RESUMO
The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.
Assuntos
Arginina Vasopressina , Ritmo Circadiano , Núcleo Supraquiasmático , Animais , Camundongos , Arginina , Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMO
Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used SIAH2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of SIAH2-deficiency on the regulation of rhythmically expressed genes in the liver. The absence of SIAH2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic transcriptome in the liver by increasing the number of day-time expressed genes, and flipping the rhythmic expression from nighttime expressed genes to the daytime. These effects are not readily explained by effects on known sexually dimorphic pathways in females. Moreover, loss of SIAH2 in females, not males, preferentially altered the expression of transcription factors and genes involved in regulating lipid and lipoprotein metabolism. Consequently, SIAH2-deficient females, but not males, displayed disrupted daily lipid and lipoprotein patterns, increased adiposity and impaired metabolic homeostasis. Overall, these data suggest that SIAH2 may be a key component of a female-specific circadian transcriptional output circuit that directs the circadian timing of gene expression to regulate physiological rhythms, at least in the liver. In turn, our findings imply that sex-specific transcriptional mechanisms may closely interact with the circadian clock to tailor overt rhythms for sex-specific needs.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Feminino , Lipídeos , Lipoproteínas , Masculino , Camundongos , Ubiquitina , Ubiquitina-Proteína Ligases/genéticaRESUMO
Individuals with Angelman syndrome (AS) suffer sleep disturbances that severely impair quality of life. Whether these disturbances arise from sleep or circadian clock dysfunction is currently unknown. Here, we explored the mechanistic basis for these sleep disorders in a mouse model of Angelman syndrome (Ube3a(m-/p+) mice). Genetic deletion of the maternal Ube3a allele practically eliminates UBE3A protein from the brain of Ube3a(m-/p+) mice, because the paternal allele is epigenetically silenced in most neurons. However, we found that UBE3A protein was present in many neurons of the suprachiasmatic nucleus--the site of the mammalian circadian clock--indicating that Ube3a can be expressed from both parental alleles in this brain region in adult mice. We found that while Ube3a(m-/p+) mice maintained relatively normal circadian rhythms of behavior and light-resetting, these mice exhibited consolidated locomotor activity and skipped the timed rest period (siesta) present in wild-type (Ube3a(m+/p+)) mice. Electroencephalographic analysis revealed that alterations in sleep regulation were responsible for these overt changes in activity. Specifically, Ube3a(m-/p+) mice have a markedly reduced capacity to accumulate sleep pressure, both during their active period and in response to forced sleep deprivation. Thus, our data indicate that the siesta is governed by sleep pressure, and that Ube3a is an important regulator of sleep homeostasis. These preclinical findings suggest that therapeutic interventions that target mechanisms of sleep homeostasis may improve sleep quality in individuals with AS. SIGNIFICANCE STATEMENT: Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternal copy of the UBE3A gene. Individuals with AS have severe sleep dysfunction that affects their cognition and presents challenges to their caregivers. Unfortunately, current treatment strategies have limited efficacy due to a poor understanding of the mechanisms underlying sleep disruptions in AS. Here we demonstrate that abnormal sleep patterns arise from a deficit in accumulation of sleep drive, uncovering the Ube3a gene as a novel genetic regulator of sleep homeostasis. Our findings encourage a re-evaluation of current treatment strategies for sleep dysfunction in AS, and suggest that interventions that promote increased sleep drive may alleviate sleep disturbances in individuals with AS.
Assuntos
Ondas Encefálicas/fisiologia , Ritmo Circadiano/genética , Homeostase/genética , Transtornos do Sono-Vigília/genética , Ubiquitina-Proteína Ligases/metabolismo , Análise de Variância , Animais , Ondas Encefálicas/genética , Modelos Animais de Doenças , Eletroencefalografia , Eletromiografia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Daily rhythms in mammals are programmed by a master clock in the suprachiasmatic nucleus (SCN). The SCN contains two main compartments (shell and core), but the role of each region in system-level coordination remains ill defined. Herein, we use a functional assay to investigate how downstream tissues interpret region-specific outputs by using in vivo exposure to long day photoperiods to temporally dissociate the SCN. We then analyze resulting changes in the rhythms of clocks located throughout the brain and body to examine whether they maintain phase synchrony with the SCN shell or core. RESULTS: Nearly all of the 17 tissues examined in the brain and body maintain phase synchrony with the SCN shell, but not the SCN core, which indicates that downstream oscillators are set by cues controlled specifically by the SCN shell. Interestingly, we also found that SCN dissociation diminished the amplitude of rhythms in core clock gene and protein expression in brain tissues by 50-75 %, which suggests that light-driven changes in the functional organization of the SCN markedly influence the strength of rhythms in downstream tissues. CONCLUSIONS: Overall, our results reveal that body clocks receive time-of-day cues specifically from the SCN shell, which may be an adaptive design principle that serves to maintain system-level phase relationships in a changing environment. Further, we demonstrate that lighting conditions alter the amplitude of the molecular clock in downstream tissues, which uncovers a new form of plasticity that may contribute to seasonal changes in physiology and behavior.
