A new perspective for schizophrenia: TAAR1 agonists reveal antipsychotic- and antidepressant-like activity, improve cognition and control body weight.

Schizophrenia is a chronic, severe and highly complex mental illness. Current treatments manage the positive symptoms, yet have minimal effects on the negative and cognitive symptoms, two prominent features of the disease with critical impact on the long-term morbidity. In addition, antipsychotic treatments trigger serious side effects that precipitate treatment discontinuation. Here, we show that activation of the trace amine-associated receptor 1 (TAAR1), a modulator of monoaminergic neurotransmission, represents a novel therapeutic option. In rodents, activation of TAAR1 by two novel and pharmacologically distinct compounds, the full agonist RO5256390 and the partial agonist RO5263397, blocks psychostimulant-induced hyperactivity and produces a brain activation pattern reminiscent of the antipsychotic drug olanzapine, suggesting antipsychotic-like properties. TAAR1 agonists do not induce catalepsy or weight gain; RO5263397 even reduced haloperidol-induced catalepsy and prevented olanzapine from increasing body weight and fat accumulation. Finally, TAAR1 activation promotes vigilance in rats and shows pro-cognitive and antidepressant-like properties in rodent and primate models. These data suggest that TAAR1 agonists may provide a novel and differentiated treatment of schizophrenia as compared with current medication standards: TAAR1 agonists may improve not only the positive symptoms but also the negative symptoms and cognitive deficits, without causing adverse effects such as motor impairments or weight gain.

Further characterization of sleep-active neuronal nitric oxide synthase neurons in the mouse brain.

We recently demonstrated that Fos is induced in a subpopulation of cortical neuronal nitric oxide synthase (nNOS)-immunoreactive neurons in three rodent species both during spontaneous sleep (SS) and recovery sleep (RS) after a period of sleep deprivation (SD); the proportion of cortical Fos(+)/nNOS neurons was significantly correlated with non-REM (NREM) sleep delta energy. The present study was undertaken to evaluate the specificity of this state-dependent activation of cortical nNOS cells. The percentage of nNOS neurons that expressed Fos during SD and RS was determined in nine subcortical brain regions and the cortex of the mouse brain; a significantly greater proportion of Fos(+)/nNOS neurons was observed during RS only in the cortex and in none of the nine subcortical regions. The proportion of calretinin-, calbindin- and parvalbumin-immunoreactive cortical interneurons that expressed Fos during SD and RS was also determined. In contrast to cortical nNOS neurons, a higher percentage of Fos(+)/calbindin neurons was found during SD than RS; there were no differences in the proportions of Fos-expressing parvalbumin or calretinin neurons between these conditions. Since the nNOS and calretinin cortical interneuron populations overlap extensively in the mouse brain, triple-labeling with these two phenotypic markers and Fos was undertaken in mice from the RS group to determine which combination of markers could best identify the rare "sleep-active" cortical interneuron population. The proportions of both Fos(+)/nNOS neurons and Fos(+)/nNOS/calretinin neurons far exceeded the proportion of Fos(+)/calretinin neurons during RS, but the proportions of these two cell types were not significantly different during RS. Thus, functional activation of nNOS neurons during sleep appears to be restricted to the cerebral cortex and cortical nNOS cells and nNOS/calretinin cells collectively define a cortical interneuron population that is activated during sleep.

Gene expression in the rat brain during prostaglandin D2 and adenosinergically-induced sleep.

Previous studies have supported the hypothesis that macromolecular synthesis occurs in the brain during sleep as a response to prior waking activities and that prostaglandin D2 (PGD2) is an endogenous sleep substance whose effects are dependent on adenosine A2a receptor-mediated signaling. We compared gene expression in the cerebral cortex, basal forebrain, and hypothalamus during PGD2-induced and adenosinergically-induced sleep to results from our previously published study of recovery sleep (RS) after sleep deprivation (SD). Immediate early gene expression in the cortex during sleep induced by PGD2- or by the selective adenosine A2a agonist CGS21680 showed limited similarity to that observed during RS while, in the basal forebrain and hypothalamus, widespread activation of immediate early genes not seen during RS occurred. In all three brain regions, PGD2 and CGS21680 reduced the expression of arc, a transcript whose expression is elevated during SD. Using GeneChips, the majority of genes induced by either PGD2 or CGS21680 were induced by both, suggesting activation of the same pathways. However, gene expression induced in the brain after PGD2 or CGS21680 treatment was distinct from that described during RS after SD and apparently involves glial cell gene activation and signaling pathways in neural-immune interactions.

