Role of orexin-A in the ventrolateral preoptic area on components of total energy expenditure.

BACKGROUND: Identifying whether components of total energy expenditure (EE) are affected by orexin receptor (OXR1 and OXR2) stimulation or antagonism with dual orexin receptor antagonists (DORAs) has relevance for obesity treatment. Orexin receptor stimulation reduces weight gain by increasing total EE and EE during spontaneous physical activity (SPA). OBJECTIVE: The purpose of this study was to determine if a DORA (TCS-1102) in the ventrolateral preoptic area (VLPO) reduced orexin-A-induced arousal, SPA, total EE and EE during sleep, rest, wake and SPA and whether the DORA alone reduced total EE and its components. We hypothesized that: (1) a DORA would reduce orexin-A induced increases in arousal, SPA, components of total EE, reductions in sleep and the EE during sleep and (2) the DORA alone would reduce baseline (non-stimulated) SPA and total EE. SUBJECTS/METHODS: Sleep, wakefulness, SPA and EE were determined after microinjection of the DORA (TCS-1102) and orexin-A in the VLPO of male Sprague-Dawley rats with a unilateral cannula targeted towards the VLPO. Individual components of total EE were determined based on time-stamped data. RESULTS: The DORA reduced orexin-A-induced increases in arousal, SPA, total EE and EE during SPA, wake, rest and sleep 1 h post injection (P<0.05). Orexin-A significantly reduced sleep and significantly increased EE during sleep 1 h post injection (P<0.05). Furthermore, the DORA alone significantly reduced total EE, EE during sleep (NREM and REM) and resting EE 2 h post injection (P<0.05). CONCLUSIONS: These data suggest that orexin-A reduces weight gain by stimulating total EE through increases in EE during SPA, rest and sleep. Residual effects of the DORA alone include decreases in total EE and EE during sleep and rest, which may promote weight gain.

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity.

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

Abnormal response of melanin-concentrating hormone deficient mice to fasting: hyperactivity and rapid eye movement sleep suppression.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that has been implicated in energy homeostasis. Pharmacological studies with MCH and its receptor antagonists have suggested additional behavioral roles for the neuropeptide in the control of mood and vigilance states. These suggestions have been supported by a report of modified sleep in the MCH-1 receptor knockout mouse. Here we found that MCH knockout (MCH(-)(/)(-)) mice slept less during both the light and dark phases under baseline conditions. In response to fasting, MCH(-)(/)(-) mice exhibited marked hyperactivity, accelerated weight loss and an exaggerated decrease in rapid eye movement (REM) sleep. Following a 6-h period of sleep deprivation, however, the sleep rebound in MCH(-)(/)(-) mice was normal. Thus MCH(-)(/)(-) mice adapt poorly to fasting, and their loss of bodyweight under this condition is associated with behavioral hyperactivity and abnormal expression of REM sleep. These results support a role for MCH in vigilance state regulation in response to changes in energy homeostasis and may relate to a recent report of initial clinical trials with a novel MCH-1 receptor antagonist. When combined with caloric restriction, the treatment of healthy, obese subjects with this compound resulted in some subjects experiencing vivid dreams and sleep disturbances.

Modafinil more effectively induces wakefulness in orexin-null mice than in wild-type littermates.

Narcolepsy-cataplexy, a disorder of excessive sleepiness and abnormalities of rapid eye movement (REM) sleep, results from deficiency of the hypothalamic orexin (hypocretin) neuropeptides. Modafinil, an atypical wakefulness-promoting agent with an unknown mechanism of action, is used to treat hypersomnolence in these patients. Fos protein immunohistochemistry has previously demonstrated that orexin neurons are activated after modafinil administration, and it has been hypothesized that the wakefulness-promoting properties of modafinil might therefore be mediated by the neuropeptide. Here we tested this hypothesis by immunohistochemical, electroencephalographic, and behavioral methods using modafinil at doses of 0, 10, 30 and 100 mg/kg i.p. in orexin-/- mice and their wild-type littermates. We found that modafinil produced similar patterns of neuronal activation, as indicated by Fos immunohistochemistry, in both genotypes. Surprisingly, modafinil more effectively increased wakefulness time in orexin-/- mice than in the wild-type mice. This may reflect compensatory facilitation of components of central arousal in the absence of orexin in the null mice. In contrast, the compound did not suppress direct transitions from wakefulness to REM sleep, a sign of narcolepsy-cataplexy in mice. Spectral analysis of the electroencephalogram in awake orexin-/- mice under baseline conditions revealed reduced power in the theta; band frequencies (8-9 Hz), an index of alertness or attention during wakefulness in the rodent. Modafinil administration only partly compensated for this attention deficit in the orexin null mice. We conclude that the presence of orexin is not required for the wakefulness-prolonging action of modafinil, but orexin may mediate some of the alerting effects of the compound.

GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and ß2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

Transient expression of neuropeptide W in postnatal mouse hypothalamus--a putative regulator of energy homeostasis.

Neuropeptide B and W (NPB and NPW) are cognate peptide ligands for NPBWR1 (GPR7), a G protein-coupled receptor. In rodents, they have been implicated in the regulation of energy homeostasis, neuroendocrine/autonomic responses, and social interactions. Although localization of these peptides and their receptors in adult rodent brain has been well documented, their expression in mouse brain during development is unknown. Here we demonstrate the transient expression of NPW mRNA in the dorsomedial hypothalamus (DMH) of postnatal mouse brain and its co-localization with neuropeptide Y (NPY) mRNA. Neurons expressing both NPW and NPY mRNAs begin to emerge in the DMH at about postnatal day 0 (P-0) through P-3. Their expression is highest around P-14, declines after P-21, and by P-28 only a faint expression of NPW and NPY mRNA remains. In P-18 brains, we detected NPW neurons in the region spanning the subincertal nucleus (SubI), the lateral hypothalamic (LH) perifornical (PF) areas, and the DMH, where the highest expression of NPW mRNA was observed. The majority of these postnatal hypothalamic NPW neurons co-express NPY mRNA. A cross of NPW-iCre knock-in mice with a Cre-dependent tdTomato reporter line revealed that more than half of the reporter-positive neurons in the adult DMH, which mature from the transiently NPW-expressing neurons, are sensitive to peripherally administrated leptin. These data suggest that the DMH neurons that transiently co-express NPW and NPY in the peri-weaning period might play a role in regulating energy homeostasis during postnatal development.

Midbrain dopaminergic neurons in the mouse: computer-assisted mapping.

The dopaminergic (DA) neurons in the midbrain play a role in cognition, affect and movement. The purpose of the present study was to map and quantify the number of DA neurons in the midbrain, within the nuclei that constitute cell groups A8, A9 and A10, in the mouse. Two strains of mice were used; the C57BL/6 strain was chosen because it is commonly used in neurobiological studies, and the FVB/N strain was chosen because it is used frequently in transgenic studies. DA neurons were identified, in every fifth 20-microns-thick coronal section, using an antibody against tyrosine hydroxylase. Cell locations were entered into a computer imaging system. The FVB/N strain has 42% more midbrain DA neurons than the C57BL/6 strain; on one side of the brain there were 15,135 +/- 356 neurons (mean +/- S.E.M.) in the FVB/N strain, and 10,645 +/- 315 neurons in the C57BL/6 strain. In both strains, approximately 11% of the neurons were located in nucleus A8 (the DA neurons in the retrorubral field), 38% in nucleus A9 (the DA neurons in the substantia nigra pars compacta, pars reticulata, and pars lateralis), and 51% in nucleus A10 (the DA neurons in midline regions such as the ventral tegmental area, central linear nucleus, and interfascicular nucleus). The number of midbrain DA cells, and their distribution within the three nuclear groups, is discussed with respect to findings in other species.

Midbrain dopaminergic neurons in the mouse: co-localization with Calbindin-D28K and calretinin.

The calcium-binding proteins Calbindin-D28k and calretinin are co-localized with dopamine in some of the midbrain dopaminergic neurons in the rat and monkey; the present study sought to examine the pattern of co-localization in the mouse. Double immunofluorescence staining procedures were used for tyrosine hydroxylase (a dopaminergic cell marker) and Calbindin-D28k or calretinin. Midbrain dopaminergic neurons were examined at four rostrocaudal levels, and the percentage of cells that contained both tyrosine hydroxylase and either of the two calcium-binding proteins was determined in nucleus A8 (retrorubral field), nucleus A9 (substantia nigra pars compacta, pars reticulata and pars lateralis) and nucleus A10 (nucleus paranigralis, ventral tegmental area, interfascicular nucleus, central linear nucleus). The two calcium-binding proteins were distributed similarly in midbrain dopaminergic neurons in the several nuclear groups that comprise nuclei A8, A9 and A10. The calcium-binding proteins were found in the majority (50-100%) of nucleus A10 neurons, whereas in nuclei A8 and A9 (except for the substantia nigra pars lateralis) less than 40% of the cells contained either calcium-binding protein. The pattern of co-localization in the mouse is similar to that reported for the rat and monkey. The calcium-binding proteins mark the population of midbrain dopaminergic neurons that are less vulnerable to degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