Assuntos
Encéfalo/fisiologia , Relógios Circadianos , Neurônios/citologia , Núcleo Supraquiasmático/citologia , Animais , Encéfalo/citologia , Ritmo Circadiano , Luz , Masculino , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , FotoperíodoRESUMO
The suprachiasmatic nucleus (SCN) contains a circadian clock that generates endogenous rhythmicity and entrains that rhythmicity with the day-night cycle. The neurochemical events that transduce photic input within the SCN and mediate entrainment by resetting the molecular clock have yet to be defined. Because GABA is contained in nearly all SCN neurons we tested the hypothesis that GABA serves as this signal in studies employing Syrian hamsters (Mesocricetus auratus). Activation of GABAA receptors was found to be necessary and sufficient for light to induce phase delays of the clock. Remarkably, the sustained activation of GABAA receptors for more than three consecutive hours was necessary to phase-delay the clock. The duration of GABAA receptor activation required to induce phase delays would not have been predicted by either the prevalent theory of circadian entrainment or by expectations regarding the duration of ionotropic receptor activation necessary to produce functional responses. Taken together, these data identify a novel neurochemical mechanism essential for phase-delaying the 'master' circadian clock within the SCN as well as identifying an unprecedented action of an amino acid neurotransmitter involving the sustained activation of ionotropic receptors.
Assuntos
Relógios Circadianos/fisiologia , Luz , Receptores de GABA-A/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Bicuculina/farmacologia , Relógios Circadianos/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , GABAérgicos/farmacologia , Masculino , Mesocricetus , Microinjeções , Muscimol/farmacologia , Tempo de Reação/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos dos fármacos , Fatores de TempoRESUMO
Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.
Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/imunologia , Inflamação/imunologia , Síndrome do Jet Lag/imunologia , Macrófagos Peritoneais/imunologia , Animais , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Síndrome do Jet Lag/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polissonografia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Resilience, the ability to overcome stressful conditions, is found in most mammals and varies significantly among individuals. A lack of resilience can lead to the development of neuropsychiatric and sleep disorders, often within the same individual. Despite extensive research into the brain mechanisms causing maladaptive behavioral-responses to stress, it is not clear why some individuals exhibit resilience. To examine if sleep has a determinative role in maladaptive behavioral-response to social stress, we investigated individual variations in resilience using a social-defeat model for male mice. Our results reveal a direct, causal relationship between sleep amount and resilience-demonstrating that sleep increases after social-defeat stress only occur in resilient mice. Further, we found that within the prefrontal cortex, a regulator of maladaptive responses to stress, pre-existing differences in sleep regulation predict resilience. Overall, these results demonstrate that increased NREM sleep, mediated cortically, is an active response to social-defeat stress that plays a determinative role in promoting resilience. They also show that differences in resilience are strongly correlated with inter-individual variability in sleep regulation.
