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1.
Mol Pharmacol ; 103(2): 100-112, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36379717

RESUMO

The endocannabinoid system (ECS) modulates synaptic function to regulate many aspects of neurophysiology. It adapts to environmental changes and is affected by disease. Thus, the ECS presents an important target for therapeutic development. Despite recent interest in cannabinoid-based treatments, few preclinical studies are conducted in human systems. Human induced pluripotent stem cells (hiPSCs) provide one possible solution to this issue. However, it is not known if these cells have a fully functional ECS. Here, we show that hiPSC-derived neuron/astrocyte cultures exhibit a complete ECS. Using Ca2+ imaging and a genetically encoded endocannabinoid sensor, we demonstrate that they not only respond to exogenously applied cannabinoids but also produce and metabolize endocannabinoids. Synaptically driven [Ca2+]i spiking activity was inhibited (EC50 = 48 ± 13 nM) by the efficacious agonist [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (Win 55,212-2) and by the endogenous ligand 2-arachidonoyl glycerol (2-AG; EC50 = 2.0 ± 0.6 µm). The effects of Win 55212-2 were blocked by a CB1 receptor-selective antagonist. Δ9-Tetrahydrocannabinol acted as a partial agonist, maximally inhibiting synaptic activity by 47 ± 14% (EC50 = 1.4 ± 1.9 µm). Carbachol stimulated 2-AG production in a manner that was independent of Ca2+ and blocked by selective inhibition of diacylglycerol lipase. 2-AG returned to basal levels via a process mediated by monoacylglycerol lipase as indicated by slowed recovery in cultures treated with 4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester (JZL 184). Win 55,212-2 markedly desensitized CB1 receptor function following a 1-day exposure, whereas desensitization was incomplete following 7-day treatment with JZL 184. This human cell culture model is well suited for functional analysis of the ECS and as a platform for drug development. SIGNIFICANCE STATEMENT: Despite known differences between the human response to cannabinoids and that of other species, an in vitro human model demonstrating a fully functional endocannabinoid system has not been described. Human induced pluripotent stem cells (hiPSCs) can be obtained from skin samples and then reprogrammed into neurons for use in basic research and drug screening. Here, we show that hiPSC-derived neuronal cultures exhibit a complete endocannabinoid system suitable for mechanistic studies and drug discovery.


Assuntos
Canabinoides , Células-Tronco Pluripotentes Induzidas , Humanos , Endocanabinoides/metabolismo , Transmissão Sináptica , Benzoxazinas , Canabinoides/farmacologia , Canabinoides/metabolismo , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo
2.
J Biol Chem ; 298(9): 102278, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35863435

RESUMO

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.


Assuntos
Potenciais de Ação , Calcineurina , Genes Precoces , Neurônios , Transcrição Gênica , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bicuculina/farmacologia , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Antagonistas de Receptores de GABA-A/farmacologia , Genes Precoces/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Potássio/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
3.
J Neurochem ; 148(4): 499-515, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30520043

RESUMO

HIV-associated neurocognitive disorder affects about half of HIV-infected patients. HIV impairs neuronal function through indirect mechanisms mainly mediated by inflammatory cytokines and neurotoxic viral proteins, such as the envelope protein gp120. HIV gp120 elicits a neuroinflammatory response that potentiates NMDA receptor function and induces the loss of excitatory synapses. How gp120 influences neuronal inhibition remains unknown. In this study, we expressed a green fluorescent protein (GFP)-tagged recombinant antibody-like protein that binds to the post-synaptic scaffolding protein gephyrin to label inhibitory synapses in living neurons. Treatment with 600 pM gp120 for 24 h increased the number of labeled inhibitory synapses. HIV gp120 evoked the release of interleukin-1ß (IL-1ß) from microglia to activate IL-1 receptors on neurons. Subsequent activation of the tyrosine kinase Src and GluN2A-containing NMDA receptors increased the number of inhibitory synapses via a process that required protein synthesis. In naïve cultures, inhibition of neuronal p38 mitogen-activated protein kinase (p38 MAPK) increased the number of inhibitory synapses suggesting that p38 MAPK produces a basal suppression of inhibitory synapses that is overcome in the presence of gp120. Direct activation of a mutant form of p38 MAPK expressed in neurons mimicked basal suppression of inhibitory synapses. This study shows for the first time that gp120-induced neuroinflammation increases the number of inhibitory synapses and that this increase overcomes a basal suppression of synaptic inhibition. Increased inhibition may be an adaptive mechanism enabling neurons to counteract excess excitatory input in order to maintain network homeostasis. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Proteína gp120 do Envelope de HIV/toxicidade , Inflamação/virologia , Neurônios/virologia , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/fisiopatologia , Animais , Células Cultivadas , Proteína gp120 do Envelope de HIV/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/virologia , Inflamação/metabolismo , Inflamação/patologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia , Sinapses/virologia
4.
Neurochem Res ; 44(1): 234-246, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29541929

