RESUMO
Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.
Assuntos
Alcoolismo , Síndrome de Abstinência a Substâncias , Camundongos , Animais , Diazepam/farmacologia , Receptores de GABA-A/metabolismo , Benzodiazepinas/farmacologia , Encéfalo/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/farmacologia , Transmissão SinápticaRESUMO
Although it is well documented that heavy consumption of alcohol during pregnancy impairs brain development, it remains controversial whether moderate consumption causes significant damage. Using a limited access, voluntary consumption paradigm, we recently demonstrated that moderate prenatal alcohol exposure (MPAE) is associated with dentate gyrus-dependent learning and memory deficits that are manifested in adulthood. Here, we identified a novel mechanism that may underlie this effect of MPAE. We found that MPAE mice exhibit deficits in NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in the dentate gyrus. Further, using semiquantitative immunoblotting techniques, we found that the levels of GluN2B subunits were decreased in the synaptic membrane, while levels of C2'-containing GluN1 and GluN3A subunits were increased, in the dentate gyrus of MPAE mice. These data suggest that MPAE alters the subunit composition of synaptic NMDARs, leading to impaired NMDAR-dependent LTP in the dentate gyrus.
Assuntos
Giro Denteado/efeitos dos fármacos , Etanol/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Giro Denteado/metabolismo , Feminino , Camundongos , GravidezRESUMO
BACKGROUND: It has been estimated that approximately 12% of women consume alcohol at some time during their pregnancy, and as many as 5% of children born in the United States are impacted by prenatal alcohol exposure (PAE). The range of physical, behavioral, emotional, and social dysfunctions that are associated with PAE are collectively termed fetal alcohol spectrum disorder (FASD). METHODS: Using a saccharin-sweetened ethanol solution, we developed a limited access model of PAE. C57BL/6J mice were provided access to a solution of either 10% (w/v) ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin (control) for 4 h/d. After establishing consistent drinking, mice were mated and continued drinking during gestation. Following parturition, solutions were decreased to 0% in a stepwise fashion over a period of 6 days. Characterization of the model included measurements of maternal consumption patterns, blood ethanol levels, litter size, pup weight, maternal care, and the effects of PAE on fear-conditioned and spatial learning, and locomotor activity. RESULTS: Mothers had mean daily ethanol intake of 7.17 ± 0.17 g ethanol/kg body weight per day, with average blood ethanol concentrations of 68.5 ± 9.2 mg/dl after 2 hours of drinking and 88.3 ± 11.5 mg/dl after 4 hours of drinking. Food and water consumption, maternal weight gain, litter size, pup weight, pup retrieval times, and time on nest did not differ between the alcohol-exposed and control animals. Compared with control offspring, mice that were exposed to ethanol prenatally displayed no difference in spontaneous locomotor activity but demonstrated learning deficits in 3 hippocampal-dependent tasks: delay fear conditioning, trace fear conditioning, and the delay nonmatch to place radial-arm maze task. CONCLUSIONS: These results indicate that this model appropriately mimics the human condition of PAE and will be a useful tool in studying the learning deficits seen in FASD.
Assuntos
Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/psicologia , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/psicologia , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Modelos Animais de Doenças , Etanol/sangue , Feminino , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Masculino , Comportamento Materno/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , GravidezRESUMO
α5 subunit GABA type A receptor (GABAAR) preferring negative allosteric modulators (NAMs) are cognitive enhancers with antidepressant-like effects. α5-NAM success in treating mouse models of neurodevelopmental disorders with excessive inhibition have led to Phase 2 clinical trials for Down syndrome. Despite in vivo efficacy, no study has examined the effects of continued α5-NAM treatment on inhibitory and excitatory synapse plasticity to identify mechanisms of action. Here we used L-655,708, an imidazobenzodiazepine that acts as a highly selective but weak α5-NAM, to investigate the impact of sustained treatment on hippocampal neuron synapse and dendrite development. We show that 2-day pharmacological reduction of α5-GABAAR signaling from DIV12-14, when GABAARs contribute to depolarization, delays dendritic spine maturation and the NMDA receptor (NMDAR) GluN2B/GluN2A developmental shift. In contrast, α5-NAM treatment from DIV19-21, when hyperpolarizing GABAAR signaling predominates, enhances surface synaptic GluN2A while decreasing GluN2B. Despite changes in NMDAR subtype surface levels and localization, total levels of key excitatory synapse proteins were largely unchanged, and mEPSCs were unaltered. Importantly, 2-day α5-NAM treatment does not alter the total surface levels or distribution of α5-GABAARs, reduce the gephyrin inhibitory synaptic scaffold, or impair phasic or tonic inhibition. Furthermore, α5-NAM inhibition of the GABAAR tonic current in mature neurons is maintained after 2-day α5-NAM treatment, suggesting reduced tolerance liability, in contrast to other clinically relevant GABAAR-targeting drugs such as benzodiazepines. Together, these results show that α5-GABAARs contribute to dendritic spine maturation and excitatory synapse development via a NMDAR dependent mechanism without perturbing overall neuronal excitability.
