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Prader-Willi syndrome (PWS) is a multisystemic disorder. Notably, many characteristic symptoms of PWS are correlated with locus coeruleus norepinephrine system (LC-NE) dysfunction, including impairment in arousal, learning, pain modulation, and stress-induced negative affective states. Although electrophysiological experiments in necdin-deficient mice, an established PWS animal model, have revealed decreased spontaneous neuronal firing activity in the LC and impaired excitability, the behavioral phenotypes related to LC-NE dysfunction remain unexplored. In this study, heterozygous necdin-deficient mice (B6.Cg-Ndntm1ky) were bred from wild-type (WT) females to generate WT (+m/+p) and heterozygous (+m/-p) animals. Compared to WT mice, Ndn + m/-p mice demonstrated impaired visual-spatial memory in the Y-maze test, reduced social interaction, impaired sexual recognition, and shorter falling latency on the Rotarod. Using the open field test (OFT) and elevated plus maze (EPM), we observed similar locomotion activity of Ndn + m/-p and WT mice, but Ndn + m/-p mice were less anxious. After acute restraint, Ndn + m/-p mice exhibited significant impairment in stress-induced anxiety. Additionally, the plasma norepinephrine surge following exposure to acute restraint stress was also impaired. Pretreatment with atomoxetine, a norepinephrine reuptake inhibitor aimed to enhance LC function, restored Ndn + m/-p mice to exhibit a normal response to acute restraint stress. Furthermore, by employing chemogenetic approaches to facilitate LC neuronal firing, post-stress anxious responses were also partially rescued in Ndn + m/-p mice. These data strongly suggest that LC dysfunction is implicated in the pathogenesis of stress-related neuropsychiatric symptoms in PWS. Manipulation of LC activity may hold therapeutic potential for patients with PWS.
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Proprioceptors are low-threshold mechanoreceptors involved in perceiving body position and strain bearing. However, the physiological response of proprioceptors to fatigue- and muscle-acidosis-related disturbances remains unknown. Here, we employed whole-cell patch-clamp recordings to probe the effect of mild acidosis on the mechanosensitivity of the proprioceptive neurons of dorsal root ganglia (DRG) in mice. We cultured neurite-bearing parvalbumin-positive (Pv+) DRG neurons on a laminin-coated elastic substrate and examined mechanically activated currents induced through substrate deformation-driven neurite stretch (SDNS). The SDNS-induced inward currents (ISDNS) were indentation depth-dependent and significantly inhibited by mild acidification (pH 7.2~6.8). The acid-inhibiting effect occurred in neurons with an ISDNS sensitive to APETx2 (an ASIC3-selective antagonist) inhibition, but not in those with an ISNDS resistant to APETx2. Detailed subgroup analyses revealed ISDNS was expressed in 59% (25/42) of Parvalbumin-positive (Pv+) DRG neurons, 90% of which were inhibited by APETx2. In contrast, an acid (pH 6.8)-induced current (IAcid) was expressed in 76% (32/42) of Pv+ DRG neurons, 59% (21/32) of which were inhibited by APETx2. Together, ASIC3-containing channels are highly heterogenous and differentially contribute to the ISNDS and IAcid among Pv+ proprioceptors. In conclusion, our findings highlight the importance of ASIC3-containing ion channels in the physiological response of proprioceptors to acidic environments.
Assuntos
Acidose , Mecanotransdução Celular , Animais , Camundongos , Parvalbuminas , Mecanorreceptores , NeuritosRESUMO
The locus coeruleus (LC) consists of noradrenergic (NA) neurons and plays an important role in controlling behaviours. Although much of the knowledge regarding LC functions comes from studying behavioural outcomes upon administration of muscarinic acetylcholine receptor (mAChR) agonists into the nucleus, the exact mechanisms remain unclear. Here, we report that the application of carbachol (CCh), an mAChR agonist, increased the spontaneous action potentials (sAPs) of both LC-NA neurons and local inhibitory interneurons (LC I-INs) in acute brain slices by activating M1/M3 mAChRs (m1/3 AChRs). Optogenetic activation of LC I-INs evoked inhibitory postsynaptic currents (IPSCs) in LC-NA neurons that were mediated by γ-aminobutyric acid type A (GABAA ) and glycine receptors, and CCh application decreased the IPSC amplitude through a presynaptic mechanism by activating M4 mAChRs (m4 AChRs). LC-NA neurons also exhibited spontaneous phasic-like activity (sPLA); CCh application increased the incidence of this activity. This effect of CCh application was not observed with blockade of GABAA and glycine receptors, suggesting that the sPLA enhancement occurred likely because of the decreased synaptic transmission of LC I-INs onto LC-NA neurons by the m4 AChR activation and/or increased spiking rate of LC I-INs by the m1/3 AChR activation, which could lead to fatigue of the synaptic transmission. In conclusion, we report that CCh application, while inhibiting their synaptic transmission, increases sAP rates of LC-NA neurons and LC I-INs. Collectively, these effects provide insight into the cellular mechanisms underlying the behaviour modulations following the administration of muscarinic receptor agonists into the LC reported by the previous studies.
