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1.
Am J Physiol Heart Circ Physiol ; 321(3): H580-H591, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34355986

RESUMO

Tumor necrosis factor-α (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA) in cardiovascular disease models, but mechanisms are incompletely understood. As previously reported, bilateral PVN TNFα (0.6 pmol, 50 nL) induced acute ramping of splanchnic SNA (SSNA) that averaged +64 ± 7% after 60 min and +109 ± 17% after 120 min (P < 0.0001, n = 10). Given that TNFα can rapidly strengthen glutamatergic transmission, we hypothesized that progressive activation of ionotropic glutamate receptors is critically involved. When compared with that of vehicle (n = 5), prior blockade of PVN AMPA or NMDA receptors in anesthetized (urethane/α-chloralose) adult male Sprague-Dawley rats dose-dependently (ED50: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 2.48 nmol; D-(-)-2-amino-5-phosphonopentanoic acid (APV), 12.33 nmol), but incompletely (Emax: NBQX, 64%; APV, 41%), attenuated TNFα-induced SSNA ramping (n = 5/dose). By contrast, combined receptor blockade prevented ramping (1.3 ± 2.1%, P < 0.0001, n = 5). Whereas separate blockade of PVN AMPA or NMDA receptors (n = 5/group) had little effect on continued SSNA ramping when performed 60 min after TNFα injection, combined blockade (n = 5) or PVN inhibition with the GABA-A receptor agonist muscimol (n = 5) effectively stalled, without reversing, the SSNA ramp. Notably, PVN TNFα increased local TNFα immunofluorescence after 120, but not 60 min. Findings indicate that AMPA and NMDA receptors each contribute to SSNA ramping to PVN TNFα, and that their collective availability and ongoing activity are required to initiate and sustain the ramping response. We conclude that acute sympathetic activation by PVN TNFα involves progressive local glutamatergic excitation that recruits downstream neurons capable of maintaining heightened SSNA, but incapable of sustaining SSNA ramping.NEW & NOTEWORTHY The proinflammatory cytokine TNFα contributes to heightened SNA in cardiovascular disease models, but mechanisms remain obscure. Here, we demonstrate that TNFα injection into the hypothalamic PVN triggers SNA ramping by mechanisms dependent on local ionotropic glutamate receptor availability, but largely independent of TNFα autoinduction. Continued SNA ramping depends on ionotropic glutamate receptor and neuronal activity in PVN, indicating that strengthening and/or increased efficacy of glutamatergic transmission is necessary for acute sympathoexcitation by PVN TNFα.


Assuntos
Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Nervos Esplâncnicos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Antagonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Masculino , Muscimol/farmacologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Nervos Esplâncnicos/efeitos dos fármacos , Nervos Esplâncnicos/fisiologia
2.
Neuroscience ; 472: 116-127, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34384844

RESUMO

Understanding the different mechanisms associated with different anesthetic targeted receptors is critical towards identifying accurate long-term outcome measures as a result of early-life anesthetic exposure. We examined changes in GABAA receptor mediated neurotransmission by a predominately GABAA receptor targeted anesthetic, sevoflurane or a predominately NMDA receptor targeted anesthetic, ketamine. Postnatal day 7 male mice were exposed to sevoflurane or ketamine and examined as adults for changes in inhibitory neurotransmission and its associated change in induced seizure activity. Paired pulse stimulation experiment showed that early-life sevoflurane treated mice had significantly less hippocampal CA1 inhibition later in life. There was significantly increased CA1 excitatory output in the sevoflurane treated group compared to the no sevoflurane treated group after the GABA agonist muscimol. Similar to our previously established data for early-life sevoflurane, here we established early-life ketamine administration resulted in neurodevelopmental behavioral changes later in life. However, muscimol did not produce a significant difference on the excitatory CA1 output between early-life ketamine group and saline group. While sevoflurane treated mice showed significantly higher induced seizure intensities and shorter latency periods to reach seizure intensity stage 5 (Racine score) compared with no sevoflurane treated mice, this phenomenon was not observed in the ketamine vs. saline treated groups. Early-life sevoflurane, but not ketamine, exposure reduced GABAergic inhibition and enhanced seizure activity later in life. The results indicate that early-life exposure to different anesthetics lead to distinct long-term effects and their unique pathways require mechanistic studies to understand induced long-lasting changes in the brain.


