RESUMO
We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD(67) mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD(67)in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD(+), Fos-ir/MCH(+), and GAD(+)/MCH(+) double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD(+) neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.
Assuntos
Distúrbios do Sono por Sonolência Excessiva/fisiopatologia , Hipotálamo/fisiologia , Neurônios/metabolismo , Sono REM/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Distúrbios do Sono por Sonolência Excessiva/genética , Distúrbios do Sono por Sonolência Excessiva/metabolismo , Eletroencefalografia , Eletromiografia , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Técnicas In Vitro , Masculino , Melaninas/genética , Melaninas/metabolismo , Neurônios/citologia , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/genéticaRESUMO
Formerly believed to contribute to behavioural waking (W) alone, dopaminergic (DA) neurons are now also known to participate in the regulation of paradoxical sleep (PS or REM) in mammals. Indeed, stimulation of postsynaptic DA1 receptors with agonists induces a reduction in the daily amount of PS. DA neurons in the ventral tegmental area were recently shown to fire in bursts during PS, but nothing is known about the activity of the other DA cell groups in relation to waking or PS. To fulfil this gap, we used a protocol in which rats were maintained in continuous W for 3h in a novel environment, or specifically deprived of PS for 3 days with some of them allowed to recover from this deprivation. A double immunohistochemical labeling with Fos and tyrosine hydroxylase was then performed. DA neurons in the substantia nigra (A9) and ventral tegmental area (A10), and its dorsocaudal extension in the periaqueductal gray (A10dc), almost never showed a Fos-immunoreactive nucleus, regardless of the experimental condition. The caudal hypothalamic (A11) group showed a moderate activation after PS deprivation and novel environment. During PS-recovery, the zona incerta (A13) group contained a significant number and percentage of double-labeled neurons. These results suggest that some DA neurons (A11) could participate in waking and/or the inhibition of PS during PS deprivation whereas others (A13) would be involved in the control of PS.
Assuntos
Dopamina/metabolismo , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sono REM/fisiologia , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Vigília/fisiologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Mapeamento Encefálico , Hipotálamo/citologia , Hipotálamo/metabolismo , Imuno-Histoquímica , Masculino , Rede Nervosa/citologia , Rede Nervosa/metabolismo , Vias Neurais/citologia , Vias Neurais/metabolismo , Ratos , Substância Negra/citologia , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/citologiaRESUMO
Noradrenaline is known to induce waking (W) and to inhibit paradoxical sleep (PS or REM). Both roles have been exclusively attributed to the noradrenergic neurons of the locus coeruleus (LC, A6), shown to be active during W and inactive during PS. However, the A1, A2, A5 and A7 noradrenergic neurons could also be responsible. Therefore, to determine the contribution of each of the noradrenergic groups in W and in PS inhibition, rats were maintained in continuous W for 3h in a novel environment or specifically deprived of PS for 3 days, with some of them allowed to recover from this deprivation. A double immunohistochemical labeling with Fos and tyrosine hydroxylase was then performed. Thirty percent of the LC noradrenergic cells were found to be Fos-positive after exposure to the novel environment and less than 2% after PS deprivation. In contrast, a significant number of double-labeled neurons (up to 40% of the noradrenergic neurons) were observed in the A1/C1, A2 and A5 groups, after both novel environment and PS deprivation. After PS recovery and in control condition, less than 1% of the noradrenergic neurons were Fos-immunoreactive, regardless of the noradrenergic group. These results indicate that the brainstem noradrenergic cell groups are activated during W and silent during PS. They further suggest that the inhibitory effect of noradrenaline on PS may be due to the A1/C1, A2 and to a lesser degree to A5 neurons but not from those of the LC as previously hypothesized.
Assuntos
Neurônios/metabolismo , Neurônios/fisiologia , Norepinefrina/fisiologia , Proteínas Oncogênicas v-fos/biossíntese , Privação do Sono/metabolismo , Sono REM/fisiologia , Animais , Imuno-Histoquímica , Masculino , Ratos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Although the main nodes of the neuronal network that regulate paradoxical sleep (PS), also called rapid eye movement sleep, have been identified in rodents, it still needs to be more thoroughly described. We have recently shown that 58% of a hypothalamic neuronal population, the melanin-concentrating hormone (MCH) neurons, are activated after a PS hypersomnia and that MCH, when injected intracerebroventricularly, induces a dose-dependent increase in PS. This suggests that MCH plays a role in PS regulation. Two subpopulations of MCH neurons have been distinguished neurochemically, one that coexpresses cocaine and amphetamine-regulated transcript (CART) and sends ascending projections to the septum and the hippocampus, the other, the non-CART MCH neurons, send descending projections to the lower brainstem and the spinal cord. In order to better characterize the PS-activated MCH neurons it is interesting to determine whether they belong to the first, the second, or both subgroups. We therefore undertook an MCH, CART, and Fos triple immunolabeling study in PS hypersomniac rats. We showed that the MCH neurons activated during PS are part of both subpopulations since we found CART and non-CART MCH-activated neurons. Based on these results and the literature, we propose that MCH could be involved in memory processes and in the inhibition of muscle tone during PS.