RESUMO

A defining feature of HIV-associated neurocognitive disorder (HAND) is the loss of excitatory synaptic connections. Synaptic changes that occur during exposure to HIV appear to result, in part, from a homeostatic scaling response. Here we discuss the mechanisms of these changes from the perspective that they might be part of a coping mechanism that reduces synapses to prevent excitotoxicity. In transgenic animals expressing the HIV proteins Tat or gp120, the loss of synaptic markers precedes changes in neuronal number. In vitro studies have shown that HIV-induced synapse loss and cell death are mediated by distinct mechanisms. Both in vitro and animal studies suggest that HIV-induced synaptic scaling engages new mechanisms that suppress network connectivity and that these processes might be amenable to therapeutic intervention. Indeed, pharmacological reversal of synapse loss induced by HIV Tat restores cognitive function. In summary, studies indicate that there are temporal, mechanistic and pharmacological features of HIV-induced synapse loss that are consistent with homeostatic plasticity. The increasingly well delineated signaling mechanisms that regulate synaptic scaling may reveal pharmacological targets suitable for normalizing synaptic function in chronic neuroinflammatory states such as HAND.


Assuntos
HIV/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Sinapses/virologia , Animais , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
5.
J Neurosci ; 37(33): 7837-7847, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28716964

RESUMO

HIV-associated neurocognitive disorder (HAND) affects approximately half of HIV-infected patients. Loss of synaptic connections is a hallmark of many neurocognitive disorders, including HAND. The HIV-1 protein transactivator of transcription (Tat) disrupts synaptic connections both in vitro and in vivo and has been linked to impaired neurocognitive function in humans. In vitro studies have shown that ifenprodil, an antagonist selective for GluN2B-containing NMDARs, reverses synapse loss when applied after Tat. Here, we tested the hypothesis that Tat-induced loss and ifenprodil-mediated rescue of synaptic spines in vivo would predict impairment and rescue of cognitive function. Using intracranial multiphoton imaging, we found that infusion of 100 ng of HIV-1 Tat into the lateral ventricle of yellow fluorescent protein-expressing transgenic mice produced a 17 ± 1% loss of dendritic spines in layer 1 of retrosplenial cortex. Repeated imaging of the same dendrites over 3 weeks enabled longitudinal experiments that demonstrated sustained spine loss after Tat infusion and transient rescue after ifenprodil administration (10 mg/kg, i.p.). Parallel trace fear conditioning experiments showed that spine loss predicted learning deficits and that the time course of ifenprodil-induced rescue of spine density correlated with restoration of cognitive function. These results show for the first time that, during exposure to an HIV-1 neurotoxin in vivo, alteration of GluN2B-containing NMDAR signaling suppresses spine density and impairs learning. Pharmacological inhibition of these NMDARs rescued spines and restored cognitive function. Drugs that rescue synapses may improve neurocognitive function in HAND.SIGNIFICANCE STATEMENT Synaptodendritic damage correlates with cognitive decline in HIV-associated neurocognitive disorder (HAND) patients. We developed an in vivo imaging approach for longitudinal tracking of spine density that enabled correlation of synaptic changes with behavioral outcomes in a model of HAND. We show for the first time that spine loss after exposure to an HIV-1 protein can be reversed pharmacologically and that loss and recovery of dendritic spines predict impairment and restoration of cognitive function, respectively. Therefore, synapse loss, the hallmark of cognitive decline in HAND, is reversible. Drugs that restore spine density may have broad application for improving cognitive function during the early phases of neurodegenerative diseases.