Rapid eye movement sleep deprivation modifies expression of long-term potentiation in visual cortex of immature rats.

During rapid eye movement (REM) sleep, activity of non-retinal origin is propagated into central visual-system pathways in a manner similar, in pattern and intensity, to central visual-system activity that is exogenously generated in waking. It has been hypothesized that REM sleep, which is more abundantly represented early in life than later, functions to provide adjunct 'afferent' input for shaping synaptic connectivity during brain maturation. Here we present data that support this proposal. Recent studies have described a developmentally regulated form of in vitro long-term potentiation (LTP) in the visual cortex that is experience- and age-dependent. In immature rats, suppression of retinal activation of the visual system by removal of visual experience (dark rearing) extends the age when the developmentally regulated form of LTP can be produced. This study tests whether suppression of REM-state activation of the visual system also lengthens the developmental period in which this specific form of LTP can be elicited. Young rats were deprived of REM sleep by the multiple-small-platforms-over-water method during the typically latest week for induction of such LTP in slices of visual cortex. After this week, we could still induce LTP in slices from nearly all the REM-sleep-deprived rats (8/9) but not from age-matched rats that had not lost REM sleep (0/5). The control rats had been housed on large platforms that allow the animals to obtain REM sleep. Only body weights and the concentration of thyrotrophin-releasing hormone in the hypothalamus distinguished home-caged, normal-sleeping controls from both groups of platform animals. On all measures, stress levels were not dissimilar in the two platforms groups. After 7 days of behavioral suppression of REM sleep in immature rats, and consequent reduction of the intense, extra-retinal activity endogenously generated during this sleep state, we found that the period was extended in which developmentally regulated synaptic plasticity (LTP) could be elicited in slices of visual neocortex. These studies support the role of REM sleep and its associated neuronal activity in brain maturation.

Evidence for a deficit in cholinergic interneurons in the striatum in schizophrenia.

Neurochemical and functional abnormalities of the striatum have been reported in schizophrenic brains, but the cellular substrates of these changes are not known. We hypothesized that schizophrenia may involve an abnormality in one of the key modulators of striatal output, the cholinergic interneuron. We measured the densities of cholinergic neurons in the striatum in schizophrenic and control brains in a blind analysis, using as a marker of this cell population immunoreactivity for choline acetyltransferase, the synthetic enzyme of acetylcholine. As an independent marker, we used immunoreactivity for calretinin, a protein which is co-localized with choline acetyltransferase in virtually all of the cholinergic interneurons of the striatum. A significant decrease in choline acetyltransferase-positive and calretinin-positive cell densities was found in the schizophrenic cases compared with controls in the striatum as a whole [for the choline acetyltransferase-positive cells: controls: 3.21 +/- 0.48 cells/mm2 (mean +/- S.D.), schizophrenics: 2.43 +/- 0.68 cells(mm2; P < 0.02]. The decrease was patchy in nature and most prominent in the ventral striatum (for the choline acetyltransferase-positive cells: controls: 3.47 +/- 0.59 cells/mm2, schizophrenics: 2.52 +/- 0.64 cells/ mm2; P < 0.005) which included the ventral caudate nucleus and nucleus accumbens region. Three of the schizophrenic cases with the lowest densities of cholinergic neurons had not been treated with neuroleptics for periods from more than a month to more than 20 years. A decrease in the number or function of the cholinergic interneurons of the striatum may disrupt activity in the ventral striatal-pallidal-thalamic-prefrontal cortex pathway and thereby contribute to abnormalities in function of the prefrontal cortex in schizophrenia.

Upregulation of estrogen receptors in the forebrain of aromatase knockout (ArKO) mice.