To many of us, it may seem obvious that sleep is restorative: we feel better after a good night's rest. However, exactly how sleep benefits the brain and body remains poorly understood. One clue may lie in neuropsychiatric disorders: these conditions such as depression and anxiety are often accompanied by disrupted sleep. Additionally, these neuropsychiatric disorders are frequently caused or worsened by stress, which can also interfere with sleep. This close association between stress and sleep has led some to hypothesize that sleep serves to overcome the adverse effects of stress on the brain, but this hypothesis remains largely untested. One type of stress that is common to all mammals is social stress, defined as stress caused by social interactions. This means that mice and other rodents can be subjected to social stress in the laboratory to test hypotheses about the effects of stress on the brain. Importantly, in both animals and humans, there are individual differences in resilience, or the ability to overcome the adverse effects of stress. Based on this information, Bush et al. set out to establish whether sleep can regulate resilience to social stress in mice. When the mice were gently kept awake during their normal sleep time, resilience decreased and so the mice were less able to overcome the negative effects of stress. Conversely, increasing sleep, by activating an area of the brain responsible for initiating sleep, increased the mice's resilience to social stress. Thus, Bush et al. showed that changes in sleep do lead to changes in resilience. To find out whether resilience can be predicted by changes in sleeping patterns, Bush et al. studied how both resilient mice and those susceptible to stress slept before and after social stress. Resilient mice would often sleep more after social stress; meanwhile, few changes were observed in susceptible mice. Surprisingly, sleep quality also predicted resilience, with resilient mice sleeping better than susceptible mice even before exposure to social stress. To determine whether the differences in sleep that predict resilience can be detected as brain activity, Bush et al. placed electrodes in two regions of the prefrontal cortex a part of the brain important for decision-making and social behaviors to measure how mice recovered lost sleep. This experiment revealed that the changes in sleep that predict resilience are prominent in the prefrontal cortex. Overall, Bush et al. reveal that sleeping more and sleeping better promote resilience to social stress. Furthermore, the results suggests that lack of sleep may lead to increased risk of stress-related psychiatric conditions. Humans are one of the few species that choose to deprive themselves of sleep: Bush, et al. provide evidence that this choice may have significant consequences on mental health. Furthermore, this work creates a new model that lays the groundwork for future studies investigating how sleep can overcome stress on the brain.
Assuntos
Movimentos Oculares , Estresse Psicológico , Animais , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Estresse Psicológico/psicologia , Córtex Pré-Frontal , Sono , MamíferosRESUMO
Chronic sleep loss, a common feature of human life in industrialized countries, is associated to cardiovascular disorders. Variations in functional parameters of coagulation might contribute to explain this relationship. By exploiting the mouse model and a specifically designed protocol, we demonstrated that seven days of partial sleep deprivation significantly decreases (-30.5%) the thrombin generation potential in plasma evaluated upon extrinsic (TF/FVIIa pathway) but not intrinsic activation of coagulation. This variation was consistent with a decrease (-49.8%) in the plasma activity levels of factor VII (FVII), the crucial physiologicalal trigger of coagulation, which was even more pronounced at the liver mRNA level (-85.7%). The recovery in normal sleep conditions for three days completely restored thrombin generation and FVII activity in plasma. For the first time, we demonstrate that chronic sleep deprivation on its own reduces, in a reversible manner, the FVII expression levels, thus influencing the TF/FVIIa activation pathway efficiency.
Assuntos
Fator VII/genética , Regulação da Expressão Gênica , Privação do Sono/sangue , Privação do Sono/fisiopatologia , Animais , Doença Crônica , Fator VII/metabolismo , Fator VIIa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombina/metabolismo , Tromboplastina/metabolismo , Fatores de Tempo , Redução de Peso/fisiologiaRESUMO
Activation of gamma-aminobutyric acid (GABA) A receptors in the suprachiasmatic nucleus (SCN) resets the circadian clock during the day and inhibits the ability of light to reset the clock at night. Light in turn acts during the day to inhibit the phase-resetting effects of GABA. Some evidence suggests that Period mRNA changes in the SCN are responsible for these interactions between light and GABA. Here, the hypothesis that light and the GABA A receptor interact by altering the expression of Period 1 and/or Period 2 mRNA in the SCN is tested. The GABA A agonist muscimol was injected near the SCN just prior to a light pulse, during the mid-subjective day and the early and late subjective night. Changes in Period 1 and Period 2 mRNA were measured in the SCN by in situ hybridization. Light-induced Period 1 mRNA was inhibited by GABA A receptor activation in the early and late subjective night, while Period 2 mRNA was only inhibited during the late night. During the subjective day, light had no effect on the ability of muscimol to suppress Period 1 mRNA hybridization signal. Thus, light and GABA A receptor activation inhibit each other's ability to induce behavioral phase shifts throughout the subjective day and night. However, only in the late night are these behavioral effects correlated with changes in Period gene expression. Together, our data support the hypothesis that the interacting effects of light and GABA are the result of the opposing actions of these stimuli on Period mRNA, but only during the subjective night.