Assuntos
GABAérgicos/farmacologia , Imidazóis/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia , Animais , Dendritos/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
γ-aminobutyric acid (GABA) begins as the key excitatory neurotransmitter in newly forming circuits, with chloride efflux from GABA type A receptors (GABAARs) producing membrane depolarization, which promotes calcium entry, dendritic outgrowth and synaptogenesis. As development proceeds, GABAergic signaling switches to inhibitory hyperpolarizing neurotransmission. Despite the evidence of impaired GABAergic neurotransmission in neurodevelopmental disorders, little is understood on how agonist-dependent GABAAR activation controls the formation and plasticity of GABAergic synapses. We have identified a weakly depolarizing and inhibitory GABAAR response in cortical neurons that occurs during the transition period from GABAAR depolarizing excitation to hyperpolarizing inhibitory activity. We show here that treatment with the GABAAR agonist muscimol mediates structural changes that diminish GABAergic synapse strength through postsynaptic and presynaptic plasticity via intracellular Ca2+ stores, ERK and BDNF/TrkB signaling. Muscimol decreases synaptic localization of surface γ2 GABAARs and gephyrin postsynaptic scaffold while ß2/3 non-γ2 GABAARs accumulate in the synapse. Concurrent with this structural plasticity, muscimol treatment decreases synaptic currents while enhancing the γ2 containing benzodiazepine sensitive GABAAR tonic current in an ERK dependent manner. We further demonstrate that GABAAR activation leads to a decrease in presynaptic GAD65 levels via BDNF/TrkB signaling. Together these data reveal a novel mechanism for agonist induced GABAergic synapse plasticity that can occur on the timescale of minutes, contributing to rapid modification of synaptic and circuit function.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/fisiologia , Animais , Azepinas/farmacologia , Benzamidas/farmacologia , Butadienos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Córtex Cerebral/citologia , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , GABAérgicos/farmacologia , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/farmacologia , Muscimol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nimodipina/farmacologia , Nitrilas/farmacologia , Técnicas de Patch-Clamp , Ratos , Sinapses/efeitos dos fármacosRESUMO
GABAA receptor subunit composition is a critical determinant of receptor localization and physiology, with synaptic receptors generating phasic inhibition and extrasynaptic receptors producing tonic inhibition. Extrasynaptically localized α5 GABAA receptors are largely responsible for tonic inhibition in hippocampal neurons. However, we show here that inhibitory synapses also contain a constant level of α5 GABAA receptors throughout neuronal development, as measured by its colocalization with gephyrin, the inhibitory postsynaptic scaffolding protein. Immunoprecipitation of the α5 subunit from both cultured neurons and adult rat brain coimmunoprecipitated gephyrin, confirming this interaction in vivo. Furthermore, the α5 subunit can interact with gephyrin independent of other synaptically localized alpha subunits, as shown by immunoprecipitation experiments in HEK cells. By replacing the α5 predicted gephyrin binding domain (Residues 370-385) with either the high affinity gephyrin binding domain of the α2 subunit or homologous residues from the extrasynaptic α4 subunit that does not interact with gephyrin, α5 GABAA receptor localization shifted into or out of the synapse, respectively. These shifts in the ratio of synaptic/extrasynaptic α5 localization disrupted dendritic outgrowth and spine maturation. In contrast to the predominant view of α5 GABAA receptors being extrasynaptic and modulating tonic inhibition, we identify an intimate association of the α5 subunit with gephyrin, resulting in constant synaptic levels of α5 GABAA R throughout circuit formation that regulates neuronal development.
Assuntos
Dendritos/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/fisiologia , Western Blotting , Proteínas de Transporte/metabolismo , Crescimento Celular , Imunofluorescência , Células HEK293 , Hipocampo/embriologia , Hipocampo/fisiologia , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Inibição Neural/fisiologia , Receptores de GABA-A/genética , TransfecçãoRESUMO
It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.