Assuntos
Distúrbios do Sono por Sonolência Excessiva/patologia , Distúrbios do Sono por Sonolência Excessiva/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Neurônios/metabolismo , Hormônios Hipofisários/metabolismo , Animais , Contagem de Células/métodos , Distúrbios do Sono por Sonolência Excessiva/etiologia , Análise de Fourier , Hipotálamo/patologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas v-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Privação do Sono/complicações , Proteína do Transcrito Regulada por Cocaína e AnfetaminaRESUMO
Locus coeruleus (LC) noradrenergic neurons are active during wakefulness, slow their discharge rate during slow wave sleep, and stop firing during paradoxical sleep (PS). A large body of data indicates that their inactivation during PS is due to a tonic GABAergic inhibition. To localize the neurons responsible for such inhibition, we first examined the distribution of retrogradely and Fos double-immunostained neurons following cholera toxin b subunit (CTb) injection in the LC of control rats, rats selectively deprived of PS for 3 days, and rats allowed to recover for 3 hours from such deprivation. We found a significant number of CTb/Fos double-labeled cells only in the recovery group. The largest number of CTb/Fos double-labeled cells was found in the dorsal paragigantocellular reticular nucleus (DPGi). It indeed contained 19% of the CTb/Fos double-labeled neurons, whereas the ventrolateral periaqueductal gray (vlPAG) contained 18.3% of these neurons, the lateral paragigantocellular reticular nucleus (LPGi) 15%, the lateral hypothalamic area 9%, the lateral PAG 6.7%, and the rostral PAG 6%. In addition, CTb/Fos double-labeled cells constituted 43% of all the singly CTb-labeled cells counted in the DPGi compared with 29% for the LPGi, 18% for the rostral PAG, and 10% or less for the other structures. Although all these populations of CTb/Fos double-labeled neurons could be GABAergic and tonically inhibit LC neurons during PS, our results indicate that neurons from the DPGi constitute the best candidate for this role.
Assuntos
Locus Cerúleo/citologia , Vias Neurais/anatomia & histologia , Neurônios/metabolismo , Norepinefrina/metabolismo , Sono REM/fisiologia , Animais , Toxina da Cólera/metabolismo , Locus Cerúleo/metabolismo , Masculino , Vias Neurais/metabolismo , Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Privação do SonoRESUMO
It is well accepted that populations of neurons responsible for the onset and maintenance of paradoxical sleep (PS) are restricted to the brainstem. To localize the structures involved and to reexamine the role of mesopontine cholinergic neurons, we compared the distribution of Fos- and choline acetyltransferase-labelled neurons in the brainstem of control rats, rats selectively deprived of PS for approximately 72 h and rats allowed to recover from such deprivation. Only a few cholinergic neurons from the laterodorsal (LDTg) and pedunculopontine tegmental nuclei were Fos-labelled after PS recovery. In contrast, a large number of noncholinergic Fos-labelled cells positively correlated with the percentage of time spent in PS was observed in the LDTg, sublaterodorsal, alpha and ventral gigantocellular reticular nuclei, structures known to contain neurons specifically active during PS. In addition, a large number of Fos-labelled cells were seen after PS rebound in the lateral, ventrolateral and dorsal periaqueductal grey, dorsal and lateral paragigantocellular reticular nuclei and the nucleus raphe obscurus. Interestingly, half of the cells in the latter nucleus were immunoreactive to choline acetyltransferase. In contrast to the well-accepted hypothesis, our results strongly suggest that neurons active during PS, recorded in the mesopontine cholinergic nuclei, are in the great majority noncholinergic. Our findings further demonstrate that many brainstem structures not previously identified as containing neurons active during PS contain cholinergic or noncholinergic neurons active during PS, and these structures may therefore play a key role during this state. Altogether, our results open a new avenue of research to identify the specific role of the populations of neurons revealed, their interrelations and their neurochemical identity.
Assuntos
Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Fibras Colinérgicas/fisiologia , Neurônios/fisiologia , Privação do Sono/fisiopatologia , Animais , Contagem de Células , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/ultraestrutura , Substância Cinzenta Periaquedutal/citologia , Substância Cinzenta Periaquedutal/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos da Rafe/citologia , Núcleos da Rafe/fisiologia , Ratos , Ratos Sprague-Dawley , Formação Reticular/citologia , Formação Reticular/fisiologia , Vigília/fisiologiaRESUMO
BACKGROUND: Peptidergic neurons containing the melanin-concentrating hormone (MCH) and the hypocretins (or orexins) are intermingled in the zona incerta, perifornical nucleus and lateral hypothalamic area. Both types of neurons have been implicated in the integrated regulation of energy homeostasis and body weight. Hypocretin neurons have also been involved in sleep-wake regulation and narcolepsy. We therefore sought to determine whether hypocretin and MCH neurons express Fos in association with enhanced paradoxical sleep (PS or REM sleep) during the rebound following PS deprivation. Next, we compared the effect of MCH and NaCl intracerebroventricular (ICV) administrations on sleep stage quantities to further determine whether MCH neurons play an active role in PS regulation. RESULTS: Here we show that the MCH but not the hypocretin neurons are strongly active during PS, evidenced through combined hypocretin, MCH, and Fos immunostainings in three groups of rats (PS Control, PS Deprived and PS Recovery rats). Further, we show that ICV administration of MCH induces a dose-dependent increase in PS (up to 200%) and slow wave sleep (up to 70%) quantities. CONCLUSION: These results indicate that MCH is a powerful hypnogenic factor. MCH neurons might play a key role in the state of PS via their widespread projections in the central nervous system.