Assuntos
Anestésicos Inalatórios , Ketamina , Animais , Encéfalo/metabolismo , Ketamina/toxicidade , Masculino , Camundongos , Receptores de GABA-A/metabolismo , Sevoflurano , Transmissão Sináptica
3.
Biomolecules ; 11(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34356653

RESUMO

Rosmarinic acid, a major component of rosemary, is a polyphenolic compound with potential neuroprotective effects. Asreducing the synaptic release of glutamate is crucial to achieving neuroprotectant's pharmacotherapeutic effects, the effect of rosmarinic acid on glutamate release was investigated in rat cerebrocortical nerve terminals (synaptosomes). Rosmarinic acid depressed the 4-aminopyridine (4-AP)-induced glutamate release in a concentration-dependent manner. The removal of extracellular calcium and the blockade of vesicular transporters prevented the inhibition of glutamate release by rosmarinic acid. Rosmarinic acid reduced 4-AP-induced intrasynaptosomal Ca2+ elevation. The inhibition of N-, P/Q-type Ca2+ channels and the calcium/calmodulin-dependent kinase II (CaMKII) prevented rosmarinic acid from having effects on glutamate release. Rosmarinic acid also reduced the 4-AP-induced activation of CaMKII and the subsequent phosphorylation of synapsin I, the main presynaptic target of CaMKII. In addition, immunocytochemistry confirmed the presence of GABAA receptors. GABAA receptor agonist and antagonist blocked the inhibitory effect of rosmarinic acid on 4-AP-evoked glutamate release. Docking data also revealed that rosmarinic acid formed a hydrogen bond with the amino acid residues of GABAA receptor. These results suggested that rosmarinic acid activates GABAA receptors in cerebrocortical synaptosomes to decrease Ca2+ influx and CaMKII/synapsin I pathway to inhibit the evoked glutamate release.


Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Ácido Glutâmico/metabolismo , Sinaptossomos/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cinamatos/química , Depsídeos/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Sinaptossomos/metabolismo
4.
Nat Commun ; 12(1): 4857, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381039

RESUMO

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord.


Assuntos
Locomoção , Neuroglia/metabolismo , Neurônios/metabolismo , Medula Espinal/crescimento & desenvolvimento , Animais , Interneurônios/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Condicionamento Físico Animal , Receptores Colinérgicos/metabolismo , Receptores de GABA-A/metabolismo , Recuperação de Função Fisiológica , Medula Espinal/citologia , Medula Espinal/fisiologia , Transmissão Sináptica , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismo
5.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437090

RESUMO

The ability to perceive and respond to environmental stimuli emerges in the absence of sensory experience. Spontaneous retinal activity prior to eye opening guides the refinement of retinotopy and eye-specific segregation in mammals, but its role in the development of higher-order visual response properties remains unclear. Here, we describe a transient window in neonatal mouse development during which the spatial propagation of spontaneous retinal waves resembles the optic flow pattern generated by forward self-motion. We show that wave directionality requires the same circuit components that form the adult direction-selective retinal circuit and that chronic disruption of wave directionality alters the development of direction-selective responses of superior colliculus neurons. These data demonstrate how the developing visual system patterns spontaneous activity to simulate ethologically relevant features of the external world and thereby instruct self-organization.


Assuntos
Fluxo Óptico , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Visão Ocular/fisiologia , Vias Visuais , Potenciais de Ação , Células Amácrinas/fisiologia , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Proteínas do Citoesqueleto/genética , Camundongos , Movimento (Física) , Mutação , Piridazinas/farmacologia , Receptores de GABA-A/metabolismo , Retina/crescimento & desenvolvimento , Análise Espaço-Temporal , Colículos Superiores/fisiologia
6.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361022

