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Adverse effects of drugs on the human nervous system are rarely possible to anticipate based on preclinical neurotoxicity data, thus propagating the centuries long single most important obstacle to drug discovery and development for disorders of the nervous system. An emerging body of evidence indicates that in vivo electrophysiology using chronically implanted high-density electrodes (ciHDE) in freely moving animals is a rigorous method with enhanced potential for use in translational research. In particular, the structure and function of the hippocampal trisynaptic circuit (HTC) is conserved from rodents to primates, including Homo sapiens, suggesting that the effects of therapeutic agents and other potential neurologically active agents, whether beneficial or adverse, are likely to translate across species when interrogated using a conserved neural circuitry platform. This review explores science advances in the rapidly moving field of in vivo ciHDE in animal models of learning and memory. For this reason we focus on the HTC, where substantial research has investigated neural circuitry level responses and specific behaviors that reflect memory permitting a test of the ground truth validity of the findings. Examples of changes in neural network activity induced by endogenous neurotoxicants associated with neurodegenerative diseases, as well as exogenous therapeutics, drugs, and neurotoxicants are presented. Several illustrative examples of relevant findings that involve longer range neural circuitry outside of the HTC are discussed. Lastly, the limitations of in vivo ciHDE as applied to preclinical neurotoxicology are discussed with a view toward leveraging circuitry level actions to enhance our ability to project the specificity of in vitro target engagement with the desired psychopharmacological or neurological outcome. At the same time, the goal of reducing or eliminating significant neurotoxic adverse events in human is the desired endpoint. We believe that this approach will lead to enhanced discovery of high value neuroactive therapeutics that target neural circuitry domains as their primary mechanism of action, thus enhancing their ultimate contribution toward discovery of precision therapeutics.
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Decades of research attempting to slow the onset of Alzheimer's disease (AD) indicates that a better understanding of memory will be key to the discovery of effective therapeutic approaches. Here, we ask whether prodromal neural network dysfunction might occur in the hippocampal trisynaptic circuit by using α5IA (an established memory enhancer and selective negative allosteric modulator of extrasynaptic tonically active α5GABA-A receptors) as a probe drug in TgF344-AD transgenic rats, a model for ß-amyloid induced early onset AD. The results demonstrate that orally bioavailable α5IA increases CA1 pyramidal cell mean firing rates during foraging and peak ripple amplitude during wakeful immobility in wild type F344 rats in a familiar environment. We further demonstrate that CA1 ripples in TgF344-AD rats are nonresponsive to α5IA by 9 months of age, prior to the onset of AD-like pathology and memory dysfunction. TgF344-AD rats express human ß-amyloid precursor protein (with the Swedish mutation) and human presenilin-1 (with a Δ exon 9 mutation) and we found high serum Aß42 and Aß40 levels by 3 months of age. When taken together, this demonstrates, to the best of our knowledge, the first evidence for prodromal α5GABA-A receptor dysfunction in the ripple-generating hippocampal trisynaptic circuit of AD-like transgenic rats. As α5GABA-A receptors are found at extrasynaptic and synaptic contacts, we posit that negative modulation of α5GABA-A receptor mediated tonic as well as phasic inhibition augments CA1 ripples and memory consolidation but that this modulatory mechanism is lost at an early stage of AD onset.
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Memory dysfunction is a symptomatic feature of many neurologic and neuropsychiatric disorders; however, the basic underlying mechanisms of memory and altered states of circuitry function associated with disorders of memory remain a vast unexplored territory. The initial discovery of endogenous neurosteroids triggered a quest to elucidate their role as neuromodulators in normal and diseased brain function. In this review, based on the perspective of our own research, the advances leading to the discovery of positive and negative neurosteroid allosteric modulators of GABA type-A (GABAA), NMDA, and non-NMDA type glutamate receptors are brought together in a historical and conceptual framework. We extend the analysis toward a state-of-the art view of how neurosteroid modulation of neural circuitry function may affect memory and memory deficits. By aggregating the results from multiple laboratories using both animal models for disease and human clinical research on neuropsychiatric and age-related neurodegenerative disorders, elements of a circuitry level view begins to emerge. Lastly, the effects of both endogenously active and exogenously administered neurosteroids on neural networks across the life span of women and men point to a possible underlying pharmacological connectome by which these neuromodulators might act to modulate memory across diverse altered states of mind.
