RESUMO
Mild traumatic brain injury (mTBI) can cause severe long-term cognitive and emotional deficits, including impaired memory, depression, and persevering fear, but the neuropathological basis of these deficits is uncertain. As medial prefrontal cortex (mPFC) and hippocampus play important roles in memory and emotion, we used multi-site, multi-electrode recordings of oscillatory neuronal activity in local field potentials (LFPs) in awake, head-fixed mice to determine if the functioning of these regions was abnormal after mTBI, using a closed-skull focal cranial blast model. We evaluated mPFC, hippocampus CA1, and primary somatosensory/visual cortical areas (S1/V1). Although mTBI did not alter the power of oscillations, it did cause increased coherence of θ (4-10 Hz) and ß (10-30 Hz) oscillations within mPFC and S1/V1, reduced CA1 sharp-wave ripple (SWR)-evoked LFP activity in mPFC, downshifted SWR frequencies in CA1, and enhanced θ-γ phase-amplitude coupling (PAC) within mPFC. These abnormalities might be linked to the impaired memory, depression, and persevering fear seen after mTBI. Treatment with the cannabinoid type-2 (CB2) receptor inverse agonist SMM-189 has been shown to mitigate functional deficits and neuronal injury after mTBI in mice. We found that SMM-189 also reversed most of the observed neurophysiological abnormalities. This neurophysiological rescue is likely to stem from the previously reported reduction in neuron loss and/or the preservation of neuronal function and connectivity resulting from SMM-189 treatment, which appears to stem from the biasing of microglia from the proinflammatory M1 state to the prohealing M2 state by SMM-189.
Assuntos
Benzofenonas/uso terapêutico , Concussão Encefálica/tratamento farmacológico , Concussão Encefálica/patologia , Encéfalo/efeitos dos fármacos , Agonistas de Receptores de Canabinoides/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Animais , Encéfalo/patologia , Mapeamento Encefálico , Ondas Encefálicas/efeitos dos fármacos , Modelos Animais de Doenças , Eletroencefalografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Análise de Componente Principal , Receptor CB2 de Canabinoide/metabolismo , Fatores de TempoRESUMO
Recent findings indicate a high level of specialization at the level of microcircuits and cell populations within brain structures with regards to the control of fear and anxiety. The hippocampus, however, has been treated as a unitary structure in anxiety and fear research despite mounting evidence that different hippocampal subregions have specialized roles in other cognitive domains. Using novel cell-type- and region-specific conditional knockouts of the GABAA receptor α2 subunit, we demonstrate that inhibition of the principal neurons of the dentate gyrus or CA3 via α2-containing GABAA receptors (α2GABAARs) is required to suppress anxiety, while the inhibition of CA1 pyramidal neurons is required to suppress fear responses. We further show that the diazepam-modulation of hippocampal theta activity shows certain parallels with our behavioral findings, suggesting a possible mechanism for the observed behavioral effects. Thus, our findings demonstrate a double dissociation in the regulation of anxiety versus fear by hippocampal microcircuitry.
Assuntos
Ansiedade , Mapeamento Encefálico , Medo , Hipocampo/fisiologia , Animais , Camundongos , Rede Nervosa , Receptores de GABA-A/genéticaRESUMO
Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory transmission and pharmacological profiles seen in different brain areas.
RESUMO
Alcoholism, stress, and anxiety are strongly interacting heritable, polygenetic traits. In a previous study, we identified a quantitative trait locus (QTL) on murine chromosome (Chr) 1 between 23.0 and 31.5 Mb that modulates genetic differences in the effects of ethanol on anxiety-related phenotypes. The goal of the present study was to extend the analysis of this locus with a focus on identifying candidate genes using newly available data and tools. Anxiety-like behavior was evaluated with an elevated zero maze following saline or ethanol injections (1.8 g/kg) in C57BL/6J, DBA2J, and 72 BXD strains. We detected significant effects of strain and treatment and their interaction on anxiety-related behaviors, although surprisingly, sex was not a significant factor. The Chr1 QTL is specific to the ethanol-treated cohort. Candidate genes in this locus were evaluated using now standard bioinformatic criteria. Collagen 19a1 (Col19a1) and family sequence 135a (Fam135a) met most criteria but have lower expression levels and lacked biological verification and, therefore, were considered less likely candidates. In contrast, two other genes, the prenylated protein tyrosine phosphate family member Ptp4a1 (protein tyrosine phosphate 4a1) and the zinc finger protein Phf3 (plant homeoDomain finger protein 3) met each of our bioinformatic criteria and are thus strong candidates. These findings are also of translational relevance because both Ptp4a1 and Phf3 have been nominated as candidates genes for alcohol dependence in a human genome-wide association study. Our findings support the hypothesis that variants in one or both of these genes modulate heritable differences in the effects of ethanol on anxiety-related behaviors.
