RESUMO
The mammalian circadian clock in the suprachiasmatic nucleus (SCN) is very robust, able to coordinate our daily physiological and behavioral rhythms with exquisite accuracy. Simultaneously, the SCN clock is highly sensitive to environmental timing cues such as the solar cycle. This duality of resiliency and sensitivity may be sustained in part by a complex intertwining of three cellular oscillators: transcription/translation, metabolic/redox, and membrane excitability. We suggest here that one of the links connecting these oscillators may be forged from copper (Cu). Cellular Cu levels are highly regulated in the brain and peripherally, and Cu affects cellular metabolism, redox state, cell signaling, and transcription. We have shown that both Cu chelation and application induce nighttime phase shifts of the SCN clock in vitro and that these treatments affect glutamate, N-methyl-D-aspartate receptor, and associated signaling processes differently. More recently we found that Cu induces mitogen-activated protein kinase-dependent phase shifts, while the mechanisms by which Cu removal induces phase shifts remain unclear. Lastly, we have found that two Cu transporters are expressed in the SCN, and that one of these transporters (ATP7A) exhibits a day/night rhythm. Our results suggest that Cu homeostasis is tightly regulated in the SCN, and that changes in Cu levels may serve as a time cue for the circadian clock. We discuss these findings in light of the existing literature and current models of multiple coupled circadian oscillators in the SCN.
Assuntos
Relógios Circadianos , Animais , Ritmo Circadiano , Cobre , Ácido Glutâmico , Receptores de N-Metil-D-Aspartato , Núcleo SupraquiasmáticoRESUMO
Epinephrine autoinjectors (EAIs) are important first aid medications for treating anaphylaxis. A 10-fold price increase over the past 12 years and evidence that expired EAIs may still contain significant doses of available epinephrine have motivated interest in the efficacy of expired EAIs as treatments of last resort. Degradation of expired EAIs, which can be caused by improper storage conditions, results in various degrees of discoloration of the epinephrine solution. Previous studies have determined that significant epinephrine remains available in expired EAIs, but these have only considered EAIs that show no discoloration. Here, we investigate the potential for colorimetric estimation of available epinephrine dose based on the degree of discoloration in expired EAIs. The correlation of available epinephrine dose and time since expiration date was poor (r = - 0.37), as determined by an industry standard UHPLC protocol. Visible absorbance of the samples integrated across the range 430-475 nm correlated well with available epinephrine dose (r = - 0.71). This wavelength corresponds to the blue channel of a typical smartphone camera Bayer filter. Smartphone camera images of the EAI solutions in various illumination conditions were analyzed to assign color indices representing the degree of discoloration. Color index of the samples showed similar correlation (|r| > 0.7) with available epinephrine dose as that of visible spectrophotometry. Smartphone imaging colorimetry is proposed as a potential point-of-use epinephrine dose estimator for expired and degraded EAIs. Graphical abstract.