Assuntos
Neurônios Adrenérgicos , Carbacol/farmacologia , Neurônios Adrenérgicos/metabolismo , Locus Cerúleo/metabolismo , Receptores de Glicina , Transmissão Sináptica/fisiologia , Receptores Muscarínicos/metabolismo , Agonistas Muscarínicos/farmacologia , Interneurônios/metabolismo , Ácido gama-Aminobutírico/fisiologiaRESUMO
BACKGROUND: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by multiple respiratory, cognitive, endocrine, and behavioral symptoms, such as central apnea, intellectual disabilities, exaggerated stress responses, and temper tantrums. The locus coeruleus noradrenergic system (LC-NE) modulates a diverse range of behaviors, including arousal, learning, pain modulation, and stress-induced negative affective states, which are possibly correlated with the pathogenesis of PWS phenotypes. Therefore, we evaluated the LC-NE neuronal activity of necdin-deficient mice, an animal model of PWS. METHODS: Heterozygous necdin-deficient mice (B6.Cg-Ndntm1ky) were bred from wild-type (WT) females to generate WT (+m/+p) and heterozygotes (+m/-p) animals, which were examined of LC-NE neuronal activity, developmental reflexes, and plethysmography. RESULTS: On slice electrophysiology, LC-NE neurons of Ndntm1ky mice with necdin deficiency showed significantly decreased spontaneous activities and impaired excitability, which was mediated by enhanced A-type voltage-dependent potassium currents. Ndntm1ky mice also exhibited the neonatal phenotypes of PWS, such as hypotonia and blunt respiratory responses to hypercapnia. CONCLUSIONS: LC-NE neuronal firing activity decreased in necdin-deficient mice, suggesting that LC, the primary source of norepinephrine in the central nervous system, is possibly involved in PWS pathogenesis.
Assuntos
Neurônios Adrenérgicos/metabolismo , Locus Cerúleo/metabolismo , Proteínas do Tecido Nervoso , Proteínas Nucleares , Síndrome de Prader-Willi/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
KEY POINTS: The locus coeruleus (LC) contains noradrenergic (NA) neurons that respond to novel stimuli in the environment with phasic activation to initiate an orienting response; phasic LC activation is also triggered by stimuli, representing the outcome of task-related decision processes, to facilitate ensuing behaviours and help optimize task performance. Here, we report that LC-NA neurons exhibit bursts of action potentials in vitro resembling phasic LC activation in vivo, and the activity is gated by inhibitory interneurons (I-INs) located in the peri-LC. We also observe that inhibition of peri-LC I-INs enhances prepulse inhibition and axons from cortical areas that play important roles in evaluating the cost/reward of a stimulus synapse on both peri-LC I-INs and LC-NA neurons. The results help us understand the cellular mechanisms underlying the generation and regulation of phasic LC activation with a focus on the role of peri-LC I-INs. ABSTRACT: Noradrenergic (NA) neurons in the locus coeruleus (LC) have global axonal projection to the brain. These neurons discharge action potentials phasically in response to either novel stimuli in the environment to initiate an orienting behaviour or stimuli representing the outcome of task-related decision processes to facilitate ensuing behaviours and help optimize task performance. Nevertheless, the cellular mechanisms underlying the generation and regulation of phasic LC activation remain unknown. We report here that LC-NA neurons recorded in brain slices exhibit bursts of action potentials that resembled the phasic activation-pause profile observed in animals. The activity was referred to as phasic-like activity (PLA) and was suppressed and enhanced by blocking excitatory and inhibitory synaptic transmissions, respectively. These results suggest the existence of a local circuit to drive PLA, and the activity could be regulated by the excitatory-inhibitory balance of the circuit. In support of this notion, we located a population of inhibitory interneurons (I-INs) in the medial part of the peri-LC that exerted feedforward inhibition of LC-NA neurons through GABAergic and glycinergic transmissions. Selective inhibition of peri-LC I-INs with chemogenetic methods could enhance PLA in brain slices and increase prepulse inhibition in animals. Moreover, axons from the orbitofrontal and prelimbic cortices, which play important roles in evaluating the cost/reward of a stimulus, synapse on both peri-LC I-INs and LC-NA neurons. These observations demonstrate functional roles of peri-LC I-INs in integrating inputs of the frontal cortex onto LC-NA neurons and gating the phasic LC output.