Assuntos
Cromossomos de Mamíferos/genética , Etanol/efeitos adversos , Estudos de Associação Genética , Locos de Características Quantitativas/genética , Estresse Fisiológico/genética , Animais , Comportamento Animal , Feminino , Proteínas Imediatamente Precoces/genética , Masculino , Camundongos , Fenótipo , Polimorfismo Genético , Proteínas Tirosina Fosfatases/genéticaRESUMO
Nitric oxide (NO) is an important molecule for the proper development and function of the central nervous system. In this study, we investigated the behavioral alterations in the neuronal NO synthase knockout mice (NOS1 KO) with a deficient NO production mechanism in the brain, characterizing it as a potential rodent model for attention deficit hyperactivity disorder (ADHD). NOS1 KO exhibited higher locomotor activity than their wildtype counterparts in a novel environment, as measured by open field (OF) test. In a 2-way active avoidance paradigm (TWAA), we found sex-dependent effects, where male KO displayed deficits in avoidance and escape behavior, sustained higher incidences of shuttle crossings, and higher incidences of intertrial interval crossings, suggesting learning, and/or performance impairments. On the other hand, female KO demonstrated few deficits in TWAA. Molsidomine (MSD), a NO donor, rescued TWAA deficits in male KO when acutely administered before training. In a passive avoidance paradigm, KO of both sexes displayed significantly shorter step-through latencies after training. Further, abnormal spontaneous motor activity rhythms were found in the KO during the dark phase of the day, indicating dysregulation of rhythmic activities. These data indicate that NOS1 KO mimics certain ADHD-like behaviors and could potentially serve as a novel rodent model for ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Modelos Animais de Doenças , Óxido Nítrico Sintase Tipo I/fisiologia , Animais , Transtorno do Deficit de Atenção com Hiperatividade/prevenção & controle , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Molsidomina/administração & dosagem , Atividade Motora/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/genéticaRESUMO
RATIONALE: Zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) with preferential binding affinity and efficacy for α1-subunit containing GABA(A) receptors (α1-GABA(A)Rs). Over the last three decades, a variety of animal models and experimental procedures have been used in an attempt to relate the behavioral profile of zolpidem and classic benzodiazepines (BZs) to their interaction with α1-GABA(A)Rs. OBJECTIVES: This paper reviews the results of rodent and non-human primate studies that have evaluated the effects of zolpidem on motor behaviors, anxiety, memory, food and fluid intake, and electroencephalogram (EEG) sleep patterns. Also included are studies that examined zolpidem's discriminative, reinforcing, and anticonvulsant effects as well as behavioral signs of tolerance and withdrawal. RESULTS: The literature reviewed indicates that α1-GABA(A)Rs play a principle role in mediating the hypothermic, ataxic-like, locomotor- and memory-impairing effects of zolpidem and BZs. Evidence also suggests that α1-GABA(A)Rs play partial roles in the hypnotic, EEG sleep, anticonvulsant effects, and anxiolytic-like of zolpidem and diazepam. These studies also indicate that α1-GABA(A)Rs play a more prominent role in mediating the discriminative stimulus, reinforcing, hyperphagic, and withdrawal effects of zolpidem and BZs in primates than in rodents. CONCLUSIONS: The psychopharmacological data from both rodents and non-human primates suggest that zolpidem has a unique pharmacological profile when compared with classic BZs. The literature reviewed here provides an important framework for studying the role of different GABA(A)R subtypes in the behavioral effects of BZ-type drugs and helps guide the development of new pharmaceutical agents for disorders currently treated with BZ-type drugs.
