RESUMO
Introducing students to the challenges and rewards of legitimate experimentation has become an essential part of many undergraduate lab courses. However, this objective can be difficult to achieve if the students find the topic uninteresting and therefore do not take ownership of the project. Additionally, the budgets of most undergraduate courses do not allow for the purchase of new equipment for student-generated projects. Here we describe a lab project where students engaged in the process of designing and building their own inexpensive apparatus. Driven by their interest in anxiety research, students in a Neuroscience Methods course developed the following protocol to build an elevated plus maze (EPM) and optional data acquisition module, for less than $100 each. The project engaged students in work that required applied critical thinking and real-world problem solving, and produced a functional EPM that was used in multiple projects beyond this course.
RESUMO
The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen-activated protein kinase pathway appears to play a central role in light-evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the mitogen-activated protein kinase effector mitogen-stress-activated kinase 1 (MSK1) in the SCN. In this study, we utilised MSK1(-/-) mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12 h light/dark cycle and exhibited circadian free-running rhythms in constant darkness. Interestingly, the free-running period in MSK1 null mice was significantly longer than in wild-type control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase-delaying response to an early night light pulse (100 lux, 15 min), and, using an 8 h phase-advancing 'jet-lag' experimental paradigm, MSK1 knockout animals exhibited a significantly delayed rate of re-entrainment. At the molecular level, early night light-evoked cAMP response element-binding protein (CREB) phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1(-/-) animals relative to wild-type mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Núcleo Supraquiasmático/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Histonas/metabolismo , Síndrome do Jet Lag , Camundongos , Camundongos Knockout , Atividade Motora , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fosforilação , Estimulação Luminosa , Fotoperíodo , Núcleo Supraquiasmático/metabolismo , Transcrição GênicaRESUMO
While the pedagogical benefits of incorporating inquiry driven labs into an undergraduate curriculum are well established, often the prohibitive costs of providing equipment for such labs limits the types of experiences that can be offered. For example, the lab portion of Advanced Neuroscience at Centenary College of Louisiana consists of a semester-long research project developed by the students. Frequently, these junior- and senior-level students generate interesting research questions that must be culled or scaled back simply due to a lack of appropriate equipment. In the most recent iteration of the class, the students wanted to examine analgesia using the tail flick test, a measure of spinal nociception. In this test a rodent subject is restrained; its tail is exposed to a heat source; and the latency to flick its tail away from the noxious stimuli is recorded. As commercial devices were far beyond the lab budget, we sought to develop an inexpensive tail flick analgesia meter that was easy to use and generated reliable data. The prototype device was tested by students in the above-mentioned class and was found to consistently produce reliable data in agreement with the literature. Here we present plans for a tail flick analgesia meter that can be constructed for $50-75, roughly 100 times cheaper than commercial devices.
RESUMO
Signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been shown to be a key intracellular signaling event that couples light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Because many of the physiological effects of the MAPK pathway are mediated by extracellular signal-regulated kinase (ERK)-regulated kinases, it was of interest to identify kinase targets of ERK in the SCN. In this study, we examined whether mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of ERK in the SCN and whether it couples to clock gene expression. Here we show that photic stimulation during the subjective night stimulates MSK1 phosphorylation at serine 360, an event required for robust kinase activation. Activated ERK and MSK1 were colocalized in SCN cell nuclei after photic stimulation. The in vivo administration of the MAP kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene] attenuated MSK1 phosphorylation. MSK1 phosphorylation was more responsive to late-night than early-night photic stimulation, indicating that MSK1 may differentially contribute to light-induced phase advancing and phase delaying of the clock. The potential connection between pituitary adenylate cyclase-activating polypeptide (PACAP) (a regulator of clock entrainment) and MSK1 phosphorylation was examined. PACAP infusion stimulated MSK1 phosphorylation, whereas PACAP receptor antagonist infusion attenuated light-induced MSK1 phosphorylation in the SCN. In reporter gene assays, MSK1 was shown to couple to mPeriod1 via a cAMP response element-binding protein-dependent mechanism. Together, these data identify MSK1 as both a downstream target of the MAPK cascade within the SCN and a regulator of clock gene expression.