Gene expression in the rat cerebral cortex: comparison of recovery sleep and hypnotic-induced sleep.

Most hypnotic medications currently on the market target some aspect of GABAergic neurotransmission. Although all such compounds increase sleep, these drugs differentially affect the activity of the cerebral cortex as measured by the electroencephalogram. Whereas benzodiazepine medications such as triazolam tend to suppress slow wave activity in the cortex, the GABA(B) ligand gamma-hydroxybutyrate greatly enhances slow wave activity and the non-benzodiazepine, zolpidem, which binds to the omega1 site on the GABA(A) receptor/Cl(-) ionophore complex, is intermediate in this regard. Our previous studies have demonstrated that a small number of genes exhibit increased expression in the cerebral cortex of the mouse and rat during recovery sleep after sleep deprivation: egr-3, fra-2, grp78, grp94, ngfi-b, and nr4a3. Using these genes as a panel of biomarkers associated with sleep, we asked whether hypnotic medications induce similar molecular changes in the rat cerebral cortex to those observed when both sleep continuity and slow wave activity are enhanced during recovery sleep. We find that, although each drug increases the expression of a subset of genes in the panel of biomarkers, no drug fully replicates the molecular changes in the cortex associated with recovery sleep. Furthermore, high levels of slow wave activity in the cortex are correlated with increased expression of fra-2 whereas the expression of grp94 is correlated with body temperature. These results demonstrate that sleep-related changes in gene expression may be affected by physiological covariates of sleep and wakefulness rather than by vigilance state per se.

Gene expression in the rat brain during sleep deprivation and recovery sleep: an Affymetrix GeneChip study.

Previous studies have demonstrated that macromolecular synthesis in the brain is modulated in association with the occurrence of sleep and wakefulness. Similarly, the spectral composition of electroencephalographic activity that occurs during sleep is dependent on the duration of prior wakefulness. Since this homeostatic relationship between wake and sleep is highly conserved across mammalian species, genes that are truly involved in the electroencephalographic response to sleep deprivation might be expected to be conserved across mammalian species. Therefore, in the rat cerebral cortex, we have studied the effects of sleep deprivation on the expression of immediate early gene and heat shock protein mRNAs previously shown to be upregulated in the mouse brain in sleep deprivation and in recovery sleep after sleep deprivation. We find that the molecular response to sleep deprivation and recovery sleep in the brain is highly conserved between these two mammalian species, at least in terms of expression of immediate early gene and heat shock protein family members. Using Affymetrix Neurobiology U34 GeneChips , we also screened the rat cerebral cortex, basal forebrain, and hypothalamus for other genes whose expression may be modulated by sleep deprivation or recovery sleep. We find that the response of the basal forebrain to sleep deprivation is more similar to that of the cerebral cortex than to the hypothalamus. Together, these results suggest that sleep-dependent changes in gene expression in the cerebral cortex are similar across rodent species and therefore may underlie sleep history-dependent changes in sleep electroencephalographic activity.

Region-specific changes in immediate early gene expression in response to sleep deprivation and recovery sleep in the mouse brain.