RESUMO

Gamma-aminobutyric acid (GABA) is considered the primary inhibitory neurotransmitter in the human cortex. However, whether GABA regulates melanogenesis has not been comprehensively elucidated. In this study, we reveal that GABA (20 mM) significantly inhibited α-melanocyte-stimulating hormone (α-MSH)-induced extracellular (from 354.9% ± 28.4% to 126.5% ± 16.0%) and intracellular melanin contents (from 236.7% ± 11.1% to 102.7% ± 23.1%) in B16F10 melanoma cells, without inducing cytotoxicity. In addition, α-MSH-induced hyperpigmentation in zebrafish larvae was inhibited from 246.3% ± 5.4% to 116.3% ± 3.1% at 40 mM GABA, displaying no apparent cardiotoxicity. We also clarify that the GABA-mediated antimelanogenic properties were related to the direct inhibition of microphthalmia-associated transcription factor (MITF) and tyrosinase expression by inhibiting cyclic adenosine monophosphate (cAMP) and cAMP response element-binding protein (CREB). Furthermore, under α-MSH stimulation, GABA-related antimelanogenic effects were mediated through the GABAA and GABAB receptors, with subsequent inhibition of Ca2+ accumulation. In B16F10 melanoma cells and zebrafish larvae, pretreatment with bicuculline, a GABAA receptor antagonist, and CGP 46381, a GABAB receptor antagonist, reversed the antimelanogenic effect of GABA following α-MSH treatment by upregulating Ca2+ accumulation. In conclusion, our results indicate that GABA inhibits α-MSH-induced melanogenesis. Hence, in addition to the health benefits of GABA in the central nervous system, it could ameliorate hyperpigmentation disorders.


Assuntos
Melaninas/biossíntese , Receptores de GABA-B/metabolismo , alfa-MSH/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Receptores de GABA-A/metabolismo , Peixe-Zebra
7.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281185

RESUMO

Our recent work on genetic epilepsy (GE) has identified common mechanisms between GE and neurodegenerative diseases including Alzheimer's disease (AD). Although both disorders are seemingly unrelated and occur at opposite ends of the age spectrum, it is likely there are shared mechanisms and studies on GE could provide unique insights into AD pathogenesis. Neurodegenerative diseases are typically late-onset disorders, but the underlying pathology may have already occurred long before the clinical symptoms emerge. Pathophysiology in the early phase of these diseases is understudied but critical for developing mechanism-based treatment. In AD, increased seizure susceptibility and silent epileptiform activity due to disrupted excitatory/inhibitory (E/I) balance has been identified much earlier than cognition deficit. Increased epileptiform activity is likely a main pathology in the early phase that directly contributes to impaired cognition. It is an enormous challenge to model the early phase of pathology with conventional AD mouse models due to the chronic disease course, let alone the complex interplay between subclinical nonconvulsive epileptiform activity, AD pathology, and cognition deficit. We have extensively studied GE, especially with gene mutations that affect the GABA pathway such as mutations in GABAA receptors and GABA transporter 1. We believe that some mouse models developed for studying GE and insights gained from GE could provide unique opportunity to understand AD. These include the pathology in early phase of AD, endoplasmic reticulum (ER) stress, and E/I imbalance as well as the contribution to cognitive deficit. In this review, we will focus on the overlapping mechanisms between GE and AD, the insights from mutations affecting GABAA receptors, and GABA transporter 1. We will detail mechanisms of E/I imbalance and the toxic epileptiform generation in AD, and the complex interplay between ER stress, impaired membrane protein trafficking, and synaptic physiology in both GE and AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Humanos , Camundongos , Receptores de GABA-A/metabolismo , Convulsões/genética , Convulsões/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo
8.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202516

RESUMO

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1ß and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.