Assuntos
Proteínas do Olho/biossíntese , Agonistas de Receptores de GABA-A , Luz , RNA Mensageiro/biossíntese , Receptores de GABA-A/fisiologia , Núcleo Supraquiasmático/metabolismo , Animais , Autorradiografia , Cricetinae , Interpretação Estatística de Dados , Proteínas do Olho/genética , Agonistas GABAérgicos/farmacologia , Hibridização In Situ , Masculino , Mesocricetus , Muscimol/farmacologia , Proteínas Circadianas Period , RNA Mensageiro/genética , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiaçãoRESUMO
There are no Food and Drug Administration approved pharmacotherapies for methamphetamine (METH) overdose, thus identifying novel drug targets to prevent this devastating adverse event is a public-health imperative. Previous research suggests that serotonin and sigma receptors may contribute to the adverse effects of METH. The present study assessed whether pretreatment with the 5-HT2A receptor antagonist M100907 or the sigma 1 (σ1) receptor antagonist BD 1047 attenuated METH-induced lethality, hyperthermia, convulsions, and seizures. Male, Swiss-Webster mice received intraperitoneal injections of M100907 (1 and 10â¯mg/kg), BD 1047 (10â¯mg/kg), or a combination of M100907 (1â¯mg/kg) and BD 1047 (10â¯mg/kg) prior to treatment with METH (78â¯mg/kg). Convulsions and lethality were assessed by observation, core body temperature was assessed by surgically implanted telemetric probes, and seizures were assessed by electroencephalography. M100907 reduced METH-elicited lethality from 67% to 33%, BD1047 reduced METH-elicited lethality from 67% to 50%, and combined administration of both agents eliminated lethality in all mice tested. Similarly, both agents and their combination reduced METH-elicited seizures and convulsions. None of the treatments decreased METH-induced hyperthermia. This research suggests that reducing METH-induced seizures is an important factor in reducing lethality associated with METH overdose. However, future studies should examine whether M100907 and BD 1047 modulate METH-induced hypertension and other adverse effects that may also contribute to METH overdose. Our data support the continued investigation of compounds that target 5-HT2A and σ1 receptors in METH-induced overdose, including their potential to yield emergency reversal agents.
Assuntos
Estimulantes do Sistema Nervoso Central/antagonistas & inibidores , Estimulantes do Sistema Nervoso Central/toxicidade , Etilenodiaminas/farmacologia , Fluorbenzenos/farmacologia , Metanfetamina/antagonistas & inibidores , Metanfetamina/toxicidade , Piperidinas/farmacologia , Receptores sigma/antagonistas & inibidores , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Eletroencefalografia/efeitos dos fármacos , Febre/induzido quimicamente , Febre/prevenção & controle , Dose Letal Mediana , Masculino , Camundongos , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Receptor Sigma-1RESUMO
Sleep loss can severely impair the ability to perform, yet the ability to recover from sleep loss is not well understood. Sleep regulatory processes are assumed to lie exclusively within the brain mainly due to the strong behavioral manifestations of sleep. Whole-body knockout of the circadian clock gene Bmal1 in mice affects several aspects of sleep, however, the cells/tissues responsible are unknown. We found that restoring Bmal1 expression in the brains of Bmal1-knockout mice did not rescue Bmal1-dependent sleep phenotypes. Surprisingly, most sleep-amount, but not sleep-timing, phenotypes could be reproduced or rescued by knocking out or restoring BMAL1 exclusively in skeletal muscle, respectively. We also found that overexpression of skeletal-muscle Bmal1 reduced the recovery response to sleep loss. Together, these findings demonstrate that Bmal1 expression in skeletal muscle is both necessary and sufficient to regulate total sleep amount and reveal that critical components of normal sleep regulation occur in muscle.
Assuntos
Fatores de Transcrição ARNTL/genética , Encéfalo/metabolismo , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Sono/genética , Fatores de Transcrição ARNTL/deficiência , Animais , Relógios Circadianos/genética , Eletrodos Implantados , Eletroencefalografia , Eletromiografia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Secretogranina II/genética , Secretogranina II/metabolismo , Vigília/genéticaRESUMO
Nitrergic neurons of the dorsal raphe nucleus (DRN) may play a role in physiological stress responses. The caudal lateral wings (CLW) are unique compared to other rostral-caudal DRN sub-regions because they contain distinct nitric oxide (NO) synthase (NOS) populations that are independent of tryptophan hydroxylase (TPH). NOS neurons in the CLW are also highly activated during acute restraint stress. However, the effects of acute stress duration on NOS activation in the CLW are unclear. Here NADPH-d, an index of NOS activity, is used to show that sub-regions of the DRN have differential NOS activation in response to 6 hours of restraint stress in rats. We report increased NOS activity through 6 hours of restraint in the caudal lateral wings and ventromedial sub-regions. These data suggest that, NOS neurons may play a dynamic role in the response to stress duration.