Previous studies have documented changes in expression of the immediate early gene (IEG) c-fos and Fos protein in the brain between sleep and wakefulness. Such expression differences implicate changes in transcriptional regulation across behavioral states and suggest that other transcription factors may also be affected. In the current study, we examined the expression of seven fos/jun family member mRNAs (c-fos, fosB, fos related antigen (fra)1, fra-2, junB, c-jun, and junD) and three other IEG mRNAs (egr-1, egr-3, and nur77) in mouse brain following short-term (6 h) sleep deprivation (SD) and 4 h recovery sleep (RS) after SD. Gene expression was quantified in seven brain regions by real-time reverse transcription-polymerase chain reaction (RT-PCR). Multivariate analysis of variance revealed statistically significant variation in cerebral cortex, basal forebrain, thalamus and cerebellum. Levels of c-fos and fosB mRNA were elevated during SD in all four of these brain regions. In the cerebral cortex, junB mRNA was also elevated during SD whereas, in the basal forebrain, fra-1 and fra-2 mRNA levels increased in this condition. During RS, the only IEG mRNA to undergo significant increase was fra-2 in the cortex. C-jun and junD mRNAs were invariant across experimental conditions. These results indicate that the expression of fos/jun family members is diverse during SD. Among other IEGs, nur77 mRNA expression across conditions was similar to c-fos and fosB, egr-1 mRNA was elevated during SD in the cortex and basal forebrain, and egr-3 mRNA was elevated in the cortex during both SD and RS. The similarity of fosB and nur77 expression to c-fos expression indicates that these genes might also be useful markers of functional activity. Along with our previous results, the increased levels of fra-2 and egr-3 mRNAs during RS reported here suggest that increased mRNA expression during sleep is rare and may be anatomically restricted.

Food- and light-entrained circadian rhythms in rats with hypocretin-2-saporin ablations of the lateral hypothalamus.

Daily feeding schedules can entrain circadian rhythms of food-anticipatory activity in mammals. The site of the circadian oscillators that drive food-entrained rhythms is unknown. Lateral hypothalamic (LH) neurons containing hypocretins (Hcrt1 and 2, also known as orexin A and B) regulate feeding, energy metabolism and arousal state, raising the possibility that they may also participate in the entrainment of activity rhythms by a daily mealtime. To examine this, Hcrt neurons in rats were ablated by LH injections of Hcrt2 conjugated to the ribosome-inactivating protein saporin. To assess photic entrainment and masking, drinking activity was recorded continuously in LD 12:12 for approximately 6 weeks, in DD for 48 h, and in LD 2:2 for 24 h. To assess food-entrainment, drinking and food cup activity were recorded for 4-7 weeks during which food was restricted to a 3-h daily meal beginning 6 h after lights-on. Lesions were assessed by immunocytochemistry or inspection of Nissl stained sections. Hcrt cell depletion ranged from 0 to 100%. Lesions were associated with hypophagia, hypodypsia and weight loss. Despite reduced mean daily drinking, there was no significant effect on the shape or amplitude of the circadian waveforms in LD, LD 2:2 or DD at approximately 6 weeks after surgery. All rats exhibited drinking or food cup activity in anticipation of the daily meal, indicative of circadian entrainment. These results indicate that the Hcrt system modulates ingestive behaviors but does not play a necessary role in the entrainment or expression of food-anticipatory circadian rhythms.

Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep.

Although sleep is thought to be restorative from prior wakeful activities, it is not clear what is being restored. To determine whether the synthesis of macromolecules is increased in the cerebral cortex during sleep, we subjected C57BL/6 mice to 6 hours of sleep deprivation and then screened the expression of 1176 genes of known function by using cDNA arrays. The expression of the heat shock proteins (HSP), endoplasmic reticulum protein (ERp72) and glucose-regulated protein (GRp78), was among the genes whose expression was significantly elevated in the cortex during sleep deprivation, whereas GRp78 and GRp94 mRNAs were elevated in the cortex during recovery sleep after sleep deprivation, as confirmed by conventional and quantitative real-time polymerase chain reaction and/or Northern analyses. A systematic evaluation of the expression of six heat shock protein family members (ERP72, GRp78, GRp94, HSP27, HSP70-1, and HSP84) in seven brain regions revealed increased mRNA levels in cortex, basal forebrain, hypothalamus, cerebellum and medulla during sleep deprivation, whereas increased mRNA levels during recovery sleep were limited to the cortex and medulla. Immunohistochemical studies identified increased numbers of GRp78-, GRp94-, and ERp72-immunoreactive cells in the dorsal and lateral cortex during sleep deprivation but, during recovery sleep, elevated numbers of these cells were found only in the lateral cortex. In the medulla, increased numbers of GRp94-immunoreactive cells were observed in nucleus tractus solitarius, dorsal motor nucleus of the vagus and the rostroventrolateral medulla during recovery sleep. The widespread increase of heat shock protein family mRNAs in brain during sleep deprivation may be a neuroprotective response to prolonged wakefulness. In contrast, the relatively limited heat shock protein family mRNA expression during recovery sleep may be related to the role of heat shock proteins in protein biogenesis and thus to the restorative function of sleep.

