neurobiology  2010;93(1):13-24.
Multiple lines of evidence in schizophrenia, from brain imaging, studies in postmortem brains, and genetic association studies, have implicated oligodendrocyte and myelin dysfunction in this disease. Recent studies suggest that oligodendrocyte and myelin dysfunction leads to changes in synaptic formation and function, which could lead to cognitive dysfunction, a core symptom of schizophrenia. Furthermore, there is accumulating data linking oligodendrocyte and myelin dysfunction with dopamine and glutamate abnormalities, both of which are found in schizophrenia. These findings implicate oligodendrocyte and myelin dysfunction as a primary change in schizophrenia, not only as secondary consequences of the illness or treatment. Strategies targeting oligodendrocyte and myelin abnormalities could therefore provide therapeutic opportunities for patients suffering from schizophrenia.
doi:10.1016/j.pneurobio.2010.09.004
PMCID: PMC3622281  PMID: 20950668
myelin; gene expression; genetic association; brain imaging; oligodendrocyte; synaptic plasticity; dopamine; glutamate
19.  NPBWR1 and NPBWR2: Implications in Energy Homeostasis, Pain, and Emotion 
Neuropeptide B/W receptor-1 (NPBWR1) and NPBWR2 had been known as orphan receptors GPR7 and 8, respectively. Endogenous peptide ligands of these receptors, neuropeptide B (NPB) and neuropeptide W (NPW), were identified in 2002 and 2003 (Fujii et al., 2002; Brezillon et al., 2003; Tanaka et al., 2003). These peptides have been implicated in regulation of feeding behavior, energy homeostasis, neuroendocrine function, and modulating inflammatory pain. In addition, strong and discrete expression of their receptors in the extended amygdala and bed nucleus of the stria terminalis suggests a potential role in regulating stress responses, emotion, anxiety, and fear. Recent studies of NPB/NPW using both pharmacological and phenotypic analyses of genetically engineered mice as well as a human study support this hypothesis.
doi:10.3389/fendo.2013.00023
PMCID: PMC3600615  PMID: 23515889
neuropeptide B; neuropeptide W; hypothalamus; limbic system; amygdala; pain; emotions
20.  Role of orexin in modulating arousal, feeding, and motivation 
Orexin deficiency results in narcolepsy in humans, dogs, and rodents, suggesting that the orexin system is particularly important for maintenance of wakefulness. However, orexin neurons are “multi-tasking” neurons that regulate sleep/wake states as well as feeding behavior, emotion, and reward processes. Orexin deficiency causes abnormalities in energy homeostasis, stress-related behavior, and reward systems. Orexin excites waking-active monoaminergic and cholinergic neurons in the hypothalamus and brain stem regions to maintain a long, consolidated waking period. Orexin neurons also have reciprocal links with the hypothalamic nuclei, which regulates feeding. Moreover, the responsiveness of orexin neurons to peripheral metabolic cues suggests that these neurons have an important role as a link between energy homeostasis and vigilance states. The link between orexin and the ventral tegmental nucleus serves to motivate an animal to engage in goal-directed behavior. This review focuses on the interaction of orexin neurons with emotion, reward, and energy homeostasis systems. These connectivities are likely to be highly important to maintain proper vigilance states.
doi:10.3389/fnbeh.2013.00028
PMCID: PMC3629303  PMID: 23616752
orexin A; orexins; orexin receptors; hypothalamus; sleep; feeding behavior; reward
21.  Differential Roles of Orexin Receptors in the Regulation of Sleep/Wakefulness 
Orexin A and orexin B are hypothalamic neuropeptides that play critical roles in the regulation of sleep/wakefulness, as well as in a variety of physiological functions such as emotion, reward, and energy homeostasis. The actions of orexins are mediated by two receptors, orexin 1 (OX1R) and orexin 2 (OX2R) receptors. OX1R and OX2R show partly overlapping but distinct distributions throughout the central nervous system, suggesting their differential roles. This review presents and discusses the current knowledge concerning the physiological roles of each orexin receptor subtype, focusing on the regulation of sleep/wakefulness.
doi:10.3389/fendo.2013.00057
PMCID: PMC3656340  PMID: 23730297
orexin; hypothalamus; sleep; monoamine; narcolepsy; arousal
22.  GABAergic neurons in the preoptic area send direct inhibitory projections to orexin neurons 
Populations of neurons in the hypothalamic preoptic area (POA) fire rapidly during sleep, exhibiting sleep/waking state-dependent firing patterns that are the reciprocal of those observed in the arousal system. The majority of these preoptic “sleep-active” neurons contain the inhibitory neurotransmitter GABA. On the other hand, a population of neurons in the lateral hypothalamic area (LHA) contains orexins, which play an important role in the maintenance of wakefulness, and exhibit an excitatory influence on arousal-related neurons. It is important to know the anatomical and functional interactions between the POA sleep-active neurons and orexin neurons, both of which play important, but opposite roles in regulation of sleep/wakefulness states. In this study, we confirmed that specific pharmacogenetic stimulation of GABAergic neurons in the POA leads to an increase in the amount of non-rapid eye movement (NREM) sleep. We next examined direct connectivity between POA GABAergic neurons and orexin neurons using channelrhodopsin 2 (ChR2) as an anterograde tracer as well as an optogenetic tool. We expressed ChR2-eYFP selectively in GABAergic neurons in the POA by AAV-mediated gene transfer, and examined the projection sites of ChR2-eYFP-expressing axons, and the effect of optogenetic stimulation of ChR2-eYFP on the activity of orexin neurons. We found that these neurons send widespread projections to wakefulness-related areas in the hypothalamus and brain stem, including the LHA where these fibers make close appositions to orexin neurons. Optogenetic stimulation of these fibers resulted in rapid inhibition of orexin neurons. These observations suggest direct connectivity between POA GABAergic neurons and orexin neurons.
doi:10.3389/fncir.2013.00192
PMCID: PMC3844858  PMID: 24348342
orexin; preoptic area; GABA; sleep; wakefulness; hypothalamus
23.  Loss of function studies in mice and genetic association link receptor protein tyrosine phosphatase α to schizophrenia 
Biological psychiatry  2011;70(7):626-635.