Assuntos
Comportamento Animal/efeitos dos fármacos , Agonistas de Receptores de GABA-A/farmacologia , Piridinas/farmacologia , Receptores de GABA-A/metabolismo , Animais , Comportamento Animal/fisiologia , Agonistas de Receptores de GABA-A/farmacocinética , Humanos , Piridinas/farmacocinética , ZolpidemRESUMO
Brain-derived neurotrophic factor (BDNF) is known to have an integral role in establishing stable memories after learning events. The neuroplasticity induced by Pavlovian fear conditioning has likewise been shown to rely on interactions between BDNF and its principal receptor, tyrosine kinase receptor B (TrkB), in the amygdala after training. Although the necessity of amygdala bdnf expression and TrkB activation for associative learning within aversive contexts has been explored, it is unclear to what extent this interaction is involved in appetitive learning. It is also unclear whether the noted increases in amygdala BDNF after fear conditioning are due to local gene transcription and translation or anterograde transmission from cortical regions. To address both of these questions, we used two lentiviral approaches in mice, using both fear conditioning and cocaine-conditioned place preference (CPP), during acquisition and extinction. First, we decreased expression of bdnf mRNA in the amygdala of homozygous floxed mice with a Cre-expressing virus. In a second set of studies, we infused a virus that expressed a dominant-negative TrkB isoform into the same region. These approaches significantly impaired consolidation of fear conditioning and cocaine-CPP, as well as extinction of CPP. Together, these data suggest that BDNF-TrkB signaling is critical for amygdala-dependent learning of both appetitive and aversive emotional memories.
Assuntos
Tonsila do Cerebelo/metabolismo , Apetite , Aprendizagem por Associação/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/fisiologia , Animais , Memória/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos MutantesRESUMO
The excessive accumulation of soluble amyloid peptides (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD), particularly in synaptic dysfunction. The role of the two major chaperone proteins, Hsp70 and Hsp90, in clearing misfolded protein aggregates has been established. Despite their abundant presence in synapses, the role of these chaperones in synapses remains elusive. Here, we report that Hsp90 inhibition by 17-AAG elicited not only a heat shock-like response but also upregulated presynaptic and postsynaptic proteins, such as synapsin I, synaptophysin, and PSD95 in neurons. 17-AAG treatment enhanced high-frequency stimulation-evoked LTP and protected neurons from synaptic damage induced by soluble Aß. In AD transgenic mice, the daily administration of 17-AAG over 7 d resulted in a marked increase in PSD95 expression in hippocampi. 17-AAG treatments in wild-type C57BL/6 mice challenged by soluble Aß significantly improved contextual fear memory. Further, we demonstrate that 17-AAG activated synaptic protein expression via transcriptional mechanisms through the heat shock transcription factor HSF1. Together, our findings identify a novel function of Hsp90 inhibition in regulating synaptic plasticity, in addition to the known neuroprotective effects of the chaperones against Aß and tau toxicity, thus further supporting the potential of Hsp90 inhibitors in treating neurodegenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Sinapses/metabolismo , Doença de Alzheimer/patologia , Animais , Western Blotting , Modelos Animais de Doenças , Imuno-Histoquímica , Potenciação de Longa Duração/fisiologia , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos , Sinapses/efeitos dos fármacos , Sinapses/patologiaRESUMO
RATIONALE: Zolpidem is a short-acting, non-benzodiazepine hypnotic that acts as a full agonist at α1-containing GABAA receptors. Overall, zolpidem purportedly has fewer instances of abuse and dependence than traditionally used benzodiazepines. However, several studies have shown that zolpidem may be more similar to benzodiazepines in terms of behavioral tolerance and withdrawal symptoms. OBJECTIVES: In the current study, we examined whether subchronic zolpidem or diazepam administration produced deficits in zolpidem's locomotor-impairing effects, anxiety-like behaviors, and changes in GABAAR subunit messenger RNA (mRNA). METHODS: Mice were given subchronic injections of either zolpidem (10 mg/kg), diazepam (20 mg/kg), or vehicle twice daily for 7 days. On day 8, mice were given a challenge dose of zolpidem (2 mg/kg) or vehicle before open field testing. Another set of mice underwent the same injection regimen but were sacrificed on day 8 for qRT-PCR analysis. RESULTS: We found that subchronic zolpidem and diazepam administration produced deficits in the acute locomotor-impairing effects of zolpidem and increased anxiety-like behaviors 1 day after drug termination. In addition, we found that subchronic treatment of zolpidem and diazepam induced distinct but overlapping GABAAR subunit mRNA changes in the cortex but few changes in the hippocampus, amygdala, or prefrontal cortex. Levels of mRNA measured in separate mice after a single injection of either zolpidem or diazepam revealed no mRNA changes. CONCLUSIONS: In mice, subchronic treatment of zolpidem and diazepam can produce deficits in the locomotor-impairing effects of zolpidem, anxiety-like withdrawal symptoms, and subunit-specific mRNA changes.