Assuntos
Neurônios Adrenérgicos , Locus Cerúleo , Potenciais de Ação , Animais , Interneurônios , Camundongos , NorepinefrinaRESUMO
The norepinephrine-releasing neurons in the locus coeruleus (LC) are well known to regulate wakefulness/arousal. They display active firing during wakefulness and a decreased discharge rate during sleep. We have previously reported that LC neurons express large numbers of GABAB receptors (GABABRs) located at peri-/extrasynaptic sites and are subject to tonic inhibition due to the continuous activation of GABABRs by ambient GABA, which is significantly higher during sleep than during wakefulness. In this study, we further showed using western blot analysis that the activation of GABABRs with baclofen could increase the level of phosphorylated extracellular signal-regulated kinase 1 (ERK1) in LC tissue. Recordings from LC neurons in brain slices showed that the inhibition of ERK1/2 with U0126 and FR180204 accelerated the decay of whole-cell membrane current induced by prolonged baclofen application. In addition, the inhibition of ERK1/2 also increased spontaneous firing and reduced tonic inhibition of LC neurons after prolonged exposure to baclofen. These results suggest a new role of GABABRs in mediating ERK1-dependent autoregulation of the stability of GABABR-activated whole-cell current, in addition to its well-known effect on gated potassium channels, to cause a tonic current in LC neurons.
Assuntos
Potenciais de Ação/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Homeostase , Neurônios/fisiologia , Receptores de GABA-B/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Baclofeno/farmacologia , Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B/farmacologia , Locus Cerúleo/citologia , Locus Cerúleo/metabolismo , Masculino , Neurônios/citologia , Neurônios/metabolismo , Nitrilas/farmacologia , Técnicas de Patch-Clamp/métodos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/farmacologiaRESUMO
Noradrenergic neurons in the locus coeruleus referred to as locus coeruleus neurons, provide the major supply of norepinephrine to the forebrain and play important roles in behavior through regulation of wakefulness and arousal. In a previous study using brain slice preparations, we reported that locus coeruleus neurons are subject to tonic inhibition mediated by γ-aminobutyric acid B receptors (GABABRs) and that the extent of tonic inhibition varies with ambient GABA levels. Since ambient GABA in the locus coeruleus was reported to fluctuate during the sleep-wakefulness cycle, here we tested whether GABABR-mediated tonic inhibition of locus coeruleus neurons could be a mechanism underlying changes in brain arousal. We first demonstrated that GABABR-mediated tonic inhibition of locus coeruleus neurons also exists in vivo by showing that local infusion of CGP35348, a GABABR antagonist, into the locus coeruleus increased the firing rate of locus coeruleus neurons in anesthetized rats. We then showed that this manipulation accelerated the behavioral emergence of rats from deep anesthesia induced by isoflurane. Together, these observations show that GABABR-mediated tonic inhibition of locus coeruleus neurons occurs in vivo and support the idea that this effect may be important in regulating the functional state of the brain.