Assuntos
Disfunção Cognitiva/prevenção & controle , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , HIV-1 , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Animais , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/metabolismo , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Piperidinas/administração & dosagem , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem
6.
J Neurosci ; 36(50): 12640-12649, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27810933

RESUMO

HIV-associated neurocognitive disorder (HAND) affects approximately half of HIV-infected patients. Infected non-neuronal cells release neurotoxic factors such as the viral protein transactivator of transcription (Tat) that potentiate NMDAR function. NMDARs regulate synaptic changes observed after exposure to HIV proteins, which may underlie cognitive impairment in HAND patients. Here, we used patch-clamp recording to measure NMDAR-mediated currents in rat hippocampal cultures after exposure to Tat. Tat (4-16 h) potentiated NMDA-evoked whole-cell current and increased the NMDAR:AMPAR ratio of evoked EPSCs. Potentiated currents adapted back to baseline amplitudes after 24 h of exposure to Tat. Pharmacological inhibition of GluN2A-containing NMDARs prevented adaptation, but inhibition of GluN2B-containing NMDARs did not. Pharmacological and genetic approaches determined that potentiated NMDARs activated the kinase Akt, which then activated the E3 ubiquitin ligase Mdm2. Inhibition of protein synthesis prevented adaptation, suggesting that Mdm2 altered gene expression, possibly through its well known target p53. Expression of GFP-tagged GluN1 subunits resulted in fluorescent puncta that colocalized with synaptic markers. Tat (24 h) caused an Mdm2-dependent loss of NMDAR puncta on a timescale similar to adaption of NMDAR function. Activation of the Mdm2 pathway degrades PSD-95, a scaffolding protein that clusters NMDARs at the synapse and enhances their function. Adaptation to the continued presence of excitotoxins that potentiate NMDARs such as HIV Tat may protect from excessive NMDAR activation while also contributing to the synaptic loss that correlates with cognitive decline in HAND. SIGNIFICANCE STATEMENT: Synaptodendritic damage correlates with cognitive decline in HIV-associated neurocognitive disorder (HAND). In a cell culture model, we show that the HIV protein transactivator of transcription (Tat) initially potentiates NMDARs that then adapt to the presence of the toxin. Adaptation of NMDAR function was mediated by a GluN2A/Akt/Mdm2 pathway not previously linked to neuroinflammatory disorders such as HAND. Activation of this pathway caused a loss of synaptic NMDAR clusters. Decreased NMDAR function may result from a homeostatic response gone awry and underlie impaired synaptic function in HAND.


Assuntos
Síndromes Neurotóxicas/fisiopatologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Humanos , Masculino , Proteína Oncogênica v-akt/metabolismo , Técnicas de Patch-Clamp , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Receptores de LDL/efeitos dos fármacos , Receptores de LDL/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética
7.
J Neurophysiol ; 115(4): 1875-85, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26843596

RESUMO

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1ß accelerated Ca(2+) clearance in a manner blocked by an IL-1ß receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1ß activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons.


Assuntos
Hipocampo/metabolismo , Interleucina-1beta/farmacologia , Neurônios/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Quinases da Família src/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ratos , Quinases da Família src/antagonistas & inibidores
8.
J Neurochem ; 132(3): 354-66, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25156524

RESUMO

HIV-associated neurocognitive disorders afflict approximately half of HIV-infected patients. HIV-infected cells within the CNS release neurotoxic viral proteins such as the transactivator of transcription (Tat). Tat caused a biphasic change in NMDAR function; NMDA-evoked increases in intracellular Ca(2+) were initially potentiated following 16 h exposure to Tat and then adapted by gradually returning to baseline by 24 h. Following Tat-induced NMDAR potentiation, a RhoA/Rho-associated protein kinase (ROCK) signaling pathway was activated; a subsequent remodeling of the actin cytoskeleton reduced NMDA-evoked increases in intracellular Ca(2+) . Pharmacologic or genetic inhibition of RhoA or ROCK failed to affect potentiation, but prevented adaptation of NMDAR function. Activation of RhoA/ROCK signaling increases the formation of filamentous actin. Drugs that prevent changes to filamentous actin blocked adaptation of NMDAR function following Tat-induced potentiation, whereas stimulating either depolymerization or polymerization of actin attenuated NMDAR function. These findings indicate that Tat activates a RhoA/ROCK signaling pathway resulting in actin remodeling and subsequent reduction of NMDAR function. Adaptation of NMDAR function may be a mechanism to protect neurons from excessive Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders.