Assuntos
Ritmo Circadiano , Luz , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Proteínas Quinases S6 Ribossômicas/fisiologia , Núcleo Supraquiasmático/efeitos da radiação , Animais , Butadienos/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Escuridão , Ativação Enzimática , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Proteínas Nucleares/biossíntese , Proteínas Circadianas Period , Fosforilação , Estimulação Luminosa , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa , Serina/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMO
The dual nature of the NMDA receptor as a mediator of excitotoxic cell death and activity-dependent cell survival likely results from divergent patterns of kinase activation, transcription factor activation, and gene expression. To begin to address this divergence, we examined cellular and molecular signaling events that couple excitotoxic and nontoxic levels of NMDA receptor stimulation to activation of the cAMP response element-binding protein (CREB)/cAMP response element (CRE) pathway in cultured cortical neurons. Pulses (10 min) of NMDA receptor-mediated synaptic activity (nontoxic) triggered sustained (up to 3 h) CREB phosphorylation (pCREB) at serine 133. In contrast, brief stimulation with an excitotoxic concentration of NMDA (50 microm) triggered transient pCREB. The duration of pCREB was dependent on calcineurin activity. Excitotoxic levels of NMDA stimulated calcineurin activity, whereas synaptic activity did not. Calcineurin inhibition reduced NMDA toxicity and converted the transient increase in pCREB into a sustained increase. In accordance with these observations, sustained pCREB (up to 3 h) did not require persistent kinase pathway activity. The sequence of stimulation with excitotoxic levels of NMDA and neuroprotective synaptic activity determined which stimulus exerted control over pCREB duration. Constitutively active and dominant-negative CREB constructs were used to implicate CREB in synaptic activity-dependent neuroprotection against NMDA-induced excitotoxicity. Together these data provide a framework to begin to understand how the neuroprotective and excitotoxic effects of NMDA receptor activity function in an antagonistic manner at the level of the CREB/CRE transcriptional pathway.
Assuntos
Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Neurônios/metabolismo , Proteínas Quinases/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Potenciais de Ação , Animais , Apoptose/efeitos dos fármacos , Bicuculina/farmacologia , Encéfalo/citologia , Encéfalo/embriologia , Sinalização do Cálcio , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Tacrolimo/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The mammalian circadian pacemaker located in the suprachiasmatic nuclei (SCN) drives a vast array of biochemical and physiological processes with 24-h periodicity. The phasing of SCN pacemaker activity is tightly regulated by photic input from the retina. Recent work has implicated protein kinase C (PKC) as a regulator of photic input, although stimulus-induced PKC activity has not been examined. Here we used a combination of biochemical, immunohistochemical and behavioral techniques to examine both the regulation and role of PKC in light-induced clock entrainment in mice. We report that photic stimulation during the subjective night, but not during the subjective day, stimulates PKC activity within the SCN. To assess the role of PKC in clock entrainment, we employed an in-vivo infusion approach to deliver the PKC inhibitor bisindolylmaleimide I to the SCN. The disruption of PKC activity significantly enhanced the phase-shifting effects of light, indicating that PKC functions as a negative regulator of light entrainment. Importantly, bisindolylmaleimide I infusion in the absence of light treatment did not phase shift the clock, demonstrating that transient disruption of basal PKC activity does not affect inherent pacemaker activity. The capacity of light to stimulate immediate early gene expression in the SCN was not substantively altered by PKC inhibition, suggesting that PKC does not couple light to rapid transcriptional activation. Rather, a combination of in-vivo and cell culture assays indicates that PKC functions as an inhibitor of PERIOD1 degradation. Thus, PKC may influence clock entrainment via a post-translational mechanism that influences clock protein stability.
Assuntos
Ritmo Circadiano/fisiologia , Proteína Quinase C/fisiologia , Animais , Western Blotting , Contagem de Células , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Densitometria , Humanos , Imuno-Histoquímica , Imunoprecipitação , Luz , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Circadianas Period , Estimulação Luminosa , Processamento de Proteína Pós-Traducional/fisiologia , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologiaRESUMO
Recent work has revealed that signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway couples light to entrainment of the circadian clock located in the suprachiasmatic nucleus (SCN). Given that many effects of the MAPK pathway are mediated by intermediate kinases, it was of interest to identify kinase targets of ERK in the SCN. One potential target is the family of 90-kDa ribosomal S6 kinases (RSKs). In this study, we examined light-induced regulation of RSK-1 in the SCN. Immunohistochemical and Western analysis were used to show that photic stimulation during the early and late night triggered the phosphorylation of RSK-1 at two sites that are targeted by ERK. This increase in the phosphorylation state of RSK-1 corresponded with an approximate fourfold increase in kinase activity. Light exposure during the subjective day did not increase the phosphorylated form of RSK-1, indicating that the capacity of light to stimulate RSK-1 activation is phase-restricted. Double immunofluorescent labelling of SCN tissue revealed the colocalized expression of the activated form of ERK with the phosphorylated form of RSK-1 following a light pulse. In vivo pharmacological inhibition of light-induced MAPK pathway activation blocked RSK-1 phosphorylation, indicating that RSK-1 activity is regulated by the MAPK pathway. PDK-1, a coregulator of RSK-1, is also expressed in the SCN and is likely to contribute to RSK-1 activity. RSK-1 phosphorylation was also rhythmically regulated within a subset of phospho-ERK-expressing cells. Together these results identify RSK-1 as a light- and clock-regulated kinase and raise the possibility that it contributes to entrainment and timing of the circadian pacemaker.