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Neurônios Adrenérgicos/efeitos dos fármacos , Neurônios Adrenérgicos/fisiologia , Anestesia , Anestésicos Inalatórios/administração & dosagem , Isoflurano/administração & dosagem , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/fisiologia , Receptores de GABA-B/fisiologia , Animais , Antagonistas de Receptores de GABA-B/administração & dosagem , Masculino , Inibição Neural/efeitos dos fármacos , Compostos Organofosforados/administração & dosagem , Ratos Sprague-DawleyRESUMO
BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in ATXN1 gene resulting in an expansion of polyglutamine repeats in the ATXN1 protein. Unfortunately, there has yet been any effective treatment so far for SCA1. This study investigated the feasibility of transplanting human umbilical mesenchymal stem cells (HUMSCs) into transgenic SCA1 mice containing an expanded uninterrupted allele with 82 repeats in the ATXN1-coding region. METHODS: 106 human umbilical mesenchymal stem cells were transplanted into the cerebella at 1 month of age. RESULTS: HUMSCs displayed significant ameliorating effects in SCA1 mice in terms of motor behaviors in balance beam test and open field test as compared with the untransplanted SCA1 mice. HUMSCs transplantation effectively reduced the cerebellar atrophy, salvaged Purkinje cell death, and alleviated molecular layer shrinkage. Electrophysiological studies showed higher amplitudes of compound motor action potentials as indicated by increasing neuronal-muscular response strength to stimuli after stem cell transplantation. At 5 months after transplantation, HUMSCs scattering in the mice cerebella remained viable and secreted cytokines without differentiating into neuronal or glia cells. CONCLUSIONS: Our findings provide hope for a new therapeutic direction for the treatment of SCA1.
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BACKGROUND: Compelling animal and clinical studies support the N-methyl-D-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia and suggest promising pharmacological agents to ameliorate negative and cognitive symptoms of schizophrenia, including sarcosine, a glycine transporter-1 inhibitor. AIMS AND METHODS: It is imperative to evaluate the therapeutic potential of sarcosine in animal models, which provide indispensable tools for testing drug effects in detail and elucidating the underlying mechanisms. In this study, a series of seven experiments was conducted to investigate the effect of sarcosine in ameliorating behavioral deficits and the underlying mechanism in pharmacological (i.e., MK-801-induced) and genetic (i.e., serine racemase-null mutant (SR-/-) mice) NMDAR hypofunction models. RESULTS: In Experiment 1, the acute administration of 500/1000 mg/kg sarcosine (i.p.) had no adverse effects on motor function and serum biochemical responses. In Experiments 2-4, sarcosine significantly alleviated MK-801-induced (0.2 mg/kg) brain abnormalities and behavioral deficits in MK-801-induced and SR-/- mouse models. In Experiment 5, the injection of sarcosine enhanced CSF levels of glycine and serine in rat brain. In Experiments 6-7, we show for the first time that sarcosine facilitated NMDAR-mediated hippocampal field excitatory postsynaptic potentials and influenced the movement of surface NMDARs at extrasynaptic sites. CONCLUSIONS: Sarcosine effectively regulated the surface trafficking of NMDARs, NMDAR-evoked electrophysiological activity, brain glycine levels and MK-801-induced abnormalities in the brain, which contributed to the amelioration of behavioral deficits in mouse models of NMDAR hypofunction.
Assuntos
Sintomas Comportamentais/tratamento farmacológico , Encefalopatias/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Glicina/agonistas , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sarcosina/farmacologia , Esquizofrenia/tratamento farmacológico , Animais , Sintomas Comportamentais/induzido quimicamente , Encefalopatias/induzido quimicamente , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Racemases e Epimerases/genética , Sarcosina/administração & dosagem , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Esquizofrenia/fisiopatologiaRESUMO
Pain-related diseases are the top leading causes of life disability. Identifying brain regions involved in persistent neuronal changes will provide new insights for developing efficient chronic pain treatment. Here, we showed that anterior nucleus of paraventricular thalamus (PVA) plays an essential role in the development of mechanical hyperalgesia in neuropathic and inflammatory pain models in mice. Increase in c-Fos, phosphorylated extracellular signal-regulated kinase, and hyperexcitability of PVA neurons were detected in hyperalgesic mice. Direct activation of PVA neurons using optogenetics and pharmacological approaches were sufficient to induce persistent mechanical hyperalgesia in naive animals. Conversely, inhibition of PVA neuronal activity using DREADDs (designer receptors exclusively activated by designer drugs) or inactivation of PVA extracellular signal-regulated kinase at the critical time window blunted mechanical hyperalgesia in chronic pain models. At the circuitry level, PVA received innervation from central nucleus of amygdala, a known pain-associated locus. As a result, activation of right central nucleus of amygdala with blue light was enough to induce persistent mechanical hyperalgesia. These findings support the idea that targeting PVA can be a potential therapeutic strategy for pain relief.