Assuntos
Diazepam/farmacologia , Moduladores GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Hipnóticos e Sedativos/farmacologia , Piridinas/farmacologia , Animais , Ansiedade/induzido quimicamente , Ansiedade/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Tolerância a Medicamentos/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de GABA-A/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia , ZolpidemRESUMO
Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25-40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50-60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2-8 weeks after blast, 50-60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3-8 weeks after blast. Thus, the TBI caused by single 50-60 psi blasts in mice exhibits the minimal neuronal loss coupled to "diffuse" axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.
RESUMO
NMDA receptors are glutamate receptor ion channels that contribute to synaptic plasticity and are important for many forms of learning and memory. In the amygdala, NMDA receptors are critical for the acquisition, retention, and extinction of classically conditioned fear responses. Although the GluN2B subunit has been implicated in both the acquisition and extinction of conditioned fear, GluN2C-knockout mice show reduced conditioned fear responses. Moreover, D-cycloserine (DCS), which facilitates fear extinction, selectively enhances the activity of GluN2C-containing NMDA receptors. To further define the contribution of GluN2C receptors to fear learning, we infused the GluN2C/GluN2D-selective potentiator CIQ bilaterally into the basolateral amygdala (3, 10, or 30 µg/side) following either fear conditioning or fear extinction training. CIQ both increased the expression of conditioned fear 24 h later and enhanced the extinction of the previously conditioned fear response. These results support a critical role for GluN2C receptors in the amygdala in the consolidation of learned fear responses and suggest that increased activity of GluN2C receptors may underlie the therapeutic actions of DCS.
Assuntos
Tonsila do Cerebelo/metabolismo , Condicionamento Psicológico/fisiologia , Extinção Psicológica/fisiologia , Medo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Retenção Psicológica/fisiologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Área Sob a Curva , Benzimidazóis/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Atividade Motora/efeitos dos fármacos , Oócitos , Receptores de N-Metil-D-Aspartato/genética , Retenção Psicológica/efeitos dos fármacos , XenopusRESUMO
We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50-60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50-60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.
Assuntos
Benzofenonas/farmacologia , Lesões Encefálicas/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Depressão/etiologia , Depressão/patologia , Modelos Animais de Doenças , Agonismo Inverso de Drogas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fenótipo , Receptor CB2 de Canabinoide/metabolismo , Transtornos da Visão/etiologia , Transtornos da Visão/patologiaRESUMO
Experience-induced changes associated with odor learning are mediated by a number of signaling molecules, including nitric oxide (NO), which is predominantly synthesized by neuronal nitric oxide synthase (nNOS) in the brain. In the current study, we investigated the role of nNOS in the acquisition and retention of conditioned olfactory fear. Mice lacking nNOS received six training trials, each consisting of an odor-CS co-terminating with a foot shock-US. Mice showed reduced freezing responses to the trained odor 24 h and 7 d after training, compared to wild-type mice. Pretraining systemic injections of the NO donor, molsidomine, rescued fear retention in nNOS knockout mice. In wild-type mice, pretraining systemic injections of L-NAME, a nonspecific nNOS blocker, disrupted odor-CS fear retention in a dose-dependent manner. To evaluate whether NO signaling is involved in generalization of fear memories, nNOS knockout mice and wild-type mice receiving L-NAME were trained to one odor and tested with a series of similar odors. In both cases, we found increased generalization, as measured by increased freezing to similar, unpaired odors. Despite the impairment in fear memory retention and generalization, neither mice receiving injections of L-NAME nor nNOS knockout mice showed any deficits in either novel odor investigation time or odor habituation, suggesting intact olfactory perception and short-term memory olfactory learning. These results support a necessary role for neuronal NO signaling in the normal expression and generalization of olfactory conditioned fear.
Assuntos
Condicionamento Psicológico/fisiologia , Memória de Longo Prazo/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Olfato , Tonsila do Cerebelo/fisiologia , Animais , Medo , Feminino , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , OdorantesRESUMO
Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.
