RESUMO
Enhancing α7 nAChR function serves as a therapeutic strategy for cognitive disorders. Here, we report the synthesis and evaluation of 2-arylamino-thiazole-5-carboxylic acid amide derivatives 6-9 that as positive allosteric modulators (PAMs) activate human α7 nAChR current expressed in Xenopus ooctyes. Among the 4-amino derivatives, a representative atypical type I PAM 6p exhibits potent activation of α7 current with an EC50 of 1.3 µM and the maximum activation effect on the current over 48-fold in the presence of acetylcholine (100 µM). The structure-activity relationship (SAR) analysis reveals that the 4-amino group is crucial for the allosteric activation of α7 currents by compound 6p as the substitution of 4-methyl group results in its conversion to compound 7b (EC50 = 2.1 µM; max effect: 58-fold) characterized as a typical type I PAM. Furthermore, both 6p and 7b are able to rescue auditory gating deficits in mouse schizophrenia-like model of acoustic startle prepulse inhibition.
Assuntos
Tiazóis , Receptor Nicotínico de Acetilcolina alfa7 , Animais , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica/efeitos dos fármacos , Relação Estrutura-Atividade , Humanos , Tiazóis/farmacologia , Tiazóis/química , Tiazóis/síntese química , Tiazóis/uso terapêutico , Camundongos , Xenopus laevis , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Ácidos Carboxílicos/síntese química , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Masculino , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismoRESUMO
The discovery of a new neurotransmitter, especially one in the central nervous system, is both important and difficult. We have been searching for new neurotransmitters for 12 y. We detected creatine (Cr) in synaptic vesicles (SVs) at a level lower than glutamate and gamma-aminobutyric acid but higher than acetylcholine and 5-hydroxytryptamine. SV Cr was reduced in mice lacking either arginine:glycine amidinotransferase (a Cr synthetase) or SLC6A8, a Cr transporter with mutations among the most common causes of intellectual disability in men. Calcium-dependent release of Cr was detected after stimulation in brain slices. Cr release was reduced in Slc6a8 and Agat mutants. Cr inhibited neocortical pyramidal neurons. SLC6A8 was necessary for Cr uptake into synaptosomes. Cr was found by us to be taken up into SVs in an ATP-dependent manner. Our biochemical, chemical, genetic, and electrophysiological results are consistent with the possibility of Cr as a neurotransmitter, though not yet reaching the level of proof for the now classic transmitters. Our novel approach to discover neurotransmitters is to begin with analysis of contents in SVs before defining their function and physiology.
Assuntos
Creatina , Deficiência Intelectual , Masculino , Humanos , Camundongos , Animais , Creatina/genética , Deficiência Intelectual/genética , Sistema Nervoso Central , Neurotransmissores , EletrofisiologiaRESUMO
Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of SLC6A17-based-ID were both unclear. Here, we report learning deficits in Slc6a17 knockout and point mutant mice. Biochemistry, proteomic, and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of Slc6a17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with seven types of genetically modified mice have shown Gln as an endogenous substrate of SLC6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.
Assuntos
Deficiência Intelectual , Vesículas Sinápticas , Animais , Camundongos , Glutamina/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Mutação , Proteômica , Vesículas Sinápticas/metabolismoRESUMO
Maternal affiliation by infants is the first social behavior of mammalian animals. We report here that elimination of the Tph2 gene essential for serotonin synthesis in the brain reduced affiliation in mice, rats, and monkeys. Calcium imaging and c-fos immunostaining showed maternal odors activation of serotonergic neurons in the raphe nuclei (RNs) and oxytocinergic neurons in the paraventricular nucleus (PVN). Genetic elimination of oxytocin (OXT) or its receptor reduced maternal preference. OXT rescued maternal preference in mouse and monkey infants lacking serotonin. Tph2 elimination from RN serotonergic neurons innervating PVN reduced maternal preference. Reduced maternal preference after inhibiting serotonergic neurons was rescued by oxytocinergic neuronal activation. Our genetic studies reveal a role for serotonin in affiliation conserved from mice and rats to monkeys, while electrophysiological, pharmacological, chemogenetic, and optogenetic studies uncover OXT downstream of serotonin. We suggest serotonin as the master regulator upstream of neuropeptides in mammalian social behaviors.