Assuntos
Dor Crônica/fisiopatologia , Hiperalgesia/fisiopatologia , Núcleos da Linha Média do Tálamo/fisiopatologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Comportamento Animal/fisiologia , Dor Crônica/metabolismo , Hiperalgesia/metabolismo , Camundongos , Núcleos da Linha Média do Tálamo/metabolismo , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Fosforilação , Estimulação Física , Proteínas Proto-Oncogênicas c-fos/metabolismoRESUMO
Attention deficit hyperactivity disorder (ADHD) has a prevalence of 7.5% in school-age children in Taiwan. A number of ADHD patients start taking medications in elementary school and continue their treatment until they are in college or adulthood. Methylphenidate is the most frequently used medication prescribed for ADHD treatment. The influence of long-term treatment of methylphenidate on neuro-development, especially dopaminergic neurons, in rats would be explored. This study investigated the impact of long-term treatment of methylphenidate on different neurons. Rats aged 1 month were divided into three groups: Normal group receiving only sucrose solution, Low-dose group receiving 2â¯mg/kg methylphenidate, and High-dose group receiving 10â¯mg/kg methylphenidate; for each group, the drug was administrated twice per day. After 7 months of the treatment period, then the alterations in number of norepinephrine, serotonergic, cholinergic and dopaminergic neurons were quantified. The number of dopaminergic neurons in the substantia nigra (SN), the serotonergic neurons in the dorsal raphe nucleus, and the cholinergic neurons in the tegmental nucleus significantly decreased as compared with Normal group, whereas the noradrenergic neurons in the locus coeruleus substantially increased. The whole-cell recording was made from dopaminergic neurons residing in the SN for examination of their firing activity. The recorded dopaminergic neurons in SN were categorized into slow and fast firing using 10â¯Hz as a classified index. The results displayed that the ratio of dopaminergic neurons with fast firing in the High-dose group was less as compared with those in the Normal and the Low-dose group. Furthermore, the amplitude of action potential of the dopaminergic neurons with slow firing was higher in the High-dose group than those in the Normal and Low-dose groups. The firing behavior of dopaminergic neurons and dopamine concentration in the brain is affected by the long-term challenge of methylphenidate.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Metilfenidato/farmacologia , Tempo , Potenciais de Ação/fisiologia , Animais , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/farmacologia , Masculino , Metilfenidato/administração & dosagem , Norepinefrina/farmacologia , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacosRESUMO
Vilse/Arhgap39 is a Rho GTPase activating protein (RhoGAP) and utilizes its WW domain to regulate Rac/Cdc42-dependent morphogenesis in Drosophila and murine hippocampal neurons. However, the function of Vilse in mammalian dendrite architecture and synaptic plasticity remained unclear. In the present study, we aimed to explore the possible role of Vilse in dendritic structure and synaptic function in the brain. Homozygous knockout of Vilse resulted in premature embryonic lethality in mice. Changes in dendritic complexity and spine density were noticed in hippocampal neurons of Camk2a-Cre mediated forebrain-specific Vilse knockout (VilseΔ/Δ) mice. VilseΔ/Δ mice displayed impaired spatial memory in water maze and Y-maze tests. Electrical stimulation in hippocampal CA1 region revealed that the synaptic transmission and plasticity were defected in VilseΔ/Δ mice. Collectively, our results demonstrate that Vilse is essential for embryonic development and required for spatial memory.
Assuntos
Região CA1 Hipocampal/fisiologia , Dendritos/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Dendritos/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Genes Letais/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasticidade Neuronal/genética , Células Piramidais/metabolismo , Memória Espacial/fisiologia , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.