Assuntos
Hiperamonemia/complicações , Hiperamonemia/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Imuno-Histoquímica , Inflamação/patologia , Modelos Biológicos , Doenças do Sistema Nervoso/patologia , Transporte Proteico , Ratos , Transdução de Sinais
9.
Molecules ; 26(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205930

RESUMO

BACKGROUND: Neurotic disturbances, anxiety, neurosis-like disorders, and stress situations are widespread. Benzodiazepine tranquillizers have been found to be among the most effective antianxiety drugs. The pharmacological action of benzodiazepines is due to their interaction with the supra-molecular membrane GABA-a-benzodiazepine receptor complex, linked to the Cl-ionophore. Benzodiazepines enhance GABA-ergic transmission and this has led to a study of the role of GABA in anxiety. The search for anxiolytics and anticonvulsive agents has involved glutamate-ergic, 5HT-ergic substances and neuropeptides. However, each of these well-known anxiolytics, anticonvulsants and cognition enhancers (nootropics) has repeatedly been reported to have many adverse side effects, therefore there is an urgent need to search for new drugs able to restore damaged cognitive functions without causing significant adverse reactions. OBJECTIVE: Considering the relevance of epilepsy diffusion in the world, we have addressed our attention to the discovery of new drugs in this field Thus our aim is the synthesis and study of new compounds with antiepileptic (anticonvulsant) and not only, activity. METHODS: For the synthesis of compounds classical organic methods were used and developed. For the evaluation of biological activity some anticonvulsant and psychotropic methods were used. RESULTS: As a result of multistep reactions 26 new, five-membered heterocyclic systems were obtained. PASS prediction of anticonvulsant activity was performed for the whole set of the designed molecules and probability to be active Pa values were ranging from 0.275 to 0.43. The studied compounds exhibit protection against pentylenetetrazole (PTZ) seizures, anti-thiosemicarbazides effect as well as some psychotropic effect. The biological assays evidenced that some of the studied compounds showed a high anticonvulsant activity by antagonism with pentylenetetrazole. The toxicity of compounds is low and they do not induce muscle relaxation in the studied doses. According to the study of psychotropic activity it was found that the selected compounds have an activating behavior and anxiolytic effects on the models of "open field" and "elevated plus maze" (EPM). The data obtained indicate the anxiolytic (anti-anxiety) activity of the derivatives of pyrimidines, especially pronounced in compounds 6n, 6b, and 7c. The studied compounds increase the latent time of first immobilization on the model of "forced swimming" (FST) and exhibit some antidepressant effect similarly to diazepam. Docking studies revealed that compound 6k bound tightly in the active site of GABAA receptor with a value of the scoring function that estimates free energy of binding (ΔG) at -7.95 kcal/mol, while compound 6n showed the best docking score and seems to be dual inhibitor of SERT transporter as well as 5-HT1A receptor. CONCLUSIONS: Тhe selected compounds have an anticonvulsant, activating behavior and anxiolytic effects, at the same time exhibit some antidepressant effect.


Assuntos
Azepinas/administração & dosagem , Azepinas/síntese química , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Convulsões/tratamento farmacológico , Animais , Ansiolíticos/administração & dosagem , Ansiolíticos/síntese química , Ansiolíticos/química , Ansiolíticos/farmacologia , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Azepinas/química , Azepinas/farmacologia , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Pentilenotetrazol/efeitos adversos , Pirimidinas/química , Pirimidinas/farmacologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Convulsões/induzido quimicamente , Convulsões/fisiopatologia
10.
Neuroimage Clin ; 31: 102701, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34090124