Assuntos
Ocitocina , Serotonina , Animais , Camundongos , Ratos , Relações Interpessoais , Mamíferos , Ocitocina/fisiologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Neurônios SerotoninérgicosRESUMO
Enhancing neuronal α7 nicotinic acetylcholine receptor (α7 nAChR) function can alleviate cognitive deficits. Here, we report the design, synthesis, and evaluation of N-(4-(trifluoromethoxy)phenyl)-1,3,5-triazin-2-amine derivatives 8-10 as a series of novel α7 nAChR positive allosteric modulators (PAMs). The representative compound 10e functions as a type I PAM with an EC50 of 3.0 µM and approximately 38-fold enhancement of α7 current in the presence of agonist acetylcholine (100 µM). It specifically enhances α7 current with high selectivity. Compound 10e shows good pharmacokinetic property in mice. Intraperitoneal injection of 10e (3 mg/kg) exhibits sufficient blood-brain barrier penetration in mice. Furthermore, 10e can also rescue the auditory gating deficit in mice with schizophrenia-like behavior. Molecular docking of 10e with homopentameric α7 nAChR reveals a new mode of action. These results support the potential of 10e for treatment for schizophrenia and Alzheimer's disease.
Assuntos
Agonistas Nicotínicos/uso terapêutico , Esquizofrenia/tratamento farmacológico , Triazinas/uso terapêutico , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Maleato de Dizocilpina , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Agonistas Nicotínicos/síntese química , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacocinética , Esquizofrenia/induzido quimicamente , Filtro Sensorial/efeitos dos fármacos , Triazinas/síntese química , Triazinas/metabolismo , Triazinas/farmacocinética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Native M-current is a low-threshold, slowly activating potassium current that exerts an inhibitory control over neuronal excitability. The M-channel is primarily co-assembled by heterotetrameric Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits that are specifically expressed in the brain and peripheral nociceptive and visceral sensory neurons in the spinal cord. Reduction of M-channel function leads to neuronal hyperexcitability that defines the fundamental mechanism of neurological disorders such as epilepsy and pain, indicating that pharmacological activation of Kv7/KCNQ/M-channels may serve the basis for the therapy. The well-known KCNQ opener retigabine (ezogabine or Potiga) was approved by FDA in 2011 as an anticonvulsant used for an adjunctive treatment of partial epilepsies. Unfortunately, retigabine was discontinued in 2017 due to its side effects of blue-colored appearance of the skin and eyes after prolonged intake. In addition, flupirtine, a structural derivative of retigabine and a centrally acting non-opioid analgesic, was also withdrawn in 2018 for liver toxicity. Fortunately, these side effects are compound-structures related and can be avoided. Thus, further identification and development of novel potent and selective Kv7 channel openers may lead to an effective therapy with improved safety window for anti-epilepsy and anti-nociception.
Assuntos
Epilepsia , Canais de Potássio KCNQ , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Humanos , Neurônios , Dor/tratamento farmacológicoRESUMO
A natural monoterpene alkaloid incarvillateine isolated from the plant Incarvillea sinensis is known to relieve inflammatory and neuropathic pain. However, the molecular target for the action of incarvillateine remains elusive. Here, we report that incarvillateine exacerbates epileptic seizures by inhibiting subtypes of γ-Aminobutyric acid type A (GABAA) receptors. Two-electrode voltage clamp recordings of α1ß3γ2, α2ß3γ2, α3ß3γ2 and α5ß3γ2 subtypes expressed in Xenopus oocytes revealed that incarvillateine inhibited the GABAA currents with IC50 of 25.1⯵M, 43.1⯵M, 105.1⯵M and 93.7⯵M, respectively. Whole-cell patch clamp recordings of hippocampal slices confirmed that incarvillateine inhibited spontaneous inhibitory postsynaptic currents (IPSCs), and miniature IPSCs and tonic currents. Moreover, inhibition of GABAA currents and spontaneous IPSCs by incarvillateine persisted even in the presence of blockers of adenosine receptors. In addition, incarvillateine enhanced epileptic discharges induced by Mg2+-free artificial cerebrospinal fluid (ACSF) in hippocampal slices. Furthermore, intracerebral ventricular injections of incarvillateine increased the severity of seizures induced by kainic acid in a dose-dependent manner. Taken together, our data demonstrate that incarvillateine aggravates seizures by inhibition of GABAA currents and GABAergic synaptic transmissions.
Assuntos
Alcaloides/efeitos adversos , Produtos Biológicos/efeitos adversos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/efeitos adversos , Monoterpenos/efeitos adversos , Receptores de GABA-A/metabolismo , Segurança , Convulsões/fisiopatologia , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Camundongos , Neurotransmissores/metabolismo , Ratos , Convulsões/induzido quimicamente , Convulsões/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
Structural modifications of nicotinamide, a form of vitamin B3, gave rise to a series of compounds (8aa-8ce) that exhibit activities as type I positive allosteric modulators (PAMs) of human α7 nAChR expressed in Xenopus oocytes in two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious PAM with an EC50â¯=â¯3.34⯱â¯1.13⯵M and the maximum activation effect of α7 current over 1474⯱â¯246% in the presence of acetylcholine (100⯵M). It is highly specific to α7 nAChR over other subtypes of nAChR and 5-HT3A receptors. The structure-activity relationship analysis identified a key skeleton of nicotinamide nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7 nAChR may be beneficial for improvement of cognitive deficit.
