RESUMO
Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by a loss-of-function mutation in CDKL5 gene, encoding a serine-threonine kinase highly expressed in the brain. CDD manifests with early-onset epilepsy, autism, motor impairment and severe intellectual disability. While there are no known treatments for CDD, the use of cannabidiol has recently been introduced into clinical practice for neurodevelopmental disorders. Given the increased clinical utilization of cannabidiol, we examined its efficacy in the CDKL5R59X knock-in (R59X) mice, a CDD model based on a human mutation that exhibits both lifelong seizure susceptibility and behavioural deficits. We found that cannabidiol pre-treatment rescued the increased seizure susceptibility in response to the chemoconvulsant pentylenetetrazol (PTZ), attenuated working memory and long-term memory impairments, and rescued social deficits in adult R59X mice. To elucidate a potential mechanism, we compared the developmental hippocampal and cortical expression of common endocannabinoid (eCB) targets in R59X mice and their wild-type littermates, including cannabinoid type 1 receptor (CB1R), transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), G-coupled protein receptor 55 (GPR55) and adenosine receptor 1 (A1R). Many of these eCB targets were developmentally regulated in both R59X and wild-type mice. In addition, adult R59X mice demonstrated significantly decreased expression of CB1R and TRPV1 in the hippocampus, and TRPV2 in the cortex, while TRPV1 was increased in the cortex. These findings support the potential for dysregulation of eCB signalling as a plausible mechanism and therapeutic target in CDD, given the efficacy of cannabidiol to attenuate hyperexcitability and behavioural deficits in this disorder.
Assuntos
Canabidiol , Proteínas Serina-Treonina Quinases , Convulsões , Animais , Canabidiol/farmacologia , Convulsões/tratamento farmacológico , Convulsões/genética , Convulsões/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Síndromes Epilépticas/genética , Síndromes Epilépticas/tratamento farmacológico , Pentilenotetrazol , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Masculino , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Endocanabinoides/metabolismo , Comportamento Animal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Espasmos Infantis , Receptores de CanabinoidesRESUMO
Pathogenic mutations in cyclin-dependent kinase-like 5 (CDKL5) result in CDKL5 deficiency disorder (CDD), a rare disease marked by early-life seizures, autistic behaviors, and intellectual disability. Although mouse models of CDD exhibit dendritic instability and alterations in synaptic scaffolding proteins, studies of glutamate receptor levels and function are limited. Here we used a novel mouse model of CDD, the Cdkl5R59X knock-in mouse (R59X), to investigate changes in synaptic glutamate receptor subunits and functional consequences. Male mice were used for all experiments to avoid the confounding effects of X-inactivation that would be present in female heterozygous mice. We showed that adult male R59X mice recapitulated the behavioral outcomes observed in other mouse models of CDD, including social deficits and memory and learning impairments, and exhibited decreased latency to seizure upon pentylenetetrazol administration. Furthermore, we observed a specific increase in GluA2-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (AMPARs) in the adult R59X hippocampus, which is accompanied electrophysiologically by increased rectification ratio of AMPAR EPSCs and elevated early-phase long term potentiation (LTP). Finally, we showed that acute treatment with the GluA2-lacking AMPAR blocker IEM-1460 decreased AMPAR currents, and rescued social deficits, working memory impairments, and seizure behavior latency in R59X mice.SIGNIFICANCE STATEMENT CDKL5 deficiency disorder (CDD) is a rare disease marked by autistic-like behaviors, intellectual disability, and seizures. While synaptic dysfunction has been observed in mouse models of CDD, there is limited information on how synaptic alterations contribute to behavioral and functional changes in CDD. Here we reveal elevated hippocampal GluA2-lacking AMPAR expression in a novel mouse model of CDD that is accompanied by changes in synaptic AMPAR function and plasticity. We also show, for the first time, that acutely targeting GluA2-lacking AMPAR dysregulation rescues core synaptic and neurobehavioral deficits in CDD.