Background
Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPα, in the control of radial neuronal migration, cortical cytoarchitecture, and oligodendrocyte differentiation. The human gene encoding RPTPα, PTPRA, maps to a chromosomal region (20p13) associated with susceptibility to psychotic illness.
Methods
We characterized neurobehavioral parameters, as well as gene expression in the central nervous system, of mice with a null mutation in the Ptpra gene. We searched for genetic association between polymorphisms in PTPRA and schizophrenia risk (2 independent cohorts; total of 1420 cases and 1377 controls), and we monitored PTPRA expression in prefrontal dorsolateral cortex of SZ patients (35 cases, 2 control groups of 35 cases)
Results
We find that Ptpra−/− mice reproduce neurobehavioral endophenotypes of human SZ: sensitization to metamphetamine-induced hyperactivity, defective sensorimotor gating, and defective habituation to a startle response. Ptpra loss of function also leads to reduced expression of multiple myelination genes, mimicking the hypomyelination-associated changes in gene expression observed in post mortem patient brains. We further report that a polymorphism at the PTPRA locus is genetically associated with SZ, and that PTPRA mRNA levels are reduced in post mortem dorsolateral prefrontal cortex of subjects with SZ.
Conclusion
The implication of this well-studied signaling protein in SZ risk and endophenotype manifestation provides novel entry points into the etiopathology of this disease.
doi:10.1016/j.biopsych.2011.06.016
PMCID: PMC3176920  PMID: 21831360
schizophrenia; tyrosine phosphatase; myelination; mouse model; RPTPα; PTPRA
24.  Haploinsufficiency of Cyfip1 Produces Fragile X-Like Phenotypes in Mice 
PLoS ONE  2012;7(8):e42422.
Background
Copy number variation (CNV) at the 15q11.2 region, which includes a gene that codes for CYFIP1 (cytoplasmic FMR1 interacting protein 1), has been implicated in autism, intellectual disability and additional neuropsychiatric phenotypes. In the current study we studied the function of Cyfip1 in synaptic physiology and behavior, using mice with a disruption of the Cyfip1 gene.
Methodology/Principal Findings
We observed that in Cyfip1 heterozygous mice metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) induced by paired-pulse low frequency stimulation (PP-LFS) was significantly increased in comparison to wildtype mice. In addition, mGluR-LTD was not affected in the presence of protein synthesis inhibitor in the Cyfip1 heterozygous mice, while the same treatment inhibited LTD in wildtype littermate controls. mGluR-agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD was also significantly increased in hippocampal slices from Cyfip1 heterozygous mice and again showed independence from protein synthesis only in the heterozygous animals. Furthermore, we observed that the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin was only effective at reducing mGluR-LTD in wildtype animals. Behaviorally, Cyfip1 heterozygous mice showed enhanced extinction of inhibitory avoidance. Application of both mGluR5 and mGluR1 antagonist to slices from Cyfip1 heterozygous mice reversed the increase in DHPG-induced LTD in these mice.
Conclusions/Significance
These results demonstrate that haploinsufficiency of Cyfip1 mimics key aspects of the phenotype of Fmr1 knockout mice and are consistent with the hypothesis that these effects are mediated by interaction of Cyfip1 and Fmrp in regulating activity-dependent translation. The data provide support for a model where CYFIP1 haploinsufficiency in patients results in intermediate phenotypes increasing risk for neuropsychiatric disorders.
doi:10.1371/journal.pone.0042422
PMCID: PMC3416859  PMID: 22900020
25.  A Single Nucleotide Polymorphism of the Neuropeptide B/W Receptor-1 Gene Influences the Evaluation of Facial Expressions 
PLoS ONE  2012;7(4):e35390.
Neuropeptide B/W receptor-1 (NPBWR1) is expressed in discrete brain regions in rodents and humans, with particularly strong expression in the limbic system, including the central nucleus of the amygdala. Recently, Nagata-Kuroiwa et al. reported that Npbwr1−/− mice showed changes in social behavior, suggesting that NPBWR1 plays important roles in the emotional responses of social interactions.
The human NPBWR1 gene has a single nucleotide polymorphism at nucleotide 404 (404A>T; SNP rs33977775). This polymorphism results in an amino acid change, Y135F. The results of an in vitro experiment demonstrated that this change alters receptor function. We investigated the effect of this variation on emotional responses to stimuli of showing human faces with four categories of emotional expressions (anger, fear, happiness, and neutral). Subjects' emotional levels on seeing these faces were rated on scales of hedonic valence, emotional arousal, and dominance (V-A-D). A significant genotype difference was observed in valence evaluation; the 404AT group perceived facial expressions more pleasantly than did the 404AA group, regardless of the category of facial expression. Statistical analysis of each combination of [V-A-D and facial expression] also showed that the 404AT group tended to feel less submissive to an angry face than did the 404AA group. Thus, a single nucleotide polymorphism of NPBWR1 seems to affect human behavior in a social context.
doi:10.1371/journal.pone.0035390
PMCID: PMC3335863  PMID: 22545105

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