Estrogens have numerous reproductive and nonreproductive functions in brain. The actions of estrogens are mediated by estrogen receptors (ERs), and estrogens are believed to down-regulate their own receptors in many tissues. Assuming this to be true, if estrogens are removed there should be an upregulation of ERs. We have developed a mouse model in which estrogen synthesis is completely eliminated by homologous recombination to delete the gene encoding aromatase cytochrome P450 (P450(arom)). The P450(arom) enzyme catalyzes the synthesis of estrogens from androgens in the brain. The localization and density of ERs was studied in the brains of aromatase knockout (ArKO) and wild type male mice by using immunohistochemistry with a peptide antibody to ERalpha (ER-21) and computer imaging. In the wild-type animals a high density of ERalpha was found in a small number of hypothalamic cells; in the medial preoptic area, periventricular, arcuate, and ventromedial nuclei. A low and medium density of ERalpha was observed in cells of the lateral preoptic area, supraoptic, bed nucleus of the stria terminalis, and in central, medial and anterior cortical amygdaloid nuclei. The number of cells containing ERalpha-immunoreactivity was significantly increased (244%) in the medial preoptic area of the ArKO mice. In neither wild type nor ArKO animals was immunoreactivity observed in the cerebral cortex or striatum. There was intense ER-immunostaining in the nucleus of neurons in both wild type and ArKO mice. These data indicate that in the absence of estrogens there is as much as a 2-fold increase in the number of cells with ERalpha-immunoreactivity in certain hypothalamic and limbic regions. Thus, estrogens can down-regulate ERalpha in brain.

Gestational caffeine modifies offspring behaviour in mice.

Dams from two strains of mice, BALB/c C57BR were tested during gestation with caffeine, at doses of about 60, 80 and 100 mg/kg/day, in their drinking water. The resulting offspring were behaviourally tested over a 6-month period commencing at age 9 months. When compared with controls, mice from dams that had received caffeine demonstrated longer latencies in a passive avoidance test, and differences were also noted for female C57BR offspring in activity and habituation measures. Having controlled as far as possible for post-natal maternal and environmental effects, the most likely conclusion is that caffeine has a direct pharmacological action on the foetus, and should therefore be classed as a behavioural teratogen in mice.

Cholecystokinin and cholecystokinin antagonists enhance postsynaptic excitability in the dentate gyrus.

The sulfated and unsulfated octapeptide cholecystokinin (CCK) sequences and the pancreatic CCK antagonists, CR 1409 and benzotript, were applied iontophoretically in the rat dentate gyrus granular layer while the response evoked by single pulse stimulation of the perforant path was recorded. The stimulating current was varied and the resulting relationship between the slope of the response (input) against the population spike amplitude (output) was used as a measure of excitability at the granule cell synapse. All four test compounds shifted the input/output curve to the left indicating an increase in postsynaptic excitability. These results thus imply that endogenous CCK acts at the central type of CCK receptor to modulate cortical input to granule cells by reducing the threshold for synaptic excitation.

Cholecystokinin and an evoked response in the dentate gyrus.

Cholecystokinin (CCK) as the sulfated (CCK-8S) and unsulfated (CCK-8U) octapeptide sequences, and CR 1409 were administered intraventricularly while the action potential (EAP) in the granular cell layer of the hippocampal dentate gyrus evoked by perforant path stimulation was recorded. No consistent effect of the test substances on the amplitude of the EAP was found at doses corresponding to those previously reported to cause an increase in the EAP when administered systemically. Similarly, no effect of CCK on the EAP could be found when the peptide was administered iontophoretically in the granular cell layer. In contrast, iontophoretically applied CCK-8S, CCK-8U and CR 1409 slightly but consistently reduced the slope of the evoked response recorded in the dentate gyrus molecular layer. These results are interpreted as indicating that the CCK receptor on granular cell dendrites is likely to be the central type that is activated by both CCK-8S and CCK-8U, but that any effects of systemically administered CCK on the EAP are probably mediated in the periphery.

Paradoxical sleep and coping with environmental change.

Long-term sleep recordings of mice from 3 inbred strains showed that the amount of time spent in sleep over successive 24-h periods varied as the subjects adapted to experimental conditions. A sinusoidal type variation, with a period of about 15 days, modulating the basic trends in sleep times is described; but the principal finding of this study relates to a monotonic decrease in Paradoxical Sleep (PS) as recording continued. Age, stimulus deprivation and fatigue effects do not appear to be causative factors for this decrement, indicating that the changes in PS might reflect a habituation process. On this basis it is hypothesized that an increase in PS time in the mouse is a specific response to a significant environmental stimulus and could, therefore, form part of the coping strategy. This hypothesis is generalized and discussed in terms of its implications, both for experimentation concerned with measuring PS times and with the possible functional significance of PS.