RESUMO

Neuroinflammation is a key component of epileptogenesis, the process leading to acquired epilepsy. In recent years, with the development of non-invasive in vivo positron emission tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of neuroinflammation, it has become possible to perform longitudinal studies to characterize neuroinflammation at different disease stages in animal models of epileptogenesis. This study aimed to utilize the prognostic capability of TSPO PET imaging at disease onset (2 weeks post-SE) to categorize epileptic rats with distinct seizure burden based on TSPO levels at disease onset and investigate their association to TSPO expression at the chronic epilepsy stage. Controls (n = 14) and kainic acid-induced status epilepticus (KASE) rats (n = 41) were scanned non-invasively with [18F]PBR111 PET imaging measuring TSPO expression. Animals were monitored using video-electroencephalography (vEEG) up to chronic disease (12 weeks post-SE), at which TSPO levels ([3H]PK11195) as well as other post-mortem abnormalities (namely synaptic density ([3H]UCB-J), neuronal loss (NeuN), and neurodegeneration (FjC)) were investigated. By applying multivariate analysis, TSPO PET imaging at disease onset identified three KASE groups with significantly different spontaneous recurrent seizures (SRS) burden (defined as rare SRS, sporadic SRS, and frequent SRS) (p = 0.003). Interestingly, TSPO levels were significantly different when comparing the three KASE groups (p < 0.0001), with the frequent SRS group characterized only by a limited focal TSPO increase at disease onset. On the contrary, TSPO measured during chronic epilepsy was found to be the highest in the frequent SRS group and correlated with seizure burden (r = 0.826, p < 0.0001). Importantly, early and chronic TSPO levels did not correlate (r = -0.05). Finally, significant pathological changes in neuronal loss, synaptic density, and neurodegeneration were found not only when compared to control animals (p < 0.01), but also between the three KASE rat categories in the hippocampus (p < 0.05). Early and chronic TSPO upregulation following epileptogenic insult appear to be driven by two superimposed dynamic processes. The former is associated with epileptogenesis as measured at disease onset, while the latter is related to seizure frequency as quantified during chronic epilepsy.


Assuntos
Epilepsia do Lobo Temporal , Receptores de GABA-A/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/diagnóstico por imagem , Epilepsia do Lobo Temporal/metabolismo , Imagem Molecular , Fenótipo , Tomografia por Emissão de Pósitrons , Ratos , Regulação para Cima
11.
Biomolecules ; 11(6)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074021

RESUMO

Epipregnanolone (3ß-hydroxy-5ß-pregnan-20-one, Epi) is an endogenous steroid with important physiological effects and high affinity for GABAA receptors. The effect of Epi on GABA-induced chloride current (IGABA) in native neurons has hardly been studied. In this work, we studied the influence of Epi on the IGABA in the Purkinje cells of rat cerebellum and pyramidal neurons of rat hippocampus with the patch clamp technique. We showed that Epi is a positive modulator of the IGABA with EC50 of 5.7 µM in Purkinje cells and 9.3 µM in hippocampal neurons. Epi-induced potentiation of the IGABA was more potent at low vs. high GABA concentrations. Isopregnanolone (3ß-hydroxy-5α-pregnan-20-one, Iso) counteracted Epi, reducing its potentiating effect by 2-2.3 times. Flumazenil, a nonsteroidal GABAA receptor antagonist, does not affect the Epi-induced potentiation. Comparison of the potentiating effects of Epi and allopregnanolone (3α-hydroxy-5α-pregnan-20-one, ALLO) showed that ALLO is, at least, a four times more potent positive modulator than Epi. The combined application of ALLO and Epi showed that the effects of these two steroids are not additive. We conclude that Epi has a dual effect on the IGABA increasing the current in the control solution and decreasing the stimulatory effect of ALLO.


Assuntos
Cerebelo/metabolismo , Agonistas de Receptores de GABA-A/farmacologia , Hipocampo/metabolismo , Pregnanolona/farmacologia , Células Piramidais/metabolismo , Receptores de GABA-A/metabolismo , Animais , Ratos , Ratos Wistar
12.
Laryngoscope ; 131(10): 2332-2340, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34156095

RESUMO

OBJECTIVES/HYPOTHESIS: Tinnitus can develop due to, or be aggravated by, stress in a rat model. To investigate stress as a possible causal factor in the development of tinnitus, we designed an animal study that included tinnitus behavior and excitatory/inhibitory neurotransmitter expression after noise exposure as well as restraint stress. STUDY DESIGN: An experimental animal study. METHODS: Wistar rats were grouped according to single or double exposure to noise and restraint stress. The noise exposure (NE) group was subjected to 110 dB sound pressure level (SPL) of 16 kHz narrow-band noise (NBN) for 1 hour, and the restraint stress (RS) group was restrained for 1 hour with or without noise exposure. Gap prepulse inhibition of the acoustic startle (GPIAS) reflex was measured at an NBN of 16 kHz to investigate tinnitus development. Various immunohistopathologic and molecular biologic studies were undertaken to evaluate possible mechanisms of tinnitus development after noise and/or restraint stress. RESULTS: The RS-only group showed a reduced GPIAS response, which is a reliable sign of tinnitus development. In the double-stimulus groups, more tinnitus-development signs of reduced GPIAS responses were observed. The expression of γ-aminobutyric acid A receptor α1 (GABAAR α1) in the hippocampus decreased in the NE│RS group. Increased N-methyl-d-aspartate receptor1 intensities in the NE│RS group and decreased GABAAR α1 intensities in the RS and NE│RS groups were observed in the CA3 region of the hippocampus. CONCLUSIONS: Tinnitus appeared to develop after stress alone in this animal study. An imbalance in excitatory and inhibitory neurotransmitters in the hippocampus may be related to the development of tinnitus after acute NE and/or stress. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2332-2340, 2021.