Assuntos
Técnicas de Transferência de Genes/instrumentação , Vírus da Leucemia Murina de Moloney/genética , Receptores de Somatostatina/metabolismo , Somatostatina/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus , Animais , Sítios de Ligação , Linhagem Celular , Marcação de Genes/instrumentação , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/química , Vírus da Leucemia Murina de Moloney/fisiologia , Ligação Proteica , Engenharia de Proteínas , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Somatostatina/química , Somatostatina/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
OBJECTIVE: Despite increasing awareness of the many important roles played by brain-derived neurotrophic factor (BDNF) activation of TrkB, a fuller understanding of this system and the use of potential TrkB-acting therapeutic agents has been limited by the lack of any identified small-molecule TrkB agonists that fully mimic the actions of BDNF at brain TrkB receptors in vivo. However, 7,8-dihydroxyflavone (7,8-DHF) has recently been identified as a specific TrkB agonist that crosses the blood-brain barrier after oral or intraperitoneal administration. The authors combined pharmacological, biochemical, and behavioral approaches in a preclinical study examining the role of 7,8-DHF in modulating emotional memory in mice. METHOD: The authors first examined the ability of systemic 7,8-DHF to activate TrkB receptors in the amygdala. They then examined the effects of systemic 7,8-DHF on acquisition and extinction of conditioned fear, using specific and well-characterized BDNF-dependent learning paradigms in several models using naive mice and mice with prior traumatic stress exposure. RESULTS: Amygdala TrkB receptors, which have previously been shown to be required for emotional learning, were activated by systemic 7,8-DHF (at 5 mg/kg i.p.). 7,8-DHF enhanced both the acquisition of fear and its extinction. It also appeared to rescue an extinction deficit in mice with a history of immobilization stress. CONCLUSIONS: These data suggest that 7,8-DHF may be an excellent agent for use in understanding the effects of TrkB activation in learning and memory paradigms and may be attractive for use in reversing learning and extinction deficits associated with psychopathology.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Condicionamento Clássico/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Flavanonas/farmacologia , Rememoração Mental/efeitos dos fármacos , Receptor trkB/agonistas , Tonsila do Cerebelo/patologia , Tonsila do Cerebelo/fisiopatologia , Animais , Aprendizagem por Associação/efeitos dos fármacos , Aprendizagem por Associação/fisiologia , Condicionamento Clássico/fisiologia , Extinção Psicológica/fisiologia , Medo/fisiologia , Humanos , Imobilização/fisiologia , Imobilização/psicologia , Immunoblotting , Injeções Intraperitoneais , Masculino , Rememoração Mental/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Psicológico/complicações , Estresse Psicológico/fisiopatologiaRESUMO
Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)-CA3-CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2-7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG-CA3-CA1 circuit.
Assuntos
Região CA2 Hipocampal/fisiologia , Aprendizagem , Plasticidade Neuronal , Células Piramidais/fisiologia , Proteínas RGS/metabolismo , Sinapses/fisiologia , Animais , Região CA2 Hipocampal/metabolismo , Memória , Camundongos , Camundongos Knockout , Células Piramidais/metabolismo , Sinapses/metabolismoRESUMO
The heterogeneity and distribution of GABA(A) receptor subunits mediates differential roles in behavior. It is thought that particular behavioral responses to benzodiazepine (BZ) ligands might be associated with an action at a regionally defined receptor subtype. However, the role of specific GABA(A) receptor subtypes in particular brain regions is less clear. Such detailed knowledge of regional alpha1-GABA(A) receptor function will advance our understanding of the neural circuitry underlying the role of GABA(A) receptors and the effects of GABA(A)-modulating drugs on behavior. By combining inducible, site-specific alpha1 subunit deletion, using a lentivirus expressing Cre-recombinase in mice with the alpha1 subunit gene flanked by loxP sites, we examine baseline and pharmacological effects of deletion of amygdala alpha1-GABA(A) receptors. We find that amygdala-specific reduction of alpha1 receptor subunits does not affect mRNA or protein levels of amygdala alpha2 or alpha3 subunit receptors. Nor does this inducible reduction affect baseline locomotion or measures of anxiety. However, we also find that this inducible, site-specific deletion does disrupt the normal sedative-locomotor inhibition as well as the anticonvulsive effects, of two distinct BZ-site ligands, diazepam and zolpidem, which is relatively alpha1-subunit selective. These data, using inducible, region and subunit-specific deletion, combined with pharmacogenetic approaches, demonstrate that amygdala expression of the alpha1-GABA(A) receptor subunit is required for normal BZ effects on sedation, locomotion, and seizure inhibition, but not for anxiolysis.