Assuntos
Neurônios Adrenérgicos/fisiologia , Neurônios Colinérgicos/fisiologia , Ácido Glutâmico/metabolismo , Núcleo Tegmental Pedunculopontino/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Neurônios Adrenérgicos/citologia , Neurônios Adrenérgicos/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/efeitos dos fármacos , Estimulação Elétrica , Feminino , Masculino , Técnicas de Patch-Clamp , Núcleo Tegmental Pedunculopontino/citologia , Núcleo Tegmental Pedunculopontino/efeitos dos fármacos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3(f/f)) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3(-/-)) and Pv-Cre::Asic3(f/f) mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Mecanotransdução Celular/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais Iônicos Sensíveis a Ácido/deficiência , Canais Iônicos Sensíveis a Ácido/genética , Animais , Gânglios Espinais/fisiologia , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Fusos Musculares/inervação , Fusos Musculares/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Parvalbuminas/metabolismo , Equilíbrio Postural/fisiologia , Propriocepção/fisiologiaRESUMO
Tian ma (Gastrodia elata, GE) is an ancient Chinese herbal medicine that has been suggested to be effective as an anticonvulsant and analgesic, and to have sedative effects against vertigo, general paralysis, epilepsy and tetanus. The primary active ingredient isolated from GE is termed gastrodin, which is the glucoside of 4-hydroxybenzyl alcohol (4-HBA). Gastrodin can abolish hypoxia-, glutamate- and N-methyl-D-aspartate (NMDA) receptor-induced toxicity in primary culture of rat cortical neurons, and reduces seizure severity in seizure-sensitive gerbils. We evaluated the effect of gastrodin on NMDA excitotoxicity in hippocampal slice cultures (HSCs) with propidium iodide (PI) fluorescence measurement. We also evaluated the effects of gastrodin for treating active in vivo temporal lobe seizures induced by lithium/pilocarpine. Seizure severity, time span to seizure onset, mortality rate and hippocampal histology for survivors were compared. The effect of gastrodin was evaluated for treating in vitro seizures induced by Mg²âº-free medium in hippocampal slices. Frequencies and amplitudes of epileptiform discharges were compared. The effect of gastrodin on synaptic transmission was evaluated on hippocampal CA1 Schaffer collaterals. Application of 25 µM gastrodin significantly suppressed NMDA excitotoxicity in CA3 but not in CA1 hippocampus and dentate gyrus. Intraventricular gastrodin accelerated seizure onset for 12 min after intraperitoneal pilocarpine injection (P = 0.051). Three of five rats (60%) in the gastrodin group, and three of four (75%) in the dimethyl sulfoxide (DMSO) group died within 3 days after status epilepticus (SE). Gastrodin also failed to inhibit epileptiform discharges in hippocampal slices induced by Mg²âº-free medium, believed to be NMDA receptor-mediated spontaneous activity. The frequencies of the spontaneous epileptiform discharges were similar under treatments with 25 µM gastrodin, 200 µM gastrodin and DMSO. For the evaluation of gastrodin on synaptic transmission, application of DMSO, 25 µM or 200 µM gastrodin had no significant effect on excitatory postsynaptic potential (EPSP) slopes. Gastrodin at 200 µM decreased paired-pulse facilitation (PPF) from 1.23 ± 0.04 to 1.12 ± 0.04 (P = 0.002). In conclusion, gastrodin failed to suppress in vivo and in vitro seizures in our study. Gastrodin showed no effect on hippocampal Schaffer collateral EPSP. These findings suggest that gastrodin does not interact with ionotropic glutamate receptors to inhibit NMDA receptor-facilitated seizures. However, gastrodin showed protective effects against NMDA toxicity on cultured hippocampal slices. Nevertheless, gastrodin is still a potential neuroprotective agent against NMDA excitotoxicity, with potential benefits for stroke and patients with epilepsy.
Assuntos
Anticonvulsivantes/farmacologia , Álcoois Benzílicos/farmacologia , Epilepsia do Lobo Temporal/fisiopatologia , Glucosídeos/farmacologia , Hipocampo/fisiopatologia , Neurônios/efeitos dos fármacos , Convulsões/fisiopatologia , Animais , Anticonvulsivantes/administração & dosagem , Álcoois Benzílicos/administração & dosagem , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiopatologia , Região CA3 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/fisiopatologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiopatologia , Eletroencefalografia/efeitos dos fármacos , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/mortalidade , Agonistas de Aminoácidos Excitatórios/farmacologia , Glucosídeos/administração & dosagem , Hipocampo/efeitos dos fármacos , Injeções Intraventriculares , Excitação Neurológica/efeitos dos fármacos , N-Metilaspartato/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamenteRESUMO
Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg2+ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg2+ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10 µM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10 µM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.