Assuntos
Região CA3 Hipocampal/patologia , Ruído/efeitos adversos , Estresse Psicológico/complicações , Zumbido/etiologia , Estimulação Acústica/efeitos adversos , Estimulação Acústica/métodos , Animais , Modelos Animais de Doenças , Humanos , Masculino , Ratos , Receptores de GABA-A/análise , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/análise , Receptores de N-Metil-D-Aspartato/metabolismo , Reflexo de Sobressalto , Estresse Psicológico/psicologia , Zumbido/diagnóstico , Zumbido/patologia , Zumbido/psicologia
13.
eNeuro ; 8(4)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34083383

RESUMO

Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABA type-A receptors (GABAARs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3-, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knock-out (KO) mice results in absence of gephyrin and gephyrin-dependent GABAARs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3- in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3- in hippocampal pyramidal cells or hippocampal interneurons. The results show that: (1) the accumulation of gephyrin and GABAARs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3- overexpression; (2) overexpression of CBSH3- in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission; and (3) these enhanced inhibitory synapses provide protection against pentylenetetrazole (PTZ)-induced seizures. The results indicate that this AAV vector carrying CBSH3- can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain.


Assuntos
Proteínas de Transporte , Pentilenotetrazol , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Pentilenotetrazol/toxicidade , Células Piramidais/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Convulsões/induzido quimicamente , Sinapses/metabolismo , Transmissão Sináptica
14.
Acta Biochim Biophys Sin (Shanghai) ; 53(8): 1076-1087, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34137445

RESUMO

Propofol is the most commonly used intravenous anesthetic worldwide. It can induce loss of consciousness prior to the occurrence of severe respiratory suppression, which is also a pharmacodynamic feature of all general anesthetics. However, the neural mechanisms underlying this natural phenomenon are controversial and highly related to patient safety. In the present study, we demonstrated that the pharmacodynamic effects of propofol (50 and 100 µM) on suppression of consciousness-related excitatory postsynaptic currents in the medial prefrontal cortex (mPFC) and centromedian nucleus of the thalamus (CMT) were lower than those in the kernel respiratory rhythmogenesis nucleus pre-Bötzinger complex (PrBo). Furthermore, we unexpectedly found that the GABAA receptor ß3 subunit is the key target for propofol's action and that it is mutually and exclusively expressed in GABAergic neurons. It is also more abundant in the mPFC and CMT, but mainly co-localized with GABAergic neurons in the PrBo. As a result, the differentiated expression pattern should mediate more neuron suppression through the activation of GABAergic neurons in the mPFC and CMT at low doses of propofol (50 µM). However, PrBo GABAergic neurons were only activated by propofol at a high dose (100 µM). These results highlight the detailed pharmacodynamic effects of propofol on consciousness-related and respiration-related nuclei and provide the distinct interaction mechanism between the ß3 subunit and GABAergic neurons in mediating the suppression of consciousness compared to the inhibition of respiration.