Assuntos
Síndromes Epilépticas/tratamento farmacológico , Síndromes Epilépticas/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Receptores de AMPA/efeitos dos fármacos , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/genética , Adulto , Animais , Comportamento Animal , Pré-Escolar , Modelos Animais de Doenças , Síndromes Epilépticas/psicologia , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Técnicas de Introdução de Genes , Humanos , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/psicologia , Masculino , Transtornos da Memória/genética , Transtornos da Memória/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Mutação/genética , Desempenho Psicomotor , Receptores de AMPA/deficiência , Receptores de AMPA/genética , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Comportamento Social , Espasmos Infantis/psicologiaRESUMO
Sensory feedback influences motor circuits and/or their projection neuron inputs to adjust ongoing motor activity, but its efficacy varies. Currently, less is known about regulation of sensory feedback onto projection neurons that control downstream motor circuits than about sensory regulation of the motor circuit neurons themselves. In this study, we tested whether sensory feedback onto projection neurons is sensitive only to activation of a motor system, or also to the modulatory state underlying that activation, using the crab Cancer borealis stomatogastric nervous system. We examined how proprioceptor neurons (gastropyloric receptors, GPRs) influence the gastric mill (chewing) circuit neurons and the projection neurons (MCN1, CPN2) that drive the gastric mill rhythm. During gastric mill rhythms triggered by the mechanosensory ventral cardiac neurons (VCNs), GPR was shown previously to influence gastric mill circuit neurons, but its excitation of MCN1/CPN2 was absent. In this study, we tested whether GPR effects on MCN1/CPN2 are also absent during gastric mill rhythms triggered by the peptidergic postoesophageal commissure (POC) neurons. The VCN and POC pathways both trigger lasting MCN1/CPN2 activation, but their distinct influence on circuit feedback to these neurons produces different gastric mill motor patterns. We show that GPR excites MCN1 and CPN2 during the POC-gastric mill rhythm, altering their firing rates and activity patterns. This action changes both phases of the POC-gastric mill rhythm, whereas GPR only alters one phase of the VCN-gastric mill rhythm. Thus sensory feedback to projection neurons can be gated as a function of the modulatory state of an active motor system, not simply switched on/off with the onset of motor activity.NEW & NOTEWORTHY Sensory feedback influences motor systems (i.e., motor circuits and their projection neuron inputs). However, whether regulation of sensory feedback to these projection neurons is consistent across different versions of the same motor pattern driven by the same motor system was not known. We found that gating of sensory feedback to projection neurons is determined by the modulatory state of the motor system, and not simply by whether the system is active or inactive.
Assuntos
Retroalimentação Sensorial/fisiologia , Movimento/fisiologia , Neurônios/fisiologia , Filtro Sensorial/fisiologia , Potenciais de Ação , Animais , Braquiúros , Geradores de Padrão Central/fisiologia , Estimulação Elétrica , Gânglios dos Invertebrados/fisiologia , Masculino , Mastigação/fisiologia , Microeletrodos , Vias Neurais/fisiologia , Periodicidade , Propriocepção/fisiologia , Técnicas de Cultura de TecidosRESUMO
In the isolated CNS, different modulatory inputs can enable one motor network to generate multiple output patterns. Thus far, however, few studies have established whether different modulatory inputs also enable a defined network to drive distinct muscle and movement patterns in vivo, much as they enable these distinctions in behavioral studies. This possibility is not a foregone conclusion, because additional influences present in vivo (e.g., sensory feedback, hormonal modulation) could alter the motor patterns. Additionally, rhythmic neuronal activity can be transformed into sustained muscle contractions, particularly in systems with slow muscle dynamics, as in the crab (Cancer borealis) stomatogastric system used here. We assessed whether two different versions of the biphasic (protraction, retraction) gastric mill (chewing) rhythm, triggered in the isolated stomatogastric system by the modulatory ventral cardiac neurons (VCNs) and postoesophageal commissure (POC) neurons, drive different muscle and movement patterns. One distinction between these rhythms is that the lateral gastric (LG) protractor motor neuron generates tonic bursts during the VCN rhythm, whereas its POC-rhythm bursts are divided into fast, rhythmic burstlets. Intracellular muscle fiber recordings and tension measurements show that the LG-innervated muscles retain the distinct VCN-LG and POC-LG neuron burst structures. Moreover, endoscope video recordings in vivo, during VCN-triggered and POC-triggered chewing, show that the lateral teeth protraction movements exhibit the same, distinct protraction patterns generated by LG in the isolated nervous system. Thus, the multifunctional nature of an identified motor network in the isolated CNS can be preserved in vivo, where it drives different muscle activity and movement patterns.