Assuntos
Actinas/fisiologia , Cálcio/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Animais , DNA/genética , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos
9.
J Neurosci ; 33(45): 17908-20, 2013 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-24198379

RESUMO

Synaptodendritic damage correlates with cognitive decline in many neurodegenerative diseases, including human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). Because HIV-1 does not infect neurons, viral-mediated toxicity is indirect, resulting from released neurotoxins such as the HIV-1 protein transactivator of transcription (Tat). We compared the effects of Tat on inhibitory and excitatory synaptic connections between rat hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding proteins gephyrin or PSD95 fused to GFP. Tat (24 h) increased the number of GFP-gephyrin puncta and decreased the number of PSD95-GFP puncta. The effects of Tat on inhibitory and excitatory synapse number were mediated via the low-density lipoprotein receptor-related protein and subsequent Ca(2+) influx through GluN2A-containing NMDA receptors (NMDARs). The effects of Tat on synapse number required cell-autonomous activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Ca(2+) buffering experiments suggested that loss of excitatory synapses required activation of CaMKII in close apposition to the NMDAR, whereas the increase in inhibitory synapses required Ca(2+) diffusion to a more distal site. The increase in inhibitory synapses was prevented by inhibiting the insertion of GABAA receptors into the membrane. Synaptic changes induced by Tat (16 h) were reversed by blocking either GluN2B-containing NMDARs or neuronal nitric oxide synthase, indicating changing roles for pathways activated by NMDAR subtypes during the neurotoxic process. Compensatory changes in the number of inhibitory and excitatory synapses may serve as a novel mechanism to reduce network excitability in the presence of HIV-1 neurotoxins; these changes may inform the development of treatments for HAND.


Assuntos
Hipocampo/efeitos dos fármacos , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagem , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Inibição Neural/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia
10.
J Neurochem ; 130(5): 642-56, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24666322

RESUMO

HIV-associated neurocognitive disorders afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors. Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)]i). NMDA-evoked responses were potentiated following 2-h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca(2+)]i peaked by 8 h and then adapted by gradually reversing to baseline by 24 h and eventually dropping below control by 48 h. Tat-induced potentiation of NMDA-evoked responses was blocked by inhibition of lipoprotein receptor-related protein (LRP) or Src tyrosine kinase. Potentiation was unaffected by inhibition of nitric oxide synthase (NOS). However, NOS activity was required for adaptation. Adaptation was also prevented by inhibition of soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate-dependent protein kinase G (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca(2+)]i via LRP-dependent activation of Src and that this potentiation adapts via activation of the NOS/sGC/PKG pathway. Adaptation may protect neurons from excessive Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders. HIV-associated neurocognitive disorders (HAND) afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors (NMDARs). We show that HIV-1 Tat evoked biphasic changes in NMDA-evoked [Ca(2+) ]i responses. Initially, Tat potentiated NMDA-evoked responses following LRP-mediated activation of Src kinase. Subsequently, Tat-induced NMDAR potentiation adapted by activation of a NOS/sGC/PKG pathway that attenuated NMDA-evoked increases in [Ca(2+)]i . Adaptation may be a novel neuroprotective mechanism to prevent excessive Ca(2+) influx. Solid and dashed arrows represent direct and potentially indirect connections, respectively.


Assuntos
Cálcio/metabolismo , Óxido Nítrico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Células Cultivadas , Infecções por HIV/metabolismo , HIV-1/metabolismo , Immunoblotting , N-Metilaspartato/metabolismo , Neurônios/metabolismo , Neurônios/virologia , Ratos , Transfecção
11.
Mol Cell Neurosci ; 54: 22-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23267846

RESUMO

Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin-proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients.