Assuntos
Agonistas Nicotínicos/uso terapêutico , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos , Regulação Alostérica , Humanos , Agonistas Nicotínicos/farmacologia , Relação Estrutura-AtividadeRESUMO
A series of novel thiazolo[4,5- d]pyrimidin-7(6 H)-ones (3aa-3eq) were designed, synthesized, and evaluated as the type I positive allosteric modulators of human α7 nAChR expressed in Xenopus ooctyes by a two-electrode voltage clamp. The structure-activity relationship analysis identified the compound 3ea as a potent and efficacious PAM with the maximum activation effect of the α7 current of over 1633% in the presence of acetylcholine (100 µM) and an EC50 = 1.26 µM. It is highly specific to α7 nAChR over other subtypes of nAChR, 5-HT3A, NMDA, and GABAA receptors. Compound 3ea showed an elimination half-life of 10.8 ± 1.5 h for 3 mg/kg, i.v., and 7.4 ± 1.1 h for 60 mg/kg, i.g. in rat. It also exhibited sufficient blood-brain barrier penetration with no significant effect on hERG channel. Most importantly, compound 3ea dose-dependently (0.1-1 mg/kg, i.p.) reversed the prepulse inhibition deficit induced by MK-801 in the mouse schizophrenia model.
Assuntos
Desenho de Fármacos , Tiazóis/química , Receptor Nicotínico de Acetilcolina alfa7/química , Acetilcolina/farmacologia , Administração Oral , Regulação Alostérica , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Meia-Vida , Humanos , Camundongos , Oocistos/metabolismo , Ratos , Esquizofrenia/tratamento farmacológico , Esquizofrenia/patologia , Relação Estrutura-Atividade , Tiazóis/metabolismo , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
The prefrontal cortex (PFC) critical for higher cognition is implicated in neuropsychiatric diseases, such as Alzheimer's disease, depression and schizophrenia. The voltage-activated Kv7/KCNQ/M-channel or M-current modulates the neuronal excitability that defines the fundamental mechanism of brain function. However, whether M-current functions to regulate the excitability of PFC neurons remains elusive. In this study, we recorded the native M-current from PFC layer V pyramidal neurons in rat brain slices and showed that it modulated the intrinsic excitability and synaptic responses of PFC pyramidal neurons. Application of a specific M-channel blocker XE991 (40 µmol/L) or opener retigabine (10 µmol/L) resulted in inhibition or activation of M-current, respectively. In the current-clamp recordings, inhibition of M-current was evidenced by the increased average spike frequency and the reduced first inter-spike interval (ISI), spike onset latency and fast afterhyperpolarization (fAHP), whereas activation of M-current caused opposite responses. Furthermore, inhibition of M-current significantly increased the amplitude of excitatory postsynaptic potentials (EPSPs) and depolarized the resting membrane potential (RMP) without affecting the miniature EPSC (mEPSC) frequency. These data demonstrate that voltage-gated neuronal Kv7/KCNQ/M-current modulates the excitability and synaptic transmission of PFC neurons, suggesting that pharmacological modulation of M-current in the PFC may exert beneficial effects on cognitive deficits implicated in the pathophysiology of neuropsychiatric disorders.
Assuntos
Antracenos/farmacologia , Canais de Potássio KCNQ/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Antracenos/química , Relação Dose-Resposta a Droga , Canais de Potássio KCNQ/metabolismo , Masculino , Bloqueadores dos Canais de Potássio/química , Córtex Pré-Frontal/metabolismo , Células Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
AIM: Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca(2+)-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. METHODS: Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. RESULTS: Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 µmol/L. Br-IQ17B is selective over other subtypes such as α4ß2 and α3ß4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [(3)H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. CONCLUSION: We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.