Assuntos
Tonsila do Cerebelo/fisiologia , Anticonvulsivantes/farmacologia , Benzodiazepinas/farmacologia , Hipnóticos e Sedativos/farmacologia , Locomoção/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Ansiolíticos/farmacologia , Linhagem Celular Transformada , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Locomoção/genética , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pentilenotetrazol , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Transfecção/métodos , ZolpidemRESUMO
The use of conventional knockout technologies has proved valuable for understanding the role of key genes and proteins in development, disease states, and complex behaviors. However, these strategies are limited in that they produce broad changes in gene function throughout the neuroaxis and do little to identify the effects of such changes on neural circuits thought to be involved in distinct functions. Because the molecular functions of genes often depend on the specific neuronal circuit in which they are expressed, restricting gene manipulation to specific brain regions and times may be more useful for understanding gene functions. Conditional gene manipulation strategies offer a powerful alternative. In this report we briefly describe two conditional gene strategies that are increasingly being used to investigate the role of genes in behavior - the Cre/loxP recombination system and lentiviral vectors. Next, we summarize a number of recent experiments which have used these techniques to investigate behavior after spatial and/or temporal and gene manipulation. These conditional gene targeting strategies provide useful tools to study the endogenous mechanisms underlying complex behaviors and to model disease states resulting from aberrant gene expression.
RESUMO
Gerbils show a neurokinin (NK)1 receptor pharmacological profile, which is similar to that observed in humans, and thus have become a commonly used species to test efficacy of NK1 receptor antagonists. The aim of this study was to determine whether systemic administration of the NK1 receptor antagonist GR-205171 produced anxiolytic-like effects in the elevated plus maze and in a novel contextual conditioned fear test using fear-potentiated startle (FPS). On the elevated plus maze, treatment with GR-205171 at 0, 0.3, 1.0, and 5.0 mg/kg doses, 30 min before testing produced anxiolytic-like effects in an increasing dose-response manner as measured by the percentage of open arm time and percentage of open arm entries. For contextual fear conditioning, gerbils were given 10 unsignaled footshocks (0.6 mA) at a 2-min variable interstimulus interval in a distinctive training context. Twenty-four hours after training, gerbils received treatment of GR-205171 at 0, 0.3, 1.0, and 5.0 mg/kg doses, 30 min before testing in which startle was elicited in the same context in which they were trained. Contextual FPS was defined as an increase in startle over pretraining baseline values. All drug dose levels (0.3, 1.0, and 5.0 mg/kg) significantly attenuated contextual FPS when compared with the vehicle control group. A control group, which received testing in a different context, showed little FPS. These findings support other evidence for anxiolytic activity of NK1 receptor antagonists and provide a novel conditioned fear test that may be an appropriate procedure to test other NK1 antagonists for preclinical anxiolytic activity in gerbils.
Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Medo/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1 , Piperidinas/uso terapêutico , Reflexo de Sobressalto/efeitos dos fármacos , Tetrazóis/uso terapêutico , Animais , Ansiolíticos/farmacologia , Relação Dose-Resposta a Droga , Gerbillinae , Masculino , Modelos Animais , Piperidinas/farmacologia , Tetrazóis/farmacologiaRESUMO
Neglect is a complex human cognitive spatial disorder typically induced by damage to prefrontal or posterior parietal association cortices. Behavioral treatments for neglect rarely generalize outside of the therapeutic context or across tasks within the same therapeutic context. Recovery, when it occurs, is spontaneous over the course of weeks to months, but often it is incomplete. A number of studies have indicated that anti-Nogo-A antibodies can be used to enhance plasticity and behavioral recovery following damage to motor cortex, and spinal cord. In the present studies the anti-Nogo-A antibodies IN-1, 7B12, or 11C7 were applied intraventricularly to adult rats demonstrating severe neglect produced by unilateral medial agranular cortex lesions in rats. The three separate anti-Nogo-A antibody groups were treated immediately following the medial agranular cortex lesions. Each of the three antibodies induced dramatic significant behavioral recovery from neglect relative to controls. Severing the corpus callosum to destroy inputs from the contralesional hemisphere resulted in reinstatement of severe neglect, pointing to a possible role of interhemispheric mechanisms in behavioral recovery from neglect.