Assuntos
Comportamento Animal/fisiologia , Neurônios Motores/fisiologia , Movimento/fisiologia , Músculo Esquelético/fisiologia , Rede Nervosa/fisiologia , Animais , Braquiúros , Gânglios dos Invertebrados/fisiologia , Contração Muscular/fisiologia , Inibição Neural/fisiologia , Vias Neurais/fisiologiaRESUMO
Rhythmically active motor circuits can generate different activity patterns in response to different inputs. In most systems, however, it is not known whether the same neurons generate the underlying rhythm for each different pattern. Thus far, information regarding the degree of conservation of rhythm generator neurons is limited to a few pacemaker-driven circuits, in most of which the core rhythm generator is unchanged across different output patterns. We are addressing this issue in the network-driven, gastric mill (chewing) circuit in the crab stomatogastric nervous system. We first establish that distinct gastric mill motor patterns are triggered by separate stimulation of two extrinsic input pathways, the ventral cardiac neurons (VCNs) and postoesophageal commissure (POC) neurons. A prominent feature that distinguishes these gastric mill motor patterns is the LG (lateral gastric) protractor motor neuron activity pattern, which is tonic during the VCN rhythm and exhibits fast rhythmic bursting during the POC rhythm. These two motor patterns also differed in their cycle period and some motor neuron phase relationships, duty cycles, and burst durations. Despite the POC and VCN motor patterns being distinct, rhythm generation during each motor pattern required the activity of the same two, reciprocally inhibitory gastric mill neurons [LG, Int1 (interneuron 1)]. Specifically, reversibly hyperpolarizing LG or Int1, but no other gastric mill neuron, delayed the start of the next gastric mill cycle until after the imposed hyperpolarization. Thus, the same circuit neurons can comprise the core rhythm generator during different versions of a network-driven rhythmic motor pattern.
Assuntos
Gânglios dos Invertebrados/fisiologia , Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Periodicidade , Potenciais de Ação/fisiologia , Animais , Braquiúros , Masculino , Rede Nervosa/fisiologiaRESUMO
The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Ovo/análise , Proteínas do Ovo/imunologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Folículo Ovariano/química , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/imunologia , Zona Pelúcida/química , Adulto , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Folículo Ovariano/citologia , Proteínas Recombinantes/imunologia , Glicoproteínas da Zona PelúcidaRESUMO
BACKGROUND: Behavioral symptoms in individuals with autism spectrum disorder (ASD) have been attributed to abnormal neuronal connectivity, but the molecular bases of these behavioral and brain phenotypes are largely unknown. Human genetic studies have implicated PCDH10, a member of the δ2 subfamily of nonclustered protocadherin genes, in ASD. PCDH10 expression is enriched in the basolateral amygdala, a brain region implicated in the social deficits of ASD. Previous reports indicate that Pcdh10 plays a role in axon outgrowth and glutamatergic synapse elimination, but its roles in social behaviors and amygdala neuronal connectivity are unknown. We hypothesized that haploinsufficiency of Pcdh10 would reduce social approach behavior and alter the structure and function of amygdala circuits. METHODS: Mice lacking one copy of Pcdh10 (Pcdh10+/-) and wild-type littermates were assessed for social approach and other behaviors. The lateral/basolateral amygdala was assessed for dendritic spine number and morphology, and amygdala circuit function was studied using voltage-sensitive dye imaging. Expression of Pcdh10 and N-methyl-D-aspartate receptor (NMDAR) subunits was assessed in postsynaptic density fractions of the amygdala. RESULTS: Male Pcdh10+/- mice have reduced social approach behavior, as well as impaired gamma synchronization, abnormal spine morphology, and reduced levels of NMDAR subunits in the amygdala. Social approach deficits in Pcdh10+/- male mice were rescued with acute treatment with the NMDAR partial agonist d-cycloserine. CONCLUSIONS: Our studies reveal that male Pcdh10+/- mice have synaptic and behavioral deficits, and establish Pcdh10+/- mice as a novel genetic model for investigating neural circuitry and behavioral changes relevant to ASD.
Assuntos
Tonsila do Cerebelo/fisiopatologia , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Caderinas/fisiologia , Comportamento Social , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/patologia , Animais , Transtorno do Espectro Autista/psicologia , Comportamento Animal/fisiologia , Caderinas/genética , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Estimulação Elétrica , Feminino , Ritmo Gama , Haploinsuficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/metabolismo , Protocaderinas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Vocalização AnimalRESUMO
BACKGROUND: Activation of NMDA receptors can induce iron movement into neurons by the small GTPase Dexras1 via the divalent metal transporter 1 (DMT1). This pathway under pathological conditions such as NMDA excitotoxicity contributes to metal-catalyzed reactive oxygen species (ROS) generation and neuronal cell death, and yet its physiological role is not well understood. RESULTS: We found that genetic and pharmacological ablation of this neuronal iron pathway in the mice increased glutamatergic transmission. Voltage sensitive dye imaging of hippocampal slices and whole-cell patch clamping of synaptic currents, indicated that the increase in excitability was due to synaptic modification of NMDA receptor activity via modulation of the PKC/Src/NR2A pathway. Moreover, we identified that lysosomal iron serves as a main source for intracellular iron signaling modulating glutamatergic excitability. CONCLUSIONS: Our data indicates that intracellular iron is dynamically regulated in the neurons and robustly modulate synaptic excitability under physiological condition. Since NMDA receptors play a central role in synaptic neurophysiology, plasticity, neuronal homeostasis, neurodevelopment as well as in the neurobiology of many diseases, endogenous iron is therefore likely to have functional relevance to each of these areas.