Assuntos
Neurônios GABAérgicos/metabolismo , Núcleos Intralaminares do Tálamo , Córtex Pré-Frontal , Propofol/farmacologia , Receptores de GABA-A/metabolismo , Mecânica Respiratória/efeitos dos fármacos , Inconsciência , Animais , Núcleos Intralaminares do Tálamo/metabolismo , Núcleos Intralaminares do Tálamo/fisiopatologia , Masculino , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Ratos , Ratos Sprague-Dawley , Inconsciência/induzido quimicamente , Inconsciência/metabolismo , Inconsciência/fisiopatologia
15.
ACS Chem Neurosci ; 12(13): 2421-2436, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34101432

RESUMO

GABA type A receptors (GABAARs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABAARs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABAAR gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABAAR desensitization gate is located at the second and third transmembrane segment. However, studies of our and others' groups indicated that other parts of the GABAAR macromolecule might be involved in this process. In the present study, we asked how selected point mutations (ß2G254V, α1G258V, α1L300V, and ß2L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α1ß2γ2 GABAAR. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABAAR.


Assuntos
Ativação do Canal Iônico , Canais Iônicos de Abertura Ativada por Ligante , Animais , Humanos , Canais Iônicos de Abertura Ativada por Ligante/genética , Mutação/genética , Técnicas de Patch-Clamp , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico
16.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064454

RESUMO

The γ-aminobutyric acid type A receptor (GABAAR) plays a major role in fast inhibitory synaptic transmission and is highly regulated by the neuromodulator dopamine. In this aspect, most of the attention has been focused on the classical intracellular signaling cascades following dopamine G-protein-coupled receptor activation. Interestingly, the GABAAR and dopamine D5 receptor (D5R) have been shown to physically interact in the hippocampus, but whether a functional cross-talk occurs is still debated. In the present study, we use a combination of imaging and single nanoparticle tracking in live hippocampal neurons to provide evidence that GABAARs and D5Rs form dynamic surface clusters. Disrupting the GABAAR-D5R interaction with a competing peptide leads to an increase in the diffusion coefficient and the explored area of both receptors, and a drop in immobile synaptic GABAARs. By means of patch-clamp recordings, we show that this fast lateral redistribution of surface GABAARs correlates with a robust depression in the evoked GABAergic currents. Strikingly, it also shifts in time the expression of long-term potentiation at glutamatergic synapses. Together, our data both set the plasma membrane as the primary stage of a functional interplay between GABAAR and D5R, and uncover a non-canonical role in regulating synaptic transmission.


Assuntos
Potenciação de Longa Duração/genética , Neurônios/metabolismo , Receptor Cross-Talk , Receptores de Dopamina D5/genética , Receptores de GABA-A/genética , Transmissão Sináptica/genética , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/citologia , Técnicas de Patch-Clamp , Peptídeos/síntese química , Peptídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D5/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/genética , Sinapses/metabolismo
17.
Cell Mol Life Sci ; 78(13): 5341-5370, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34061215

RESUMO

GABAA receptors are ligand-gated chloride channels and ionotropic receptors of GABA, the main inhibitory neurotransmitter in vertebrates. In this review, we discuss the major and diverse roles GABAA receptors play in the regulation of neuronal communication and the functioning of the brain. GABAA receptors have complex electrophysiological properties that enable them to mediate different types of currents such as phasic and tonic inhibitory currents. Their activity is finely regulated by membrane voltage, phosphorylation and several ions. GABAA receptors are pentameric and are assembled from a diverse set of subunits. They are subdivided into numerous subtypes, which differ widely in expression patterns, distribution and electrical activity. Substantial variations in macroscopic neural behavior can emerge from minor differences in structure and molecular activity between subtypes. Therefore, the diversity of GABAA receptors widens the neuronal repertoire of responses to external signals and contributes to shaping the electrical activity of neurons and other cell types.


Assuntos
Eletrofisiologia , Neurônios/fisiologia , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Humanos , Neurônios/citologia
18.
Biomed Pharmacother ; 140: 111746, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062412