Assuntos
Anafilaxia/tratamento farmacológico , Diagnóstico por Imagem/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos/normas , Epinefrina/análise , Autoadministração/métodos , Smartphone/instrumentação , Epinefrina/administração & dosagem , Humanos , Injeções Intramusculares , Autoadministração/instrumentaçãoRESUMO
BACKGROUND: Alcohol tolerance and withdrawal-induced effects are criteria for alcohol use disorders listed by the DSM-V. Although tolerance and withdrawal have been studied over many decades, there is still uncertainty regarding mechanistic distinctions that characterize these different forms of ethanol (EtOH)-induced plasticity. Previously, we demonstrated that the suprachiasmatic nucleus (SCN) circadian clock develops both acute and rapid tolerance to EtOH inhibition of glutamate-induced circadian phase shifts. Here, we demonstrate that chronic EtOH tolerance and withdrawal-induced glutamate hypersensitivity occur in vitro and that rapid tolerance, chronic tolerance, and glutamate hypersensitivity have distinct cellular changes. METHODS: We use single-unit extracellular electrophysiological recordings to determine whether chronic tolerance to EtOH inhibition of glutamatergic phase shifts and withdrawal-induced glutamate hypersensitivity develop in the SCN. We use Western blotting to compare phosphorylation state and total expression of N-methyl-D-aspartate (NMDA) receptor subunits and associated proteins in the SCN after mice were exposed to varying EtOH consumption paradigms. RESULTS: Chronic tolerance developed after a minimum of 8 days of 4 h/d EtOH access, as indicated by a decreased sensitivity to EtOH inhibition of glutamate-induced phase shifts. We also observed an increased sensitivity to glutamate-induced phase shifts in SCN tissue following withdrawal. We demonstrated an increase in the ratio of NR2B:NR2A NMDA receptor subunit expression after 21 days, but not after 10 days of EtOH drinking. This increase persisted during EtOH withdrawal, along with an increase in NR2B Y1472 phosphorylation, mature brain-derived neurotrophic factor, and phosphorylated TrkB. CONCLUSIONS: These results demonstrate that multiple tolerance forms and withdrawal-induced glutamate hypersensitivity occur in the SCN and that these different forms of EtOH-induced plasticity are accompanied by distinct changes in cellular physiology. Importantly, this study further demonstrates the power of using the SCN as a model system to investigate EtOH-induced plasticity.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Relógios Circadianos/efeitos dos fármacos , Tolerância a Medicamentos , Etanol/farmacologia , Ácido Glutâmico/metabolismo , Núcleo Supraquiasmático/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Masculino , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMO
BACKGROUND: Circadian rhythms of physiology and behavior are driven by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus. This clock is synchronized to environmental day/night cycles by photic input, which is dependent on the presence of mature brain-derived neurotrophic factor (BDNF) in the SCN. Mature BDNF is produced by the enzyme plasmin, which is converted from plasminogen by the enzyme tissue-type plasminogen activator (tPA). In this study, we evaluate circadian function in mice lacking functional tPA. RESULTS: tPA-/- mice have normal circadian periods, but show decreased nocturnal wheel-running activity. This difference is eliminated or reversed on the second day of a 48-h fast. Similarly, when placed on daily cycles of restricted food availability the genotypic difference in total wheel-running activity disappears, and tPA-/- mice show equivalent amounts of food anticipatory activity to wild type mice. CONCLUSIONS: These data suggest that tPA regulates nocturnal wheel-running activity, and that tPA differentially affects SCN-driven nocturnal activity rhythms and activity driven by fasting or temporal food restriction.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Locomoção , Ativador de Plasminogênio Tecidual/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ingestão de Alimentos , Jejum , Privação de Alimentos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Núcleo Supraquiasmático/metabolismo , Ativador de Plasminogênio Tecidual/genéticaRESUMO
Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.
Assuntos
Relógios Circadianos , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inativadores de Plasminogênio/farmacologia , Proteólise , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologia , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Melatonin supplementation has been used as a therapeutic agent for several diseases, yet little is known about the underlying mechanisms by which melatonin synchronizes circadian rhythms. G-protein signaling plays a large role in melatonin-induced phase shifts of locomotor behavior and melatonin receptors activate G-protein-coupled inwardly rectifying potassium (GIRK) channels in Xenopus oocytes. The present study tested the hypothesis that melatonin influences circadian phase and electrical activity within the central clock in the suprachiasmatic nucleus (SCN) through GIRK channel activation. Unlike wild-type littermates, GIRK2 knock-out (KO) mice failed to phase advance wheel-running behavior in response to 3 d subcutaneous injections of melatonin in the late day. Moreover, in vitro phase resetting of the SCN circadian clock by melatonin was blocked by coadministration of a GIRK channel antagonist tertiapin-q (TPQ). Loose-patch electrophysiological recordings of SCN neurons revealed a significant reduction in the average action potential rate in response to melatonin. This effect was lost in SCN slices treated with TPQ and SCN slices from GIRK2 KO mice. The melatonin-induced suppression of firing rate corresponded with an increased inward current that was blocked by TPQ. Finally, application