Assuntos
Região CA1 Hipocampal/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Tiazolidinedionas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Anilidas/farmacologia , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Regulação da Expressão Gênica , Magnésio/farmacologia , Microtomia , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/antagonistas & inibidores , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Rosiglitazona , Convulsões/tratamento farmacológico , Convulsões/genética , Convulsões/metabolismo , Convulsões/patologia , Transmissão Sináptica/efeitos dos fármacos , Tiazolidinedionas/antagonistas & inibidores , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: The descending noradrenergic (NAergic) system is one of the important endogenous analgesia systems. It has been suggested that noxious stimuli could activate descending NAergic system; nevertheless, the underlying neuronal circuit remains unclear. As NAergic neurons in the A7 catecholamine cell group (A7) are a part of the descending NAergic system and the lateral parabrachial nucleus (LPB) is an important brainstem structure that relays ascending nociceptive signal, we aimed to test whether LPB neurons have direct synaptic contact with NAergic A7 neurons. RESULTS: Stereotaxic injections of an anterograde tracer, biotinylated dextran-amine (BDA), were administered to LPB in rats. The BDA-labeled axonal terminals that have physical contacts with tyrosine hydroxylase-positive (presumed noadrenergic) neurons were identified in A7. Consistent with these morphological observations, the excitatory synaptic currents (EPSCs) were readily evoked in NAergic A7 neurons by extracellular stimulation of LPB. The EPSCs evoked by LPB stimulation were blocked by CNQX, a non-NMDA receptor blocker, and AP5, a selective NMDA receptor blocker, showing that LPB-A7 synaptic transmission is glutamatergic. Moreover, the amplitude of LPB-A7 EPSCs was significantly attenuated by DAMGO, a selective µ-opioid receptor agonist, which was associated with an increase in paired-pulse ratio. CONCLUSIONS: Taken together, the above results showed direct synaptic connections between LPB and A7 catecholamine cell group, the function of which is subject to presynaptic modulation by µ-opioid receptors.
Assuntos
Neurônios Adrenérgicos , Catecolaminas/metabolismo , Núcleos Parabraquiais , Sinapses/fisiologia , Neurônios Adrenérgicos/citologia , Neurônios Adrenérgicos/fisiologia , Animais , Masculino , Núcleos Parabraquiais/citologia , Núcleos Parabraquiais/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
KEY POINTS: Noradrenaline (NA)-releasing neurons in the locus coeruleus (LC) provide NA to the forebrain and play important roles in regulating many brain functions. LC neurons are subject to tonic inhibition mediated by GABAB receptors (GABAB Rs) and that the extent of the effect varies with ambient GABA levels. GABAB R-mediated tonic inhibition can effectively tune the spontaneous firing rate (SFR) of LC neurons; it is developmentally regulated and is responsible for maintaining a constant SFR of LC neurons during development. In male, but not female rats, chronic perinatal treatment with citalopram, a selective serotonin reuptake inhibitor, results in downregulation of GABAB R-mediated tonic inhibition of LC neurons that partially accounts for increased SFR in male, but not female, rats receiving such treatment. Our results show that GABAB R-mediated tonic inhibition could be an important player in the development of normal and abnormal behaviours/brain functions associated with the LC-NA system. Noradrenaline (NA)-releasing neurons in the locus coeruleus (LC) provide NA to the forebrain. Their activity is believed to be a key factor regulating the wakefulness/arousal level of the brain. In this study, we found that the activity of NA-releasing neurons in the LC (LC neurons) was subject to γ-aminobutyric acid (GABA) tonic inhibition through GABAB receptors (GABAB Rs), but not GABAA receptors. The intensity of GABAB R tonic inhibition was found to depend on ambient GABA levels, as it was dramatically increased by blockade of GABA reuptake. It also varied with the function of GABAB Rs. The GABAB R activity on LC neurons was found to increase with postnatal age up to postnatal days 8-10, resulting in increased tonic inhibition. Interestingly, there was no significant difference in the spontaneous activity of LC neurons at different postnatal ages unless GABAB R tonic inhibition was blocked. These results show that, during postnatal development, there is a continuous increase in GABAB R tonic inhibition that maintains the activity of LC neurons at a proper level. In male, but not female, rats, chronic perinatal treatment with citalopram, a selective serotonin reuptake inhibitor, reduced GABAB R activity and tonic inhibition, which might result in the significantly higher spontaneous activity of LC neurons seen in these animals. In conclusion, our results show that GABAB R-mediated tonic inhibition has a direct impact on the spontaneous activity of LC neurons and that the extent of the effect varies with ambient GABA levels and functionality of GABAB R signalling.