Assuntos
Núcleo Dorsal da Rafe/metabolismo , Neurônios Nitrérgicos/metabolismo , Estresse Fisiológico , Animais , Ativação Enzimática , Imobilização , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Long-EvansRESUMO
Brain and muscle-ARNT-like factor (Bmal1/BMAL1) is an essential transcriptional/translational factor of circadian clocks. Loss of function of Bmal1/BMAL1 is highly disruptive to physiological and behavioral processes. In light of these previous findings, we examined if transgenic overexpression of Bmal1/BMAL1 in skeletal muscle could alter metabolic processes. First, we characterized in vivo and ex vivo metabolic phenotypes of muscle overexpressed mice (male and female) compared to wild-type littermates (WT). Second, we examined in vivo and ex vivo metabolic processes in the presence of positive and negative homeostatic challenges: high-intensity treadmill running (positive) and acute sleep deprivation (negative). In vivo measures of metabolic processes included body composition, respiratory exchange ratio (RER; VCO2/VO2), energy expenditure, total activity counts, and food intake collected from small animal indirect calorimetry. Ex vivo measure of insulin sensitivity in skeletal muscle was determined from radioassays. RER was lower for muscle overexpressed females compared to female WTs. There were no genotype-dependent differences in metabolic phenotypes for males. With homeostatic challenges, muscle overexpressed mice had lower energy expenditure after high-intensity treadmill running. Acute sleep deprivation reduced insulin sensitivity in skeletal muscle in overexpressed male mice, but not male WTs. The present study contributes to a body of evidence showing pleiotropic, non-circadian, and homeostatic effects of altered Bmal1/BMAL1 expression on metabolic processes, demonstrating a critical need to further investigate the broad and complex actions of Bmal1/BMAL1 on physiology and behavior.
Assuntos
Relógios Circadianos/fisiologia , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Sono/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Relógios Circadianos/genética , Feminino , Homeostase/fisiologia , Masculino , Camundongos Transgênicos , Músculo Esquelético/metabolismoRESUMO
STUDY OBJECTIVES: Episodes of brief limb ischemia (remote preconditioning) in mice induce tolerance to modeled ischemic stroke (focal brain ischemia). Since stroke outcomes are in part dependent on sleep-wake history, we sought to determine if sleep is critical for the neuroprotective effect of limb ischemia. METHODS: EEG/EMG recording electrodes were implanted in mice. After a 24 h baseline recording, limb ischemia was induced by tightening an elastic band around the left quadriceps for 10 minutes followed by 10 minutes of release for two cycles. Two days following remote preconditioning, a second 24 h EEG/EMG recording was completed and was immediately followed by a 60-minute suture occlusion of the middle cerebral artery (modeled ischemic stroke). This experiment was then repeated in a model of circadian and sleep abnormalities (Bmal1 knockout [KO] mice sleep 2 h more than wild-type littermates). Brain infarction was determined by vital dye staining, and sleep was assessed by trained identification of EEG/EMG recordings. RESULTS: Two days after limb ischemia, wild-type mice slept an additional 2.4 h. This additional sleep was primarily comprised of non-rapid eye movement (NREM) sleep during the middle of the light-phase (i.e., naps). Repeating the experiment but preventing increases in sleep after limb ischemia abolished tolerance to ischemic stroke. In Bmal1 knockout mice, remote preconditioning did not increase daily sleep nor provide tolerance to subsequent focal ischemia. CONCLUSIONS: These results suggest that sleep induced by remote preconditioning is both sufficient and necessary for its neuroprotective effects on stroke outcome.