of ramelteon, a potent melatonin receptor agonist, significantly decreased firing rate and increased inward current within SCN neurons in a GIRK-dependent manner. These results are the first to show that GIRK channels are necessary for the effects of melatonin and ramelteon within the SCN. This study suggests that GIRK channels may be an alternative therapeutic target for diseases with evidence of circadian disruption, including aberrant melatonin signaling. SIGNIFICANCE STATEMENT: Despite the widespread use of melatonin supplementation for the treatment of sleep disruption and other neurological diseases such as epilepsy and depression, no studies have elucidated the molecular mechanisms linking melatonin-induced changes in neuronal activity to its therapeutic effects. Here, we used behavioral and electrophysiological techniques to address this scientific gap. Our results show that melatonin and ramelteon, a potent and clinically relevant melatonin receptor agonist, significantly affect the neurophysiological function of suprachiasmatic nucleus neurons through activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Given the importance of GIRK channels for neuronal excitability (with >600 publications on these channels to date), our study should generate broad interest from neuroscientists in fields such as epilepsy, addiction, and cognition.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Melatonina/farmacologia , Núcleo Supraquiasmático/fisiologia , Animais , Venenos de Abelha/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Indenos/farmacologia , Masculino , Melatonina/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio/farmacologia , Núcleo Supraquiasmático/efeitos dos fármacosRESUMO
BACKGROUND: Ethanol (EtOH) triggers cellular adaptations that induce tolerance in many brain areas, including the suprachiasmatic nucleus (SCN), the site of the master circadian clock. EtOH inhibits light-induced phase shifts in the SCN in vivo and glutamate-induced phase shifts in vitro. The in vitro phase shifts develop acute tolerance to EtOH, occurring within minutes of initial exposure, while the in vivo phase shifts exhibit no evidence of chronic tolerance. An intermediate form, rapid tolerance, is not well studied but may predict subsequent chronic tolerance. Here, we investigated rapid tolerance in the SCN clock. METHODS: Adult C57BL/6 mice were provided 15% EtOH or water for one 12-hour lights-off period. For in vitro experiments, SCN-containing brain slices were prepared in the morning and treated for 10 minutes with glutamate +/- EtOH the following night. Single-cell neuronal firing rates were recorded extracellularly during the subsequent day to determine SCN clock phase. For in vivo experiments, mice receiving EtOH 24 hours previously were exposed to a 30-minute light pulse immediately preceded by intraperitoneal saline or 2 g/kg EtOH injection. Mice were then placed in constant darkness and their phase-shifting responses measured. RESULTS: In vitro, the SCN clock from EtOH-exposed mice exhibited rapid tolerance, with a 10-fold increase in EtOH needed to inhibit glutamate-induced phase shifts. Co-application of brain-derived neurotrophic factor prevented EtOH inhibition, consistent with experiments using EtOH-naïve mice. Rapid tolerance lasts 48 to 96 hours, depending on whether assessing in vitro phase advances or phase delays. Similarly, in vivo, prior EtOH consumption prevented EtOH's acute blockade of photic phase delays. Finally, immunoblot experiments showed no changes in SCN glutamate receptor subunit (NR2B) expression or phosphorylation in response to rapid tolerance induction. CONCLUSIONS: The SCN circadian clock develops rapid tolerance to EtOH as assessed both in vivo and in vitro, and the tolerance lasts for several days. These data demonstrate the utility of the circadian system as a model for investigating cellular mechanisms through which EtOH acts in the brain.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Relógios Circadianos/efeitos dos fármacos , Tolerância a Medicamentos , Etanol/farmacologia , Núcleo Supraquiasmático/efeitos dos fármacos , Ciclos de Atividade/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
While soluble forms of amyloid-ß (Aß) and Tau work together to drive healthy neurons into a disease state, how their interaction may control the prion-like propagation and neurotoxicity of Tau is not fully understood. The cross-linking via disulfide bond formation is crucial for Tau oligomers to obtain stable conformers and spread between cells. This work thus focuses on how Aß42 regulates this critical process. By studying the interactions between Aß42 and TauPHF43, a construct that mimics the Tau R3 isoform, has a similar length to Aß42, and contains one cysteine (Cys-322), we discovered that fresh Aß42 could protect Tau against the formation of disulfide cross-linked dimers. We showed that the monomeric and small Aß oligomers (the "nonamyloidogenic Aß") efficiently disassembled tau dimers and heparin-induced Tau oligomers to recover Tau monomers. Interestingly, Aß serves the role of an antioxidant to prevent disulfide bond formation, as supported by the experiments of Aß with cystine. Furthermore, using cyclosporine A (CycA), a macrocyclic ß-sheet disruptor, we demonstrated that targeting amyloidogenic Aß with CycA does not affect the TauPHF43 disassembly driven by Aß42. Separately, we assessed the initial toxicity of Aß42 and TauPHF43 in acute brain slices and found that Aß42 is more toxic than TauPHF43 or the two peptides combined. Our work highlights a potential protective role of Aß42 monomers in AD that was previously overlooked while focusing on the mechanism behind Aß42 aggregation leading to tau dysfunction.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Neurônios , Fragmentos de Peptídeos/químicaRESUMO
Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.