Assuntos
Hipocampo/citologia , Neurônios/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Animais , Células Cultivadas , HIV-1 , Hipocampo/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas Relacionadas a Receptor de LDL/antagonistas & inibidores , Neurônios/metabolismo , Piperazinas/farmacologia , Piperidinas/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinaptofisina/genética , Sinaptofisina/metabolismo
12.
Front Pharmacol ; 15: 1369757, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38533258

RESUMO

Introduction: Antiretroviral (ARV) drugs have improved prognoses for people living with HIV. However, HIV-associated neurocognitive disorders (HAND) persist despite undetectable viral loads. Some ARVs have been linked to neuropsychiatric effects that may contribute to HAND. Synapse loss correlates with cognitive decline in HAND and synaptic deficits may contribute to the neuropsychiatric effects of ARV drugs. Methods: Using an automated high content assay, rat hippocampal neurons in culture expressing PSD95-eGFP to label glutamatergic synapses and mCherry to fill neuronal structures were imaged before and after treatment with 25 clinically used ARVs. Results and Discussion: At a concentration of 10 µM the protease inhibitors nelfinavir and saquinavir, the non-nucleoside reverse transcriptase inhibitors etravirine and the 8-OH metabolite of efavirenz, the integrase inhibitor bictegravir, and the capsid inhibitor lenacapavir produced synaptic toxicity. Only lenacapavir produced synapse loss at the nanomolar concentrations estimated free in the plasma, although all 4 ARV drugs induced synapse loss at Cmax. Evaluation of combination therapies did not reveal synergistic synaptic toxicity. Synapse loss developed fully by 24 h and persisted for at least 3 days. Bictegravir-induced synapse loss required activation of voltage-gated Ca2+ channels and bictegravir, etravirine, and lenacapavir produced synapse loss by an excitotoxic mechanism. These results indicate that select ARV drugs might contribute to neuropsychiatric effects in combination with drugs that bind serum proteins or in disease states in which synaptic function is altered. The high content imaging assay used here provides an efficient means to evaluate new drugs and drug combinations for potential CNS toxicity.

13.
J Neurophysiol ; 109(6): 1494-504, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23274309

RESUMO

Neurons adapt to seizure activity structurally and functionally to attenuate hyperactive neural circuits. Homer proteins provide a scaffold in the postsynaptic density (PSD) by binding to ligands through an EVH1 domain and to other Homer proteins by a coiled-coil domain. The short Homer isoform 1a (H1a) has a ligand-binding domain but lacks a coiled-coil domain and thus acts in a dominant-negative manner to uncouple Homer scaffolds. Here, we show that treating rat hippocampal cultures with bicuculline and 4-aminopyridine (Bic+4-AP) evoked epileptiform activity and synchronized Ca(2+) spiking, measured with whole cell current-clamp and fura-2-based digital imaging; Bic+4-AP increased H1a mRNA through the activation of metabotropic glutamate receptor 5 (mGluR5). Treatment with Bic+4-AP for 4 h attenuated burst firing and induced synapse loss. Synaptic changes were measured using a confocal imaging-based assay that quantified clusters of PSD-95 fused to green fluorescent protein. Treatment with an mGluR5 antagonist blocked H1a expression, synapse loss, and burst attenuation. Overexpression of H1a inhibited burst firing similar to Bic+4-AP treatment. Furthermore, knockdown of H1a using a short hairpin RNA (shRNA) strategy reduced synapse loss and burst attenuation induced by Bic+4-AP treatment. Thus an epileptiform stimulus applied to hippocampal neurons in culture induced burst firing and H1a expression through the activation of mGluR5; a 4-h exposure to this stimulus resulted in synapse loss and burst attenuation. These results suggest that H1a expression functions in a negative-feedback manner to reduce network excitability by regulating the number of synapses.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Bicuculina/farmacologia , Proteínas de Transporte/metabolismo , Convulsivantes/farmacologia , Neurônios/fisiologia , Sinapses/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/genética , Proteína 4 Homóloga a Disks-Large , Retroalimentação Fisiológica , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Proteínas de Arcabouço Homer , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transcrição Gênica/efeitos dos fármacos
14.
J Neurosci ; 31(7): 2361-70, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21325503