Assuntos
Isquemia Encefálica/terapia , Precondicionamento Isquêmico/métodos , Neuroproteção/fisiologia , Sono/fisiologia , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/fisiopatologia , Eletroencefalografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos Sprague-Dawley , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Micellar electrokinetic chromatography allows the efficient separation of biogenic amines and amino acids in biological samples. Analytes of interest, sample composition, and sample matrix may vary between studies, which necessitates optimization of separations to meet the requirements and conditions of an experiment. Factorial analysis is an efficient tool to accomplish this type of optimization involving multiple interacting factors. The present study describes an optimization procedure for separation of the inhibitory neurotransmitter GABA utilizing capillary electrophoresis with laser induced fluorescence detection. Standards labeled with the flourogenic reagent 3-(2-furoyl)quinoline-2 carboxaldehyde were separated with varying concentrations of borate buffer, beta-cyclodextrin, sodium dodecyl sulfate and pH. The optimized separation method had a correlation coefficient between concentration of GABA and fluorescent signal of 0.98, and was linear in the desired concentration range of 25 nM-10 microM. Glutamic acid, aspartic acid and taurine were also quantified using this separation. When applied to microdialysate collected from the region of the suprachiasmatic nucleus, this separation was able to measure daily variations in GABA levels. The factorial design experiment has proven to be a useful tool, allowing adjustments in the separation of neurotransmitters based on individual requirements.
Assuntos
Cromatografia Capilar Eletrocinética Micelar/métodos , Análise Fatorial , Lasers , Espectrometria de Fluorescência/métodos , Ácido gama-Aminobutírico/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Circadian rhythms are reset by light during the night or by nonphotic stimuli during the day. Neuropeptide Y (NPY), which appears to mediate at least some nonphotic phase shifts by its actions in the suprachiasmatic nucleus (SCN), induces phase advances during the day and inhibits light-induced phase advances during the night. In this study, we used a highly selective Y5-like agonist to test whether activation of NPY Y5 receptors is sufficient to mimic NPY during the day and late night in Syrian hamsters. We also tested whether NPY in the early night reduces light-induced phase delays in a dose-dependent manner. Microinjection of a selective Y5 receptor agonist, (Ala(31), Aib(32))-NPY, into the SCN significantly inhibited light-induced phase advances during the late night, but did not induce phase advances during the day. In addition, concentrations of NPY ranging from 0.23 to 23 mM did not attenuate light-induced phase delays in the early night. These results suggest that activation of Y5-like receptors is sufficient to inhibit light-induced phase advances during the late night but is not sufficient to induce phase advances during the day. Furthermore, this study provided no evidence that NPY can inhibit light-induced phase shifts early in the night.
Assuntos
Ritmo Circadiano/fisiologia , Escuridão , Luz , Receptores de Neuropeptídeo Y/fisiologia , Núcleo Supraquiasmático/fisiologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Atividade Motora/efeitos da radiação , Neuropeptídeo Y/farmacologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos da radiaçãoRESUMO
Here, we provide a detailed account of how to denervate white and brown adipose tissue (WAT and BAT) and how to measure sympathetic nervous system (SNS) activity to these and other tissues neurochemically. The brain controls many of the functions of WAT and BAT via the SNS innervation of the tissues, especially lipolysis and thermogenesis, respectively. There is no clearly demonstrated parasympathetic innervation of WAT or the major interscapular BAT (IBAT) depot. WAT and BAT communicate with the brain neurally via sensory nerves. We detail the surgical denervation (eliminating both innervations) of several WAT pads and IBAT. We also detail more selective chemical denervation of the SNS innervation via intra-WAT/IBAT 6-hydroxy-dopamine (a catecholaminergic neurotoxin) injections and selective chemical sensory denervation via intra-WAT/IBAT capsaicin (a sensory nerve neurotoxin) injections. Verifications of the denervations are provided (HPLC-EC detection for SNS, ELIA for calcitonin gene-related peptide (proven sensory nerve marker)). Finally, assessment of the SNS drive to WAT/BAT or other tissues is described using the alpha-methyl-para-tyrosine method combined with HPLC-EC, a direct neurochemical measure of SNS activity. These methods have proven useful for us and for other investigators interested in innervation of adipose tissues. The chemical denervation approach has been extended to nonadipose tissues as well.