Assuntos
Relógios Circadianos/efeitos dos fármacos , Cocaína/farmacologia , Estimulação Luminosa , Transdução de Sinais/efeitos dos fármacos , Núcleo Supraquiasmático/efeitos dos fármacos , Animais , Relógios Circadianos/fisiologia , Inibidores da Captação de Dopamina/farmacologia , Flufenazina/farmacologia , Lidocaína/farmacologia , Masculino , Metergolina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Atividade Motora/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/fisiologiaRESUMO
It is well established that the damaging effects of drugs of abuse, such as cocaine, can extend beyond the user to their offspring. While most preclinical models of the generational effects of cocaine abuse have focused on maternal effects, we, and others, report distinct effects on offspring sired by fathers treated with cocaine prior to breeding. However, little is known about the effects of paternal cocaine use on first generation (F1) offspring's social behaviors. Here, we expand upon our model of oral self-administered paternal cocaine use to address the idea that paternal cocaine alters first generation offspring social behaviors through modulation of the oxytocin system. F1 cocaine-sired males displayed unaltered social recognition vs. non-cocaine sired controls but showed increased investigation times that were not related to altered olfaction. Paternal cocaine did not alter F1 male-aggression behavior or depression-like behaviors, but cocaine-sired males did display decreased anxiety-like behaviors. Female F1 behavior was similarly examined, but there were no effects of paternal cocaine. Cocaine-sired male mice also exhibited localized oxytocin receptor expression differences vs. controls in several brain regions regulating social behavior. These results provide evidence for effects of paternal cocaine exposure on social behaviors in male offspring with associated alterations in central oxytocin transmission.
Assuntos
Cocaína , Animais , Encéfalo/metabolismo , Cocaína/farmacologia , Pai , Feminino , Humanos , Masculino , Camundongos , Ocitocina/metabolismo , Comportamento Paterno/fisiologia , Receptores de Ocitocina/metabolismo , Comportamento SocialRESUMO
Atomic structures of amyloid oligomers that capture the neurodegenerative disease pathology are essential to understand disease-state causes and finding cures. Here we investigate the G6W mutation of the cytotoxic, hexameric amyloid model KV11. The mutation results into an asymmetric dodecamer composed of a pair of 30° twisted antiparallel ß-sheets. The complete break between adjacent ß-strands is unprecedented among amyloid fibril crystal structures and supports that our structure is an oligomer. The poor shape complementarity between mated sheets reveals an interior channel for binding lipids, suggesting that the toxicity may be due to a perturbation of lipid transport rather than a direct disruption of membrane integrity. Viability assays on mouse suprachiasmatic nucleus, anterior hypothalamus, and cerebral cortex demonstrated selective regional vulnerability consistent with Alzheimer's disease. Neuropeptides released from the brain slices may provide clues to how G6W initiates cellular injury.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Doença de Alzheimer/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Animais , Encéfalo/metabolismo , Camundongos , Modelos Moleculares , Doenças Neurodegenerativas/metabolismo , Fragmentos de Peptídeos/químicaRESUMO
Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing â¼50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg(-1)·day(-1) vs. 13 g·kg(-1)·day(-1) and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40-60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20-30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites.