Assuntos
Ferro/metabolismo , Lisossomos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas ras/metabolismo , Animais , Citosol/efeitos dos fármacos , Citosol/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hidrazinas , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Quelantes de Ferro/farmacologia , Lisossomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Receptores de AMPA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Quinases da Família src/metabolismoRESUMO
Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas do Olho , Regulação da Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/biossíntese , Hipotálamo/metabolismo , Células Fotorreceptoras de Invertebrados , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Relógios Biológicos/genética , Relógios Biológicos/fisiologia , Proteínas CLOCK , Proteínas de Ciclo Celular , Linhagem Celular , Ritmo Circadiano/genética , Criptocromos , Estro/genética , Estro/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Flavoproteínas/genética , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Proteínas de Fluorescência Verde , Hipotálamo/citologia , Proteínas Luminescentes/genética , Camundongos , Camundongos Mutantes , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Most physiological systems show daily variations in functional output, entrained to the day-night cycle. Humans exhibit a daily rhythm in urinary voiding (micturition), and disruption of this rhythm (nocturia) has significant clinical impact. However, the underlying mechanisms are not well-understood. Recently, a circadian rhythm in micturition was demonstrated in rodents, correlated with functional changes in urodynamics, providing the opportunity to address this issue in an animal model. Smooth muscle cells from mouse bladder have been proposed to express a functional and autonomous circadian clock at the molecular level. In this study, we addressed whether a semi-intact preparation of mouse urinary bladder smooth muscle (UBSM) exhibited measurable differences in contractility between day and night. UBSM tissue strips were harvested at four time points over the diurnal cycle, and spontaneous (phasic) and nerve-evoked contractions were assessed using isometric tension recordings. During the active period (ZT12-24) when micturition frequency is higher in rodents, UBSM strips had no significant differences in maximal- (high K(+)) or nerve-evoked contractions compared to strips harvested from the resting period (ZT0-12). However, a diurnal rhythm in phasic contraction was observed, with higher amplitudes at ZT10. Consistent with the enhanced phasic amplitudes, expression of the BK K(+) channel, a key suppressor of UBSM excitability, was lower at ZT8. Higher expression of BK at ZT20 was correlated with an enhanced effect of the BK antagonist paxilline (PAX) on phasic amplitude, but PAX had no significant time-of-day dependent effect on phasic frequency or nerve-evoked contractions. Overall, these results identify a diurnal difference for one contractile parameter of bladder muscle. Taken together, the results suggest that autonomous clocks in UBSM make only a limited contribution to the integrated control of diurnal micturition patterns.
RESUMO
Multisystemic therapy (MST) is effective for decreasing or preventing delinquency and other externalizing behaviors and increasing prosocial or adaptive behaviors. The purpose of this project was to review the literature examining the efficacy of MST for other child psychological and health problems reflecting non-externalizing behaviors, specifically difficulties related to child maltreatment, serious psychiatric illness [Serious psychiatric illness was defined throughout the current review paper as the "presence of symptoms of suicidal ideation, homicidal ideation, psychosis, or threat of harm to self or others due to mental illness severe enough to warrant psychiatric hospitalization based on the American Academy of Child and Adolescent Psychiatry (Level of care placement criteria for psychiatric illness. American Academy of Child and Adolescent Psychiatry, Washington, DC, 1996) level of care placement criteria for psychiatric illness" (Henggeler et al. in J Am Acad Child Psy 38:1331-1345, p. 1332, 1999b). Additionally, youth with "serious emotional disturbance (SED)" defined as internalizing and/or externalizing problems severe enough to qualify for mental health services in public school who were "currently in or at imminent risk of a costly out-of-home placement" (Rowland et al. in J Emot Behav Disord 13:13-23, pp. 13-14, 2005) were also included in the serious psychiatric illness category.], and health problems (i.e., obesity and treatment adherence for diabetes). PubMed, Web of Science, MEDLINE, and PsycINFO databases; Clinicaltrials.gov; DARE; Web of Knowledge; and Cochrane Central Register of Controlled Trials were searched; and MST developers were queried to ensure identification of all relevant articles. Of 242 studies identified, 18 met inclusion criteria for review. These were combined in a narrative synthesis and critiqued in the context of review questions. Study quality ratings were all above mean scores reported in prior reviews. Mixed support was found for the efficacy of MST versus other treatments. In many cases, treatment effects for MST or comparison groups were not sustained over time. MST was efficacious for youth with diverse backgrounds. No studies discussed efficacy of MST provided in different treatment settings. Four studies found MST more cost-effective than a comparison treatment, leading to fewer out-of-home placements for youth with serious psychiatric illness or lower treatment costs for youth with poorly controlled diabetes.