Assuntos
Citalopram/farmacologia , Locus Cerúleo/fisiologia , Neurônios/fisiologia , Receptores de GABA-B/fisiologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Animais Recém-Nascidos , Feminino , Técnicas In Vitro , Locus Cerúleo/citologia , Masculino , Ratos Sprague-DawleyRESUMO
AKT1 (also known as protein kinase B, α), a serine/threonine kinase of AKT family, has been implicated in both schizophrenia and methamphetamine (Meth) use disorders. AKT1 or its protein also has epistatic effects on the regulation of dopamine-dependent behaviors or drug effects, especially in the striatum. The aim of this study is to investigate the sex-specific role of Akt1 in the regulation of Meth-induced behavioral sensitization and the alterations of striatal neurons using Akt1(-/-) mice and wild-type littermates as a model. A series of 4 Experiments were conducted. Meth-induced hyperlocomotion and Meth-related alterations of brain activity were measured. The neural properties of striatal medium spiny neurons (MSNs) were also characterized. Further, 17ß-estradiol was applied to examine its protective effect in Meth-sensitized male mice. Our findings indicate that (1) Akt1(-/-) males were less sensitive to Meth-induced hyperlocomotion during Meth challenge compared with wild-type controls and Akt1(-/-) females, (2) further sex differences were revealed by coinjection of Meth with raclopride but not SCH23390 in Meth-sensitized Akt1(-/-) males, (3) Meth-induced alterations of striatal activity were confirmed in Akt1(-/-) males using microPET scan with (18)F-flurodeoxyglucose, (4) Akt1 deficiency had a significant impact on the electrophysiological and neuromorphological properties of striatal MSNs in male mice, and (5) subchronic injections of 17ß-estradiol prevented the reduction of Meth-induced hyperactivity in Meth-sensitized Akt1(-/-) male mice. This study highlights a sex- and region-specific effect of Akt1 in the regulation of dopamine-dependent behaviors and implies the importance of AKT1 in the modulation of sex differences in Meth sensitivity and schizophrenia.
Assuntos
Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Metanfetamina/efeitos adversos , Neostriado/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Psicoses Induzidas por Substâncias/fisiopatologia , Racloprida/farmacologia , Esquizofrenia/fisiopatologia , Animais , Antipsicóticos/administração & dosagem , Benzazepinas/administração & dosagem , Benzazepinas/farmacologia , Modelos Animais de Doenças , Estradiol/administração & dosagem , Estrogênios/administração & dosagem , Feminino , Hipercinese/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neostriado/citologia , Neostriado/fisiopatologia , Neurônios/efeitos dos fármacos , Psicoses Induzidas por Substâncias/tratamento farmacológico , Psicoses Induzidas por Substâncias/etiologia , Racloprida/administração & dosagem , Esquizofrenia/tratamento farmacológico , Fatores SexuaisRESUMO
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare pediatric neuro-metabolic disease in children. Due to the lack of an animal model, its pathogenetic mechanism is poorly understood. To study the role of AADC in brain development, a zebrafish model of AADC deficiency was generated. We identified an aadc gene homolog, dopa decarboxylase (ddc), in the zebrafish genome. Whole-mount in situ hybridization analysis showed that the ddc gene is expressed in the epiphysis, locus caeruleus, diencephalic catecholaminergic clusters, and raphe nuclei of 36-h post-fertilization (hpf) zebrafish embryos. Inhibition of Ddc by AADC inhibitor NSD-1015 or anti-sense morpholino oligonucleotides (MO) reduced brain volume and body length. We observed increased brain cell apoptosis and loss of dipencephalic catecholaminergic cluster neurons in ddc morphants (ddc MO-injected embryos). Seizure-like activity was also detected in ddc morphants in a dose-dependent manner. ddc morphants had less sensitive touch response and impaired swimming activity that could be rescued by injection of ddc plasmids. In addition, eye movement was also significantly impaired in ddc morphants. Collectively, loss of Ddc appears to result in similar phenotypes as that of ADCC deficiency, thus zebrafish could be a good model for investigating pathogenetic mechanisms of AADC deficiency in children.