Assuntos
Dissuasores de Álcool/farmacologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Preferências Alimentares/efeitos dos fármacos , Preferências Alimentares/fisiologia , Taurina/análogos & derivados , Acamprosato , Animais , Ritmo Circadiano/fisiologia , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Líquidos/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Animais , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiologia , Núcleo Tegmental Pedunculopontino/efeitos dos fármacos , Núcleo Tegmental Pedunculopontino/fisiologia , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/fisiologia , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia , Taurina/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologiaRESUMO
BACKGROUND: Alcohol dependence is associated with impaired circadian rhythms and sleep. Ethanol administration disrupts circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol dependence on the circadian timing system. In this study, we extend previous work in C57BL/6J mice to: (i) characterize the suprachiasmatic nucleus (SCN) pharmacokinetics of acute systemic ethanol administration, (ii) explore the effects of acute ethanol on photic and nonphotic phase-resetting, and (iii) determine if the SCN is a direct target for photic effects. METHODS: First, microdialysis was used to characterize the pharmacokinetics of acute intraperitoneal (i.p.) injections of 3 doses of ethanol (0.5, 1.0, and 2.0 g/kg) in the mouse SCN circadian clock. Second, the effects of acute i.p. ethanol administration on photic phase delays and serotonergic ([+]8-OH-DPAT-induced) phase advances of the circadian activity rhythm were assessed. Third, the effects of reverse-microdialysis ethanol perfusion of the SCN on photic phase-resetting were characterized. RESULTS: Peak ethanol levels from the 3 doses of ethanol in the SCN occurred within 20 to 40 minutes postinjection with half-lives for clearance ranging from 0.6 to 1.8 hours. Systemic ethanol treatment dose-dependently attenuated photic and serotonergic phase-resetting. This treatment also did not affect basal SCN neuronal activity as assessed by Fos expression. Intra-SCN perfusion with ethanol markedly reduced photic phase delays. CONCLUSIONS: These results confirm that acute ethanol attenuates photic phase-delay shifts and serotonergic phase-advance shifts in the mouse. This dual effect could disrupt photic and nonphotic entrainment mechanisms governing circadian clock timing. It is also significant that the SCN clock is a direct target for disruptive effects of ethanol on photic shifting. Such actions by ethanol could underlie the disruptive effects of alcohol abuse on behavioral, physiological, and endocrine rhythms associated with alcoholism.
Assuntos
Depressores do Sistema Nervoso Central/toxicidade , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Etanol/toxicidade , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Depressores do Sistema Nervoso Central/farmacocinética , Depressores do Sistema Nervoso Central/farmacologia , Grupos Controle , Relação Dose-Resposta a Droga , Etanol/farmacocinética , Etanol/farmacologia , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise , Atividade Motora/efeitos dos fármacos , Estimulação Luminosa , Fotoperíodo , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Núcleo Supraquiasmático/efeitos dos fármacos , Fatores de TempoRESUMO
Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and membrane interface techniques. The rise of organ-on-chip technologies has demonstrated the value of microfluidic perfusion culture systems for sampling and analysis of complex biology under well-controlled in vitro or ex vivo conditions. A number of approaches to microfluidic brain slice culture have been developed, however these typically involve complex design, fabrication, or operational parameters in order to meet the high oxygen demands of brain slices. Here, we present proof-of-principle for a novel approach to microfluidic brain slice culture. In this system, which we term a microfluidic bubble perfusion device, principles of droplet microfluidics were employed to generate droplets of perfusion media dispersed between bubbles of carbogen gas, and brain tissue slices were perfused with the resulting monodispersed droplets and bubbles. The challenge of tissue immobilization in the flow system was addressed using a two-part cytocompatible carbohydrate-based tissue adhesive. Best practices are discussed for perfusion chamber designs that maintain segmented flow throughout the course of perfusion. Control of droplet and bubble volumes was possible across the range of ca. 4-15 µL, bubble generation frequency was well controlled in the range ca. 1-7 bubbles per min, and bubble duty cycle was well controlled across the range ca. 20-80%. Murine hypothalamic tissue slices containing the suprachiasmatic nuclei were successfully maintained for durations of 8-10 hours, with tissue remaining viable for the duration of perfusion as assessed by Ca2+ imaging and propidium iodide (PI) staining.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Animais , Encéfalo , Dispositivos Lab-On-A-Chip , Camundongos , PerfusãoRESUMO