Assuntos
Tecido Adiposo Marrom/inervação , Tecido Adiposo Branco/inervação , Sistema Nervoso Simpático , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/cirurgia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/cirurgia , Encéfalo/efeitos dos fármacos , Encéfalo/cirurgia , Capsaicina/administração & dosagem , Humanos , Norepinefrina/administração & dosagem , Oxidopamina/administração & dosagem , Termogênese/efeitos dos fármacosRESUMO
Sex differences in spontaneous sleep amount are largely dependent on reproductive hormones; however, in mice some sex differences in sleep amount during the active phase are preserved after gonadectomy and may be driven by non-hormonal factors. In this study, we sought to determine whether or not these sex differences are driven by sex chromosome complement. Mice from the four core genotype (FCG) mouse model, whose sex chromosome complement (XY, XX) is independent of phenotype (male or female), were implanted with electroencephalographic (EEG) and electromyographic (EMG) electrodes for the recording of sleep-wake states and underwent a 24-hr baseline recording followed by six hours of forced wakefulness. During baseline conditions in mice whose gonads remained intact, males had more total sleep and non-rapid eye movement sleep than females during the active phase. Gonadectomized FCG mice exhibited no sex differences in rest-phase sleep amount; however, during the mid-active-phase (nighttime), XX males had more spontaneous non-rapid eye movement (NREM) sleep than XX females. The XY mice did not exhibit sex differences in sleep amount. Following forced wakefulness there was a change in the factors regulating sleep. XY females slept more during their mid-active phase siestas than XX females and had higher NREM slow wave activity, a measure of sleep propensity. These findings suggest that the process that regulates sleep propensity is sex-linked, and that sleep amount and sleep propensity are regulated differently in males and females following sleep loss.
Assuntos
Sono REM/genética , Cromossomo X/fisiologia , Cromossomo Y/fisiologia , Animais , Ritmo Delta , Feminino , Genótipo , Masculino , Camundongos , Camundongos Transgênicos , Caracteres Sexuais , Privação do Sono , VigíliaRESUMO
STUDY OBJECTIVES: Electroencephalographic slow wave activity (SWA) during non-rapid eye movement (NREM) sleep results from the synchronous oscillation of cortical neurons and is the standard measurement of sleep homeostasis. SWA is not a direct measure of sleep pressure accumulation, but rather a measure of the NREM-sleep response to accumulated sleep pressure. Currently, no practical standard for the direct measurement of sleep pressure accumulation exists. Recently, it was demonstrated that rat cortical neurons undergo oscillations during wake that are similar to the cortical oscillations responsible for SWA. Furthermore, these oscillations increase in number as time awake increases. Here we hypothesize that period-amplitude analysis of the electroencephalogram (EEG), which treats the EEG as a series of discrete waves, can measure these cortical oscillations, and thus, is a measure of sleep-pressure accumulation during extended wake. DESIGN: Mice were sleep deprived for 24 h by confinement to a slowly rotating wheel in order to assess wake-dependent changes in EEG wave incidence. MEASUREMENTS AND RESULTS: Continuous period-amplitude analysis of the waking EEG across 24 h of sleep deprivation revealed that the incidence of 2 to 6 Hz waves increased exponentially over the deprivation period. This increase in wave incidence appeared to occur in two phases with exponential time constants of approximately 0.12 h and 3 h. Further analysis revealed that the changes in wave incidence were significantly correlated with two established markers of sleep pressure, SWA and NREM sleep latency. CONCLUSIONS: The data suggest that wave incidence is an effective method of measuring sleep homeostasis in the waking EEG that provides better temporal resolution than spectral power analysis.
Assuntos
Encéfalo/fisiopatologia , Eletroencefalografia , Privação do Sono/fisiopatologia , Animais , Eletroencefalografia/métodos , Eletromiografia , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vigília/fisiologiaRESUMO
Shift work and trans-time zone travel lead to insufficient sleep and numerous pathologies. Here, we examined sleep/wake dynamics during chronic exposure to environmental circadian disruption (ECD), and if chronic partial sleep loss associated with ECD influences the induction of shift-related inflammatory disorder. Sleep and wakefulness were telemetrically recorded across three months of ECD, in which the dark-phase of a light-dark cycle was advanced weekly by 6 h. A three month regimen of ECD caused a temporary reorganization of sleep (NREM and REM) and wake processes across each week, resulting in an approximately 10% net loss of sleep each week relative to baseline levels. A separate group of mice were subjected to ECD or a regimen of imposed wakefulness (IW) aimed to mimic sleep amounts under ECD for one month. Fos-immunoreactivity (IR) was quantified in sleep-wake regulatory areas: the nucleus accumbens (NAc), basal forebrain (BF), and medial preoptic area (MnPO). To assess the inflammatory response, trunk blood was treated with lipopolysaccharide (LPS) and subsequent release of IL-6 was measured. Fos-IR was greatest in the NAc, BF, and MnPO of mice subjected to IW. The inflammatory response to LPS was elevated in mice subjected to ECD, but not mice subjected to IW. Thus, the net sleep loss that occurs under ECD is not associated with a pathological immune response.