RESUMO

The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, plasticity, and synaptic transmission. Here, we examined the modulation of the plasma membrane Ca(2+) ATPase (PMCA) by tyrosine kinases. In rat sensory neurons grown in culture, the PMCA was under tonic inhibition by a member of the Src family of tyrosine kinases (SFKs). Ca(2+) clearance accelerated in the presence of selective tyrosine kinase inhibitors. Tonic inhibition of the PMCA was attenuated in cells expressing a dominant-negative construct or shRNA directed to message for the SFKs Lck or Fyn, but not Src. SFKs did not appear to phosphorylate the PMCA directly but instead activated focal adhesion kinase (FAK). Expression of constitutively active FAK enhanced and dominant-negative or shRNA knockdown of FAK attenuated tonic inhibition. Antisense knockdown of PMCA isoform 4 removed tonic inhibition of Ca(2+) clearance, indicating that FAK acts on PMCA4. The hyaluronan receptor CD44 activates SFK-FAK signaling cascades and is expressed in sensory neurons. Treating neurons with a CD44-blocking antibody or short hyaluronan oligosaccharides, which are produced during injury and displace macromolecular hyaluronan from CD44, attenuated tonic PMCA inhibition. Ca(2+)-activated K(+) channels mediate a slow afterhyperpolarization in sensory neurons that was inhibited by tyrosine kinase inhibitors and enhanced by knockdown of PMCA4. Thus, we describe a novel kinase cascade in sensory neurons that enables the extracellular matrix to alter Ca(2+) signals by modulating PMCA-mediated Ca(2+) clearance. This signaling pathway may influence the excitability of sensory neurons following injury.


Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Receptores de Hialuronatos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células Receptoras Sensoriais/metabolismo , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Receptores de Hialuronatos/imunologia , Indóis/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Técnicas de Patch-Clamp/métodos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/classificação , Proteínas Tirosina Quinases/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo , Transfecção/métodos
15.
Biochem Biophys Res Commun ; 424(1): 76-81, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22732411

RESUMO

The plasma membrane Ca(2+) ATPase (PMCA) is responsible for maintaining basal intracellular Ca(2+) concentration ([Ca(2+)](i)) and returning small increases in [Ca(2+)](i) back to resting levels. The carboxyl terminus of some PMCA splice variants bind Homer proteins; how binding affects PMCA function is unknown. Here, we examined the effects of altered expression of Homer proteins on PMCA-mediated Ca(2+) clearance from rat hippocampal neurons in culture. The kinetics of PMCA-mediated recovery from the [Ca(2+)](i) increase evoked by a brief train of action potentials was determined in the soma of single neurons using indo-1-based photometry. Exogenous expression of Homer 1a, Homer 1c or Homer 2a did not affect PMCA function. However, shRNA mediated knockdown of Homer 1 slowed PMCA mediated Ca(2+) clearance by 28% relative to cells expressing non-silencing shRNA. The slowed recovery rate in cells expressing Homer 1 shRNA was reversed by expression of a short Homer 2 truncation mutant. These results indicate that constitutively expressed Homer proteins tonically stimulate PMCA function in hippocampal neurons. We propose a model in which binding of short or long Homer proteins to the carboxyl terminus of the PMCA stimulates Ca(2+) clearance rate. PMCA-mediated Ca(2+) clearance may be stimulated following incorporation of the pump into Homer organized signaling domains and following induction of the Homer 1a immediate early gene.


Assuntos
Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Hipocampo/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Proteínas de Arcabouço Homer , Neurônios/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Ratos
16.
J Neurosci ; 30(8): 3072-81, 2010 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-20181604

RESUMO

At hippocampal excitatory synapses, endocannabinoids (eCBs) mediate two forms of retrograde synaptic inhibition that are induced by postsynaptic depolarization or activation of metabotropic glutamate receptors (mGluRs). The homer family of molecular scaffolds provides spatial organization to regulate postsynaptic signaling cascades, including those activated by mGluRs. Expression of the homer 1a (H1a) immediate-early gene produces a short homer protein that lacks the domain required for homer oligomerization, enabling it to uncouple homer assemblies. Here, we report that H1a differentially modulates two forms of eCB-mediated synaptic plasticity, depolarization-induced suppression of excitation (DSE) and metabotropic suppression of excitation (MSE). EPSCs were recorded from cultured hippocampal neurons and DSE evoked by a 15 s depolarization to 0 mV and MSE evoked by a type I mGluR agonist. Expression of H1a enhanced DSE and inhibited MSE at the same synapse. Many physiologically important stimuli initiate H1a expression including brain-derived neurotrophic factor (BDNF). Treating hippocampal cultures with BDNF increased transcription of H1a and uncoupled homer 1c-GFP (green fluorescent protein) clusters. BDNF treatment blocked MSE and enhanced DSE. Thus, physiological changes in H1a expression gate the induction pathway for eCB-mediated synaptic plasticity by uncoupling mGluR from eCB production.


Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Proteínas de Transporte/metabolismo , Endocanabinoides , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Moduladores de Receptores de Canabinoides/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Proteínas de Arcabouço Homer , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Vias Neurais/citologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
17.
Mol Pharmacol ; 80(3): 357-66, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21670103

RESUMO

HIV-1 infection of the central nervous system is associated with dendritic and synaptic damage that correlates with cognitive decline in patients with HIV-1-associated dementia (HAD). HAD is due in part to the release of viral proteins from infected cells. Because cannabinoids modulate neurotoxic and inflammatory processes, we investigated their effects on changes in synaptic connections induced by the HIV-1 envelope glycoprotein gp120. Morphology and synapses between cultured hippocampal neurons were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to green fluorescent protein (PSD95-GFP). Twenty-four-hour treatment with gp120 IIIB decreased the number of PSD95-GFP puncta by 37 ± 4%. The decrease was concentration-dependent (EC50 = 153 ± 50 pM). Synapse loss preceded cell death as defined by retention of DsRed2 fluorescence gp120 activated CXCR4 on microglia to evoke interleukin-1ß (IL-1ß) release. Pharmacological studies determined that sequential activation of CXCR4, the IL-1ß receptor, and the N-methyl-d-aspartate receptor was required. Expression of alternative reading frame polypeptide, which inhibits the ubiquitin ligase murine double minute 2, protected synapses, implicating the ubiquitin-proteasome pathway. Cannabimimetic drugs are of particular relevance to HAD because of their clinical and illicit use in patients with AIDS. The cannabinoid receptor full agonist [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt] (Win55,212-2) inhibited gp120-induced IL-1ß production and synapse in a manner reversed by a cannabinoid type 2 receptor antagonist. In contrast, Win55,212-2 did not inhibit synapse loss elicited by exposure to the HIV-1 protein Tat. These results indicate that cannabinoids prevent the impairment of network function produced by gp120 and, thus, might have therapeutic potential in HAD.


Assuntos
Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Receptor CB2 de Canabinoide/agonistas , Sinapses/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1 , Imuno-Histoquímica , Ratos , Receptores CXCR4/agonistas , Receptores de Interleucina-1/agonistas , Receptores de N-Metil-D-Aspartato/agonistas
18.
eNeuro ; 7(3)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32471847

RESUMO

Despite the success of antiretroviral therapy in suppressing viral load, nearly half of the 37 million people infected with HIV experience cognitive and motor impairments, collectively classified as HIV-associated neurocognitive disorders (HAND). In the CNS, HIV-infected microglia release neurotoxic agents that act indirectly to elicit excitotoxic synaptic injury. HIV trans-activator of transcription (Tat) protein is one such neurotoxin that is thought to play a major role in the neuropathogenesis of HAND. The endocannabinoid (eCB) system provides on-demand neuroprotection against excitotoxicity, and exogenous cannabinoids attenuate neurotoxicity in animal models of HAND. Whether this neuroprotective system is altered in the presence of HIV is unknown. Here, we examined the effects of Tat on the eCB system in rat primary hippocampal cultures. Using whole-cell patch-clamp electrophysiology, we measured changes in retrograde eCB signaling following exposure to Tat. Treatment with Tat significantly reduced the magnitude of depolarization-induced suppression of excitation (DSE) in a graded manner over the course of 48 h. Interestingly, Tat did not alter this form of short-term synaptic plasticity at inhibitory terminals. The Tat-induced decrease in eCB signaling resulted from impaired CB1 receptor (CB1R)-mediated presynaptic inhibition of glutamate release. This novel loss-of-function was particularly dramatic for low-efficacy agonists such as the eCB 2-arachidonoylglycerol (2-AG) and Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient in marijuana. Our observation that HIV Tat decreases CB1R function in vitro suggests that eCB-mediated neuroprotection may be reduced in vivo; this effect of Tat may contribute to synaptodendritic injury in HAND.