BACKGROUND: Chronic ethanol abuse is associated with disrupted circadian rhythms and sleep. Ethanol administration impairs circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol abuse on circadian timing. Here, we extend previous studies to explore the effects of chronic forced ethanol on photic phase-resetting, photic entrainment, and daily locomotor activity patterns in C57BL/6J mice. METHODS: First, microdialysis was used to characterize the circadian patterns of ethanol uptake in the suprachiasmatic (SCN) circadian clock and correlate this with systemic ethanol levels and episodic drinking of 10 or 15% ethanol. Second, the effects of chronic forced ethanol drinking and withdrawal on photic phase-delays of the circadian activity rhythm were assessed. Third, the effects of chronic ethanol drinking on entrainment to a weak photic zeitgeber (1 minute of 25 lux intensity light per day) were assessed. This method was used to minimize any masking actions of light that could mask ethanol effects on clock entrainment. RESULTS: Peak ethanol levels in the SCN and periphery occurred during the dark phase and coincided with the time when light normally induces phase-delays in mice. These delays were dose-dependently inhibited by chronic ethanol and its withdrawal. Chronic ethanol did not impede re-entrainment to a shifted light cycle but affected entrainment under the weak photic zeitgeber and disrupted the daily pattern of locomotor activity. CONCLUSIONS: These results confirm that chronic ethanol consumption and withdrawal markedly impair circadian clock photic phase-resetting. Ethanol also disturbs the temporal structure of nighttime locomotor activity and photic entrainment. Collectively, these results suggest a direct action of ethanol on the SCN clock.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Ritmo Circadiano/fisiologia , Etanol/toxicidade , Atividade Motora/fisiologia , Fotoperíodo , Animais , Ritmo Circadiano/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Estimulação Luminosa/métodos , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologiaRESUMO
BACKGROUND: Alcohol abuse leads to marked disruptions of circadian rhythms, and these disturbances in themselves can increase the drive to drink. Circadian clock timing is regulated by light, as well as by nonphotic influences such as food, social interactions, and wheel running. We previously reported that alcohol markedly disrupts photic and nonphotic modes of circadian rhythm regulation in Syrian hamsters. As an extension of this work, we characterize the hedonic interrelationship between wheel running and ethanol (EtOH) intake and the effects of environmental circadian disruption (long-term exposure to constant light [LL]) on the drive to drink. METHODS: First, we tested the effect of wheel running on chronic free-choice consumption of a 20% (v/v) EtOH solution and water. Second, the effect of this alcohol drinking on wheel running in alcohol-naive animals was investigated. Third, we assessed the influence of LL, known to suppress locomotor activity and cause circadian rhythm disruption, on EtOH consumption and wheel-running behavior. RESULTS: Inhibitory effects of wheel running on EtOH intake and vice versa were observed. Exposure to LL, while not affecting EtOH intake, induced rhythm splitting in 75% of the animals. Notably, the splitting phenotype was associated with lower levels of EtOH consumption and preference prior to, and throughout, the period of LL exposure. CONCLUSIONS: These results are evidence that exercise may offer an efficacious clinical approach to reducing EtOH intake. Also, predisposition for light-induced (or other) forms of circadian disruption may modulate the drive to drink.
Assuntos
Consumo de Bebidas Alcoólicas/psicologia , Transtornos Cronobiológicos/psicologia , Atividade Motora/efeitos dos fármacos , Fotoperíodo , Animais , Comportamento de Escolha , Cricetinae , Luz , MesocricetusRESUMO
The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha(2)-antiplasmin inhibition of glutamate-induced shifts. alpha(2)-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential 'gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Fibrinolisina/metabolismo , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Ritmo Circadiano/fisiologia , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Núcleo Supraquiasmático/citologia , Ativador de Plasminogênio Tecidual/farmacologia , Vitronectina/deficiênciaRESUMO
Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by approximately 20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 +/- 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 +/- 0.1 and 0.3 +/- 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 +/- 0.3 h, while hamsters deprived of EtOH for 2-3 days showed enhanced shifts (2.1 +/- 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting.