Transcriptional regulation of the mouse fatty acid amide hydrolase gene.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that inactivates a family of fatty acid amide molecules which are implicated in physiological processes such as pain and sleep. We cloned a 1.9 kb fragment of the 5'-untranslated region of the mouse FAAH gene into the pGL3 basic luciferase reporter vector and showed that this sequence has promoter activity in vitro. By primer extension analysis, we have determined the transcription start site to be 200 bases upstream of the ATG initiation codon and found that a TATA motif was absent. A number of putative response elements, including those for estrogen and glucocorticoids, were identified in this sequence. We have demonstrated that the estrogen and glucocorticoid receptors down-regulate transcriptional activity independent of their ligand. These data should help in understanding the mechanisms of FAAH gene transcription.

Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat.

Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.

Hypocretin (orexin) in the rat pineal gland: a central transmitter with effects on noradrenaline-induced release of melatonin.

Hypocretin-1 (HCRT-1) and hypocretin 2 (HCRT-2), also known as orexin-A and orexin-B, are two neuropeptides derived from the same precursor. Hypocretinergic neurons have been found exclusively in the hypothalamic dorsolateral area. These neurons are implicated in sleep and feeding through activation of specific G-protein-coupled orexin-1 and orexin-2 receptor (OR-R1 and OR-R2). The purpose of this study was to determine the existence of the HCRT peptides in the central input of the rat pineal gland. Further, OR-R1 and OR-R2 expression was determined in the pineal gland and the effect of HCRT-2 on melatonin synthesis and secretion was analysed in dissociated rat pinealocytes. A large contingent of HCRT-positive nerve fibres and terminals were observed in the epithalamus, many of which entered into the pineal parenchyma. A significant number of nerve fibres endowed with positive boutons were identified in the pineal stalk, though the number of positive fibres decreased along the extension of the stalk. So far, no positive fibres have been found in the superficial pineal gland. RT-PCR analysis revealed the expression of OR-R2 mRNA, whereas OR-R1-receptor mRNA was not detected. When tested alone, HCRT-2 had no effect on secretion of melatonin from cultured rat pinealocytes. However, HCRT-2 partially inhibited (by a maximum of 30%) the beta-adrenergic-induced melatonin secretion. The same effect was seen on activation of N-acetyltransferase activity. The distribution and the large number of HCRT-positive fibres together with the effect on noradrenaline-mediated melatonin release through specific receptors suggests that these peptides may be significant central transmitters in pineal function, probably mediating homeostatic signals to the pineal gland.

Modulation of the promoter region of prepro-hypocretin by alpha-interferon.

Hypocretins 1 and 2 (also called orexins A and B, respectively) are hypothalamic neuropeptides that have recently been shown to be involved in the sleep disorder narcolepsy and possibly in the normal regulation of sleep and wake functions. These two peptides are derived from a single precursor molecule called prepro-hypocretin, also known as prepro-orexin. We have cloned a 450 bp fragment from the 5'-flanking region of the human prepro-hypocretin gene and demonstrated that this fragment has promoter activity in vitro. Deletions at the 5' end from -450 to -188 reduced the promoter activity by approximately 50%. Further deletion from the 5'-end to -69 almost completely abolished promoter activity. The 450 bp fragment contains a number of potential transcription factor binding sites, including an interferon (IFN) response element. Our studies demonstrate that alpha-IFN strongly inhibits the promoter activity of both 450 and 188 bp fragments in a dose-dependent manner. The inhibitory effect of alpha-IFN is consistent with recent studies which suggest that hypocretin 1/orexin A may be involved in modulating arousal states and with the literature indicating involvement of immune-related molecules in sleep regulation.