Assuntos
Terapia Comportamental/métodos , Sintomas Comportamentais/terapia , Maus-Tratos Infantis/terapia , Controle Interno-Externo , Transtornos Mentais/terapia , Adolescente , Terapia Comportamental/economia , Terapia Comportamental/normas , Criança , HumanosRESUMO
Neuronal network flexibility enables animals to respond appropriately to changes in their internal and external states. We are using the isolated crab stomatogastric nervous system to determine how extrinsic inputs contribute to network flexibility. The stomatogastric system includes the well-characterized gastric mill (chewing) and pyloric (filtering of chewed food) motor circuits in the stomatogastric ganglion. Projection neurons with somata in the commissural ganglia (CoGs) regulate these rhythms. Previous work characterized a unique gastric mill rhythm that occurred spontaneously in some preparations, but whose origin remained undetermined. This rhythm includes a distinct protractor phase activity pattern, during which a key gastric mill circuit neuron (LG neuron) and the projection neurons MCN1 and CPN2 fire in a pyloric rhythm-timed activity pattern instead of the tonic firing pattern exhibited by these neurons during previously studied gastric mill rhythms. Here we identify a new extrinsic input, the post-oesophageal commissure (POC) neurons, relatively brief stimulation (30 s) of which triggers a long-lasting (tens of minutes) activation of this novel gastric mill rhythm at least in part via its lasting activation of MCN1 and CPN2. Immunocytochemical and electrophysiological data suggest that the POC neurons excite MCN1 and CPN2 by release of the neuropeptide Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). These data further suggest that the CoG arborization of the POC neurons comprises the previously identified anterior commissural organ (ACO), a CabTRP Ia-containing neurohemal organ. This endocrine organ thus appears to also have paracrine actions, including activation of a novel and lasting gastric mill rhythm.
Assuntos
Braquiúros/fisiologia , Animais , Braquiúros/anatomia & histologia , Estimulação Elétrica , Sistema Nervoso Entérico/anatomia & histologia , Sistema Nervoso Entérico/fisiologia , Técnicas In Vitro , Masculino , Mastigação/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Estômago/inervação , Estômago/fisiologiaRESUMO
Orexin A, a recently discovered hypothalamic peptide, has been shown to have a stimulatory effect on release of gonadotropin-releasing hormone (GnRH) from rat hypothalamic explants in vitro. However, it is presently unclear whether in vivo this effect is mediated directly at the level of the GnRH neuron, or via multiple afferent neuronal connections. Therefore, in the present study, we investigated the direct action of orexin A on GnRH neurons using the immortalized GnRH-secreting GT1-7 hypothalamic cells. Orexin-1 receptor (OX1R) expression was detected in GT1-7 cells by RT-PCR and Western blot. Results showed that 0.1-1 nM orexin A, when administered in culture media for 4 h, can significantly stimulate GnRH mRNA expression in GT1-7 cells (p < 0.05). Administration of 1 microM OX1R antagonist, SB-334867, completely blocked the observed orexin A responses in these cells, indicating that orexin A stimulation of GnRH neurons is specifically through OX1R. Moreover, 0.1 nM orexin A stimulated GnRH release after 30-45 min. To examine possible signal transduction pathways involved in mediating these effects, a MEK inhibitor (UO-126), PKC inhibitor (calphostin C), and PKA inhibitor (H-89), were used, with each blocking orexin A-induced GnRH transcription and release from immortalized cells. Collectively, our results show that orexin A is capable of directly stimulating GnRH transcription and neuropeptide release from these immortalized hypothalamic neurons, and that the effects of orexin A appear to be mediated via the OX1R, coupled with activation of the PKC-, MAPK- and PKA-signaling pathways. It is suggested that the stimulatory effect of orexin A on GnRH transcription and release may also occur directly at the level of GnRH neurons in vivo.