Assuntos
Consumo de Bebidas Alcoólicas , Comportamento Animal/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Etanol/toxicidade , Fotoperíodo , Núcleo Supraquiasmático/efeitos dos fármacos , Animais , Cricetinae , Etanol/farmacocinética , Masculino , Mesocricetus , Microdiálise , Estimulação Luminosa , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia , Núcleo Supraquiasmático/metabolismoRESUMO
BACKGROUND: Tolerance to ethanol is observed over a variety of time courses, from minutes to days. Acute tolerance, which develops over 5 to 60 minutes, has been observed for both behavioral and neurophysiological variables and may involve changes in signaling through NMDA, GABA, or other receptors. Previous work has shown that both acute and chronic ethanol treatments modulate photic and nonphotic phase resetting of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Although not specifically tested, the data thus far do not point to the development of chronic tolerance to the modulatory effects of ethanol. Here we investigated whether acute tolerance the ethanol occurs with respect to in vitro phase modulation of the SCN clock. METHODS: Mouse brain slices containing the SCN were pretreated with ethanol for varying lengths of time, followed by treatment concurrent with either glutamate or the serotonin agonist, 8-hydroxy-DPAT (DPAT). The phase of the SCN circadian clock was assessed the following day through extracellular recordings of SCN neuronal activity. SCN neuronal activity normally peaks during mid-day, and this rhythm can be shifted by treatment with either glutamate or DPAT. RESULTS: While concurrent treatment of SCN-containing brain slices with ethanol and glutamate blocks glutamate-induced phase delays of the SCN clock, pretreating the slices with ethanol for > or =15 minutes prevents this inhibition. Likewise, while concurrent treatment with ethanol and DPAT enhances DPAT-induced phase advances of the SCN clock, pretreating the slices with ethanol for > or =30 minutes prevents this enhancement. CONCLUSIONS: Both the inhibiting and enhancing effects of ethanol on in vitro SCN clock phase resetting show acute tolerance. Additional experiments are needed to determine whether more slowly developing forms of tolerance also occur with respect to the SCN circadian clock.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Etanol/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Tolerância a Medicamentos , Eletrofisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
Paternal cocaine use causes phenotypic alterations in offspring behavior and associated neural processing. In rodents, changes in first generation (F1) offspring include drug reward behavior, circadian timing, and anxiety responses. This study, utilizing a murine (C57BL/6J) oral cocaine model, examines the effects of paternal cocaine exposure on fundamental characteristics of offspring reward responses, including: 1) the extent of cocaine-induced effects after different durations of sire drug withdrawal; 2) sex- and drug-dependent differences in F1 reward preference; 3) effects on second generation (F2) cocaine preference; and 4) corresponding changes in reward area (nucleus accumbens) mRNA expression. We demonstrate that paternal cocaine intake over a single Ë40-day spermatogenic cycle significantly decreased cocaine (but not ethanol or sucrose) preference in a sex-specific manner in F1 mice from sires mated 24 h after drug withdrawal. However, F1 offspring of sires bred 4 months after withdrawal did not exhibit altered cocaine preference. Altered cocaine preference also was not observed in F2's. RNASeq analyses of F1 accumbens tissue revealed changes in gene expression in male offspring of cocaine-exposed sires, including many genes not previously linked to cocaine addiction. Enrichment analyses highlight genes linked to CNS development, synaptic signaling, extracellular matrix, and immune function. Expression correlation analyses identified a novel target, Fam19a4, that may negatively regulate many genes in the accumbens, including genes already identified in addiction. Collectively, these results reveal that paternal cocaine effects in F1 offspring may involve temporally limited epigenetic germline effects and identify new genetic targets for addiction research.