Assuntos
Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Masculino , Técnicas de Cultura de Órgãos , Células PC12 , Ratos , Ratos Sprague-Dawley , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/fisiologiaRESUMO
In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Potássio/metabolismo , Canais de Potássio Shal/metabolismo , Motivos de Aminoácidos , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Transporte de Íons/fisiologia , Proteínas Interatuantes com Canais de Kv/genética , Estrutura Terciária de Proteína , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/genéticaRESUMO
GABAergic neurons in the medial amygdala (MeA) have been indicated in information processing in reproductive behavior and fear/anxiety. However, basic knowledge of their physiological and morphological properties is still very limited, probably due to the technical challenge to selectively record the GABAergic neurons. In this study, I characterized properties of the MeA GABAergic neurons by performing whole-cell patch clamp recordings from brain slices of adult knock-in mice selectively expressing green fluorescence protein (GFP) in GABAergic neurons. The majority (73%) of GABAergic neurons exhibiting low threshold calcium spike were classified as type I neurons, with morphological properties of being bitufted or stellate, and dendrites either aspiny or covered by various shapes of spines. Axonal collaterals of some neurons were observed near somata as well as in other amygdaloid nuclei. Neurons incapable of generate low threshold calcium spikes were divided into two types. Type II neurons (11%) exhibited hyperpolarization-activated sag and higher input resistance (>400 MΩ). Most Type II neurons exhibited asymmetric dendritic trees extending towards the superficial layer covered with long neck dendritic spines. The axons of type II neurons formed large collaterals and projected to other amygdaloid nuclei. Type III neurons (16%) lack prominent hyperpolarization-activated sag and possessed lower input resistance (<400 MΩ). These neurons were local interneurons with smooth multipolar dendritic trees. Since both MeA and nearby amygdaloid nuclei are involved in fear/anxiety processing, two types of MeA GABAergic projection neurons and a third type of interneurons that might participate in anxiety-related behavior were revealed by my present study.
Assuntos
Tonsila do Cerebelo/fisiologia , Forma Celular/fisiologia , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Potenciais de Ação/fisiologia , Tonsila do Cerebelo/citologia , Animais , Axônios/fisiologia , Cálcio/metabolismo , Dendritos/fisiologia , Estimulação Elétrica , Neurônios GABAérgicos/citologia , Interneurônios/citologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Transmissão Sináptica/fisiologiaRESUMO
The gut-derived orexigenic peptide hormone ghrelin enhances neuronal firing in the substantia nigra pars compacta, where dopaminergic neurons modulate the function of the nigrostriatal system for motor coordination. Here we describe a novel mechanism by which ghrelin enhances firing of nigral dopaminergic neurons by inhibiting voltage-gated potassium Kv7/KCNQ/M-channels through its receptor GHS-R1a and activation of the PLC-PKC pathway. Brain slice recordings of substantia nigra pars compacta neurons reveal that ghrelin inhibits native Kv7/KCNQ/M-currents. This effect is abolished by selective inhibitors of GHS-R1a, PLC and PKC. Transgenic suppression of native Kv7/KCNQ/M-channels in mice or channel blockade with XE991 abolishes ghrelin-induced hyperexcitability. In vivo, intracerebroventricular ghrelin administration causes increased dopamine release and turnover in the striatum. Microinjection of ghrelin or XE991 into substantia nigra pars compacta results in contralateral dystonic posturing, and attenuation of catalepsy elicited by systemic administration of the D2 receptor antagonist haloperidol. Our findings indicate that the ghrelin/KCNQ signalling is likely a common pathway utilized by the nervous system.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Grelina/farmacologia , Canais de Potássio KCNQ/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Catalepsia/metabolismo , Catalepsia/fisiopatologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Grelina/administração & dosagem , Haloperidol , Injeções Intraventriculares , Canais de Potássio KCNQ/metabolismo , Camundongos , Camundongos Transgênicos , Nistatina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Receptores de Grelina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Substância Negra/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
Honokiol, a major bioactive constituent of the bark of Magnolia officinalis has been confirmed to have the neuroprotective effect on ischemic stroke in rats. This study was designed to observe the therapeutic time window of honokiol microemulsion on cerebral ischemia-reperfusion injury to support its potential for future clinical trials and further explore the underlying mechanisms. Honokiol microemulsion (50µg/kg, i.v. at 0, 1 or 3h after reperfusion) significantly reduced neurological deficit, infarct volume and brain water content in rats subjected to cerebral ischemia-reperfusion, and honokiol (0.1-10µM) significantly attenuated oxygen-glucose deprivation- or glutamate-induced injury of fetal rat cortical neurons. In co-immunoprecipitation and western blot test, honokiol decreased the intensity of nNOS related to PSD95 but failed to affect that of PSD95 related to NR2B in NR2B-PSD95-nNOS complex, and it also inhibited the translocation of nNOS from cytosol to membrane without affecting total nNOS expression, and then markedly decreased NO production in cortical neurons. Besides, the results of whole-cell patch-clamp recordings showed that honokiol reversibly inhibited the NMDA current by about 64%. In conclusion, honokiol has a therapeutic window of at least 5h after the onset of cerebral ischemia or 3h after reperfusion in rats, which may be in part ascribed to the disruption of the PSD95-nNOS interaction leading to the inhibition of neurotoxic NO production.