Assuntos
Ritmo Circadiano/fisiologia , Luz , Proteínas Quinases S6 Ribossômicas 90-kDa/biossíntese , Núcleo Supraquiasmático/enzimologia , Animais , Proteínas CLOCK , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transativadores/biossíntese , Transativadores/genéticaRESUMO
Signaling via the p42/p44 mitogen activated protein kinase (MAPK) pathway has been implicated as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). To examine how photic input dynamically regulates the activation state of the MAPK pathway, we monitored extracellular signal-regulated kinase (ERK) activation using different light stimulus paradigms. Compared with control animals not exposed to light, a 15 min light exposure during the early night triggered a marked increase in ERK activation and the translocation of ERK from the cytosol to the nucleus. ERK activation peaked 15 min after light onset, then returned to near basal levels within approximately 45 min. The MAPK pathway could be reactivated multiple times by light pulses spaced 45 min apart, indicating that the MAPK cascade rapidly resets and resolves individual light pulses into discrete signaling events. Under conditions of constant light (120 min), the time course for ERK activation, nuclear translocation, and inactivation was similar to the time course observed after a 15-min light treatment. The parallels between the ERK inactivation profiles elicited by a 15 and a 120 min light exposure suggest that SCN cells contain a MAPK pathway signal-termination mechanism that limits the duration of pathway activation. This concept was supported by the observation that the small G protein Ras, a regulator of the MAPK pathway, remained in the active, GTP-bound, state under conditions of constant light (120-min duration), indicating that photic information was relayed to the SCN and that SCN cells maintained their responsiveness for the duration of the light treatment. The SCN expressed both nuclear MAPK phosphatases (MKP-1 and MKP-2) and the cytosolic MAPK phosphatase Mkp-3, thus providing mechanisms by which light-induced ERK activation is terminated. Collectively, these observations provide important new information regarding the regulation of the MAPK cascade, a signaling intermediate that couples light to resetting of the SCN clock.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Núcleo Supraquiasmático/enzimologia , Núcleo Supraquiasmático/fisiologia , Animais , Western Blotting , Ritmo Circadiano/fisiologia , Fosfatase 1 de Especificidade Dupla , Ativação Enzimática/fisiologia , Genes ras , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Estimulação Luminosa , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Translocação Genética , Vias Visuais/fisiologiaRESUMO
In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus function as the major biological clock. SCN-dependent rhythms of physiology and behavior are regulated by changes in the environmental light cycle. Currently, the second messenger signaling events that couple photic input to clock entrainment have yet to be well characterized. Recent work has revealed that photic stimulation during the night triggers rapid activation of the p42/44 mitogen activated protein kinase (MAPK) pathway in the SCN. The MAPK signal transduction pathway is a potent regulator of numerous classes of transcription factors and has been shown to play a role in certain forms of neuronal plasticity. These observations led us to examine the role of the MAPK pathway in clock entrainment. Here we report that pharmacological disruption of light-induced MAPK pathway activation in the SCN uncouples photic input from clock entrainment, as assessed by locomotor activity phase. In the absence of photic stimulation, transient disruption of MAPK signaling in the SCN did not alter clock-timing properties. We also report that signaling via the Ca(2+)/calmodulin kinase pathway functions upstream of the MAPK pathway, coupling light to activation of the MAPK pathway. Together these results delineate key intracellular signaling events that underlie light-induced clock entrainment.
Assuntos
Luz , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Núcleo Supraquiasmático/efeitos da radiação , Animais , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 3 Ativada por Mitógeno , Núcleo Supraquiasmático/enzimologia , Núcleo Supraquiasmático/fisiologiaRESUMO
Signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been identified as an intermediate event coupling light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Given this observation, it was of interest to determine where within the entrainment process the MAPK pathway was functioning. In this study, we examined the role of the MAPK pathway as a regulator of light-induced gene expression in the SCN. Towards this end, we characterized the effect pharmacological disruption of the MAPK cascade has on the expression of the immediate-early genes c-Fos, JunB and EGR-1. We report that uncoupling light from MAPK pathway activation attenuated the expression of all three gene products. In the absence of photic stimulation, inhibition of the MAPK pathway did not alter basal gene product expression levels. Light-induced activation of cAMP response element (CRE)-dependent transcription, as assessed using a CRE-LacZ transgenic mouse strain, was also disrupted by blocking MAPK pathway activation. These results reveal that the MAPK cascade functions as one of the first transduction steps leading from light to rapid transcriptional activation, an essential event in the entrainment process. MAPK pathway-dependent gene expression in the SCN may result, in part, from stimulation of CRE-dependent transcription.