Inhibition of striatal energy metabolism produces cell loss in the ipsilateral substantia nigra.

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 micromol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.

Electrophysiological evidence for a functional differentiation between subtypes of the 5-HT1 receptor.

Serotonin (5-HT) neurons in the dorsal (DRN) and median (MRN) raphe nuclei, and dopamine (DA) neurons in the substantia nigra (SN) were recorded extracellularly in the anesthetized rat. Compounds which have a relatively high affinity for the 5-HT1A or 5-HT1B subtypes of the 5-HT1 receptor were administered and their effect on the firing rate of the monoamine cells was determined. 5-HT1A ligands were more potent in inhibiting impulse activity in the DRN than in the MRN, but had little effect in the SN. In contrast, 5-HT1B ligands increased the firing rate of MRN 5-HT units at low doses, and were also effective inhibitors of DA cell firing in the SN. These results could be correlated with recently described differences in the distribution of the 5-HT1A and 5-HT1B receptor subtypes, and were interpreted as indicating possible functional differentiation between these subtypes. In particular, agonist activity at the 5-HT1B autoreceptor site may decrease 5-HT release, suggesting a presynaptic locus for this receptor in the somatodendritic region. The site also appears to be implicated in 5-HT modulation of nigral DA impulse flow.

Biochemical and pharmacological characterization of CGS 12066B, a selective serotonin-1B agonist.

CGS 12066B is a novel pyrroloquinoxaline with selectivity for the serotonin-1B (5HT1B) recognition site as assessed by binding, biochemical and electrophysiological studies. The compound had an IC50 value of 51 nM at the 5HT1B recognition site as determined using the binding of [3H]5HT in the presence of 1 microM spiperone. At the 5HT1A receptor the compound had an IC50 value of 876 nM, providing a 5HT1A/5HT1B ratio of 17 in contrast to the putative 5HT1B selective agent trifluoromethylphenylpiperazine (TFMPP) which had a corresponding ratio of 3.6. The compound had minimal affinity for alpha 1-, alpha 2- and beta-adrenoceptors and for dopamine D-1 and D-2 receptors. CGS 12066B, in contrast to TFMPP, which was inactive, was found to inhibit dorsal raphe cell firing with an ED50 value of 358 nmol/kg i.v. The corresponding values for the 5HT1A selective agonists 8-OH-DPAT and ipsapirone were 1.3 and 33 nmol/kg. CGS 12066B was also effective in decreasing rat brain 5-HTP concentrations and inhibiting in vitro 5HT release. The data obtained indicate that CGS 12066B is a reasonably active 5HT1B site agonist, which due to its selectivity as compared to compounds such as TFMPP, will be a useful tool for evaluating the physiological role of such receptors in the mammalian CNS.

Cholecystokinin modulates neurotransmission through the dentate gyrus.

The sulfated and unsulfated cholecystokinin (CCK) octapeptide sequences and the pancreatic CCK antagonists, CR 1409 and benzotript, were administered iontophoretically while dentate gyrus granule cell activity was recorded in the anesthetized rat. During application of the compounds, the peri-stimulus time histogram (PSTH) was constructed of granule cell activity coupled to stimulation of the sciatic nerve. CCK and CR 1409, but not benzotript, were found to change significantly the PSTH by enhancing and prolonging the response to sensory stimulation. These results are interpreted as indicating that CCK can modulate impulse flow through the dentate gyrus.

Increased sleep time in the offspring of caffeine-treated dams from two inbred strains of mice.

Dams from two inbred strains of mice (C57BR and BALB/c) were treated with caffeine in solution in their drinking water during gestation. Doses of caffeine used corresponded to about 60, 80 or 100 mg/kg/day; controls received tap water. The offspring (as adults) revealed a significantly increased sleep time following caffeine treatment, but primarily as slow wave sleep in the males of the BALB/c strain and paradoxical sleep in the females of the C57BR strain. BALB/c females and C57BR males were relatively unaffected. These results, and in particular the sex differences, are discussed in terms of a possible central site of action of caffeine.