Prepro-hypocretin (prepro-orexin) expression is unaffected by short-term sleep deprivation in rats and mice.

The hypocretin/orexin ligand-receptor system has recently been implicated in the sleep disorder narcolepsy. During the dark (active) period, null mutants of the prepro-orexin (prepro-hypocretin) gene have cataplectic attacks and increased levels of both rapid eye movement (REM) and non-REM (NREM) sleep. Intracerebroventricular injection of one of the encoded neuropeptides, orexin-A, early in the light period increases wakefulness and reduces REM sleep in the rat, suggesting that this system may be involved in the normal regulation of sleep and wakefulness. To further test this hypothesis, we measured hypocretin (hcrt) mRNA levels by both Northern hybridization and Taqman analysis in mouse and rat hypothalamus after short-term (6 h) sleep deprivation (SD) and 2-4 hours after recovery from SD. Although our SD procedures effectively induced a sleep debt and increased c-fos mRNA expression in the cortex and hypothalamus as described by other investigators, we found that hcrt mRNA levels were not significantly changed in either species either after SD or after recovery from SD. If the hcrt system is involved in normal regulation of sleep and wakefulness, longer periods of SD may be necessary to affect hcrt mRNA levels or changes may occur at the protein rather than mRNA level. Alternatively, this system may also be involved in another function that counterbalances any SD-induced changes in hcrt mRNA levels.

The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders.

The molecules originally described as the hypocretins and subsequently as the orexins were initially implicated in the control of food intake. Recent observations implicate this newly-described neurotransmitter system in the sleep disorder narcolepsy and, potentially, in the regulation of normal sleep processes. This article reviews the research that led to the isolation of the hypocretin/orexin peptides, their receptors and the activity of these molecules as we currently understand them. A model is proposed in which the cells that make these peptides might be involved in arousal state control.

HSP70 expression is increased during the day in a diurnal animal, the golden-mantled ground squirrel Spermophilus lateralis.

Heat shock protein 70 (HSP70) gene expression was studied in a seasonal hibernator, the diurnal ground squirrel, Spermophilus lateralis. RNA transcripts of 2.7 and 2.9 kb hybridizing to an HSP70 cDNA were expressed in both brain and peripheral tissues of pre-hibernation euthermic animals; higher levels of expression were observed during the day than during nighttime samples. A decline in the expression of both transcripts occurred in all tissues examined during hibernation that remained low throughout the hibernation season, including the interbout euthermic periods and regardless of time of day. Quantitative comparisons showed pre-hibernation nighttime HSP70 expression to be as low as that observed during hibernation, despite the drastic increase in metabolic state and nearly 30 degrees C difference in body temperature. In contrast to HSP70, some mRNAs, such as beta-actin and HSP60, remained relatively constant, while others, such as glyceraldehyde 3-phosphate dehydrogenase, increased in specific tissues during the hibernation season. These results indicate that the expression of a highly conserved gene involved in protection from cellular stress, HSP70, can vary with an animal's arousal state.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Gene expression in the brain across the hibernation cycle.

The purpose of this study was to characterize changes in gene expression in the brain of a seasonal hibernator, the golden-mantled ground squirrel, Spermophilus lateralis, during the hibernation season. Very little information is available on molecular changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern analysis. Although some small differences were observed across hibernation states, none of the 29 had significant changes. However, a more direct approach, investigating expression of putative hibernation-responsive genes by Northern analysis, revealed an increase in expression of transcription factors c-fos, junB, and c-Jun, but not junD, commencing during late torpor and peaking during the arousal phase of individual hibernation bouts. In contrast, prostaglandin D2 synthase declined during late torpor and arousal but returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine decarboxylase, and a sleep-related transcript SD464 did not change significantly during individual hibernation bouts. We also observed no decline in total RNA or total mRNA during torpor; such a decline had been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that accompany hibernation involve only modest reprogramming of gene expression or steady-state mRNA levels.