RESUMO

BACKGROUND/AIMS: Asthma is a common chronic respiratory disease. It has been reported that Pingchuan formula (PCF) can control asthma attacks by reducing airway inflammation, muscle spasm and mucus secretion. However, PCF's mechanism for reducing airway mucus hypersecretion remains unclear. This study aimed to investigate the effect of PCF on airway mucus secretion in asthmatic mice and to explore changes in the PNEC-GABA-IL13-Muc5ac axis. METHODS: Male Babl/c mice were used to establish the asthma model via sensitisation with OVA. Mice were randomly divided into Normal, OVA, DEX, and PCF groups. After treatment, lung histopathology was observed with H&E and PAS staining. BALF levels of IL-5 and IL-13 were detected using ELISA. The levels of mRNA and protein expression for GAD1, GABAARß1, GABAARα1 and Muc5ac in the lung tissue were measured by RT-PCR and Western blot assays. PNECs were observed with AgNOR staining. RESULTS: PCF treatment effectively reduced goblet cell (P < 0.01) and PNEC (P < 0.05) proliferation, lung tissue inflammation and airway mucus hypersecretion. In addition, PCF also markedly downregulated mRNA and protein expression of GAD1, GABAARß1, GABAARα1 and Muc5ac (P < 0.05, compared with OVA), thus inhibiting the GABA-IL-13 pathway in the lung tissue of asthmatic mice. CONCLUSION: These findings suggest that PCF controls asthma attacks by reducing airway inflammation and mucus hypersecretion via the PNEC-GABA-IL13-Muc5ac axis.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Animais , Antiasmáticos/farmacologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Proliferação de Células/efeitos dos fármacos , Citocinas/imunologia , Medicamentos de Ervas Chinesas/farmacologia , Células Caliciformes/efeitos dos fármacos , Interleucina-13/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , Mucina-5AC/metabolismo , Muco/metabolismo , Células Neuroendócrinas/efeitos dos fármacos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo
19.
Mol Pharmacol ; 100(1): 19-31, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958479

RESUMO

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABAA receptor. The sites are located in the membrane-spanning domains of the receptor at the ß-α subunit interface (site I) and within the α (site II) and ß subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1ß2γ2L GABAA receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.


Assuntos
Substituição de Aminoácidos , Pregnanolona/farmacologia , Receptores de GABA-A/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Pregnanolona/química , Ratos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo
20.
Mol Pharmacol ; 100(1): 73-82, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33958481

RESUMO

Communication between neuronal cells, which is central to brain function, is performed by several classes of ligand-gated ionotropic receptors. The gold-standard technique for measuring rapid receptor response to agonist is manual patch-clamp electrophysiology, capable of the highest temporal resolution of any current electrophysiology technique. We report an automated high-precision patch-clamp system that substantially improves the throughput of these time-consuming pharmacological experiments. The patcherBotPharma enables recording from cells expressing receptors of interest and manipulation of them to enable millisecond solution exchange to activate ligand-gated ionotropic receptors. The solution-handling control allows for autonomous pharmacological concentration-response experimentation on adherent cells, lifted cells, or excised outside-out patches. The system can perform typical ligand-gated ionotropic receptor experimentation protocols autonomously, possessing a high success rate in completing experiments and up to a 10-fold reduction in research effort over the duration of the experiment. Using it, we could rapidly replicate previous data sets, reducing the time it took to produce an eight-point concentration-response curve of the effect of propofol on GABA type A receptor deactivation from likely weeks of recording to ∼13 hours of recording. On average, the rate of data collection of the patcherBotPharma was a data point every 2.1 minutes that the operator spent interacting with the patcherBotPharma The patcherBotPharma provides the ability to conduct complex and comprehensive experimentation that yields data sets not normally within reach of conventional systems that rely on constant human control. This technical advance can contribute to accelerating the examination of the complex function of ion channels and the pharmacological agents that act on them. SIGNIFICANCE STATEMENT: This work presents an automated intracellular pharmacological electrophysiology robot, patcherBotPharma, that substantially improves throughput and reduces human time requirement in pharmacological patch-clamp experiments. The robotic system includes millisecond fluid exchange handling and can perform highly efficient ligand-gated ionotropic receptor experiments. The patcherBotPharma is built using a conventional patch-clamp rig, and the technical advances shown in this work greatly accelerate the ability to conduct high-fidelity pharmacological electrophysiology.


Assuntos
Neurônios/citologia , Técnicas de Patch-Clamp/instrumentação , Receptores de GABA-A/metabolismo , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Cultura Primária de Células , Ratos , Robótica
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