Assuntos
Infecções por HIV , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Animais , Endocanabinoides , Plasticidade Neuronal , Ratos , Receptor CB1 de Canabinoide , Sinapses
19.
J Neurosci ; 28(48): 12604-13, 2008 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-19036954

RESUMO

Human immunodeficiency virus (HIV)-1 infection of the CNS produces changes in dendritic morphology that correlate with cognitive decline in patients with HIV-1 associated dementia (HAD). Here, we investigated the effects of HIV-1 transactivator of transcription (Tat), a protein released by virus-infected cells, on synapses between hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein (PSD95-GFP). Tat (24 h) decreased the number of PSD95-GFP puncta by 50 +/- 7%. The decrease was concentration-dependent (EC(50) = 6 +/- 2 ng/ml) and preceded cell death. Tat acted via the low-density lipoprotein receptor-related protein (LRP) because the specific LRP blocker, receptor associated protein (RAP), prevented the Tat-induced decrease in the number of PSD95-GFP puncta. Ca(2+) influx through the NMDA receptor was necessary for Tat-induced synapse loss. Expression of an ubiquitin ligase inhibitor protected synapses, implicating the ubiquitin-proteasome pathway. In contrast to synapse loss, Tat induced cell death (48 h) required activation of nitric oxide synthase. The ubiquitin ligase-inhibitor nutlin-3 prevented synapse loss but not cell death induced by Tat. Thus, the pathways diverged, consistent with the hypothesis that synapse loss is a mechanism to reduce excess excitatory input rather than a symptom of the neuron's demise. Furthermore, application of RAP to cultures treated with Tat for 16 h reversed synapse loss. These results suggest that the impaired network function and decreased neuronal survival produced by Tat involve distinct mechanisms and that pharmacologic targets, such as LRP, might prove useful in restoring function in HAD patients.


Assuntos
Complexo AIDS Demência/patologia , Encéfalo/patologia , HIV/metabolismo , Degeneração Neural/patologia , Sinapses/patologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Complexo AIDS Demência/fisiopatologia , Animais , Bioensaio/métodos , Encéfalo/fisiopatologia , Encéfalo/virologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Regulação para Baixo/fisiologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Hipocampo/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Degeneração Neural/fisiopatologia , Degeneração Neural/virologia , Neurônios/patologia , Neurônios/virologia , Ratos , Receptores de LDL/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Membranas Sinápticas/metabolismo , Membranas Sinápticas/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
20.
Mol Pharmacol ; 75(4): 892-900, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19118122

RESUMO

Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive ingredient in marijuana, acts as a partial agonist on presynaptic cannabinoid type 1 (CB1) receptors to inhibit neurotransmitter release. Here, we report that THC inhibits excitatory neurotransmission between cultured rat hippocampal neurons in a manner highly sensitive to stimulus rate. THC (1 microM) inhibited excitatory postsynaptic currents (EPSCs) and whole-cell I(Ca) evoked at 0.1 Hz but at 0.5 Hz THC had little effect. The cannabinoid receptor full agonists [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt] (Win55212-2) (100 nM) and 2-arachidonylglycerol (1 microM) inhibited EPSCs independent of stimulation at 0.1 or 0.5 Hz. THC occupied CB1 receptors at 0.5 Hz, but the receptors failed to couple to presynaptic Ca(2+) channels. Consequently, 1 microM THC blocked the inhibition of EPSC amplitude by Win55212-2 when EPSCs were evoked at 0.5 Hz. A depolarizing prepulse to 0 mV reversed THC inhibition of I(Ca), but reversal of the inhibition produced by Win55212-2 required a pulse to +80 mV, suggesting that the voltage-dependent reversal of Gbetagamma inhibition of voltage-gated Ca(2+) channels accounts for the frequency-dependence of cannabinoid action. THC blocked depolarization-induced suppression of EPSCs evoked at 0.5 Hz, indicating that it inhibited retrograde endocannabinoid signaling in a frequency-dependent manner. Thus, THC displayed a state-dependent switching from agonist to antagonist that may account for its complex actions in vivo.


Assuntos
Dronabinol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Animais , Moduladores de Receptores de Canabinoides/antagonistas & inibidores , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Receptor CB1 de Canabinoide/fisiologia , Transmissão Sináptica/fisiologia , Fatores de Tempo
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