Assuntos
Compostos de Bifenilo/uso terapêutico , Depressores do Sistema Nervoso Central/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lignanas/uso terapêutico , Proteínas de Membrana/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Bisbenzimidazol , Western Blotting , Hipóxia Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Corantes , Citosol/metabolismo , Proteína 4 Homóloga a Disks-Large , Glucose/deficiência , Ácido Glutâmico/toxicidade , Imunoprecipitação , Técnicas In Vitro , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , L-Lactato Desidrogenase/metabolismo , Masculino , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Translocação GenéticaRESUMO
BACKGROUND: Dysfunction of brain-gut interaction is thought to underlie visceral hypersensitivity which causes unexplained abdominal pain syndromes. However, the mechanism by which alteration of brain function in the brain-gut axis influences the perception of visceral pain remains largely elusive. In this study we investigated whether altered brain activity can generate visceral hyperalgesia. RESULTS: Using a forebrain specific αCaMKII promoter, we established a line of transgenic (Tg) mice expressing a dominant-negative pore mutant of the Kv7.2/KCNQ2 channel which suppresses native KCNQ/M-current and enhances forebrain neuronal excitability. Brain slice recording of hippocampal pyramidal neurons from these Tg mice confirmed the presence of hyperexcitable properties with increased firing. Behavioral evaluation of Tg mice exhibited increased sensitivity to visceral pain induced by intraperitoneal (i.p.) injection of either acetic acid or magnesium sulfate, and intracolon capsaicin stimulation, but not cutaneous sensation for thermal or inflammatory pain. Immunohistological staining showed increased c-Fos expression in the somatosensory SII cortex and insular cortex of Tg mice that were injected intraperitoneally with acetic acid. To mimic the effect of cortical hyperexcitability on visceral hyperalgesia, we injected KCNQ/M channel blocker XE991 into the lateral ventricle of wild type (WT) mice. Intracerebroventricular injection of XE991 resulted in increased writhes of WT mice induced by acetic acid, and this effect was reversed by co-injection of the channel opener retigabine. CONCLUSIONS: Our findings provide evidence that forebrain hyperexcitability confers visceral hyperalgesia, and suppression of central hyperexcitability by activation of KCNQ/M-channel function may provide a therapeutic potential for treatment of abdominal pain syndromes.
Assuntos
Hiperalgesia/metabolismo , Canal de Potássio KCNQ2/antagonistas & inibidores , Canal de Potássio KCNQ2/metabolismo , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Dor Visceral/metabolismo , Animais , Antracenos/farmacologia , Capsaicina/farmacologia , Carbamatos/farmacologia , Hiperalgesia/induzido quimicamente , Canal de Potássio KCNQ2/genética , Sulfato de Magnésio/farmacologia , Camundongos , Camundongos Transgênicos , Fenilenodiaminas/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/metabolismo , Dor Visceral/induzido quimicamenteRESUMO
The medial amygdala (MeA) is a critical center for processing pheromonal signals that regulate social and reproductive behaviors, but the fundamental cellular mechanisms underlying signal processing in the MeA have remained largely unknown. Some studies suggest that the MeA belongs to the striatum and provides inhibitory output to hypothalamic areas including the ventromedial hypothalamus (VMH). By combining tract tracing, genetic labeling of GABAergic neurons, and immunostaining against markers for glutamatergic synapses, we found that a majority of MeA neurons projecting to the VMH are glutamatergic. Whole cell patch-clamp recordings revealed that VMH-projecting neurons form a homogeneous population in terms of morphological and intrinsic properties. Nearly all cells possess I(h) and I(T) and in some cases they can give rise to postinhibitory rebound spikes. Morphological analysis of neurobiotin-filled cells revealed neurons with long dendritic arbors that extend to the MeA external layer and within the amygdala. Thus the VMH-projecting neurons in the MeA differ from the medium spiny neurons, the principal neurons of striatum, in terms of intrinsic physiological properties and morphology. In contrast, they resemble a subset of pyramidal cells in deep piriform cortex. Similar to pyramidal cells in piriform cortex, the VMH-projecting neurons in the MeA received direct excitatory input from their upstream sensory areas and inhibitory input from local GABAergic neurons. We conclude that pheromonal signals relayed to the VMH are processed by unique cortical, but not striatal, circuitry in the MeA.