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Learning and memory deficits associated with age-related mild cognitive impairment have long been attributed to impaired processing within the hippocampus. Hyperactivity within the hippocampal CA3 region that is associated with aging is mediated in part by a loss of functional inhibitory interneurons and thought to underlie impaired performance in spatial memory tasks, including the abnormal tendency in aged animals to pattern complete spatial representations. Here, we asked whether the spatial firing patterns of simultaneously recorded CA3 and CA1 neurons in young and aged rats could be manipulated pharmacologically to selectively reduce CA3 hyperactivity and thus, according to hypothesis, the associated abnormality in spatial representations. We used chronically implanted high-density tetrodes to record the spatial firing properties of CA3 and CA1 units during animal exploration for food in familiar and novel environments. Aged CA3 place cells have higher firing rates, larger place fields, less spatial information content, and respond less to a change from a familiar to a novel environment than young CA3 cells. We also find that the combination of levetiracetam (LEV) + valproic acid (VPA), previously shown to act as a cognitive enhancer in tests of spatial memory, attenuate CA3 place cell firing rates, reduce place field area, and increase spatial information content in aged but not young adult rats. This is consistent with drug enhancing the specificity of neuronal firing with respect to spatial location. Contrary to expectation, however, LEV + VPA reduces place cell discrimination between novel and familiar environments, i.e., spatial correlations increase, independent of age even though drug enhances performance in cognitive tasks. The results demonstrate that spatial information content, or the number of bits of information encoded per action potential, may be the key correlate for enhancement of spatial memory by LEV + VPA.
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Envejecimiento/efectos de los fármacos , Región CA3 Hipocampal/efectos de los fármacos , Fármacos del Sistema Nervioso Central/administración & dosificación , Piracetam/análogos & derivados , Percepción Espacial/efectos de los fármacos , Ácido Valproico/administración & dosificación , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Envejecimiento/fisiología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/fisiología , Región CA3 Hipocampal/fisiología , Discriminación en Psicología/efectos de los fármacos , Discriminación en Psicología/fisiología , Sinergismo Farmacológico , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Levetiracetam , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Piracetam/administración & dosificación , Ratas Long-Evans , Percepción Espacial/fisiología , Memoria Espacial/efectos de los fármacos , Memoria Espacial/fisiologíaRESUMEN
Anxiety disorders are a major public health concern. Here, we examine the familiar area of anxiolysis in the context of a systems-level understanding that will hopefully lead to revealing an underlying pharmacological connectome. The introduction of benzodiazepines nearly half a century ago markedly improved the treatment of anxiety disorders. These agents reduce anxiety rapidly by allosterically enhancing the postsynaptic actions of GABA at inhibitory type A GABA receptors but side effects limit their use in chronic anxiety disorders. Selective serotonin reuptake inhibitors and serotonin/norepinephrine reuptake inhibitors have emerged as an effective first-line alternative treatment of such anxiety disorders. However, many individuals are not responsive and side effects can be limiting. Research into a relatively new class of agents known as neurosteroids has revealed novel modulatory sites and mechanisms of action that are providing insights into the pathophysiology of certain anxiety disorders, potentially bridging the gap between the GABAergic and serotonergic circuits underlying anxiety. However, translating the pharmacological activity of compounds targeted to specific receptor subtypes in rodent models of anxiety to effective therapeutics in human anxiety has not been entirely successful. Since modulating any one of several broad classes of receptor targets can produce anxiolysis, we posit that a systems-level discovery platform combined with an individualized medicine approach based on noninvasive brain imaging would substantially advance the development of more effective therapeutics.
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Ansiolíticos/farmacología , Trastornos de Ansiedad/tratamiento farmacológico , Diseño de Fármacos , Animales , Ansiolíticos/efectos adversos , Trastornos de Ansiedad/fisiopatología , Encéfalo/fisiopatología , Humanos , Terapia Molecular Dirigida , Receptores de GABA-A/metabolismoRESUMEN
Fast excitatory synaptic transmission that is contingent upon N-methyl d-aspartate receptor (NMDAR) function contributes to core information flow in the central nervous system and to the plasticity of neural circuits that underlie cognition. Hypoactivity of excitatory NMDAR-mediated neurotransmission is hypothesized to underlie the pathophysiology of schizophrenia, including the associated cognitive deficits. The neurosteroid pregnenolone (PREG) and its metabolites pregnenolone sulfate (PregS) and allopregnanolone in serum are inversely associated with cognitive improvements after oral PREG therapy, raising the possibility that brain neurosteroid levels may be modulated therapeutically. PregS is derived from PREG, the precursor of all neurosteroids, via a single sulfation step and is present at low nanomolar concentrations in the central nervous system. PregS, but not PREG, augments long-term potentiation and cognitive performance in animal models of learning and memory. In this report, we communicate the first observation that PregS, but not PREG, is a potent (EC50 â¼2 pM) enhancer of intracellular Ca(2+) that is contingent upon neuronal activity, NMDAR-mediated synaptic activity, and L-type Ca(2+) channel activity. Low picomolar PregS similarly activates cAMP response element-binding protein (CREB) phosphorylation (within 10 minutes), an essential memory molecule, via an extracellular-signal-regulated kinase/mitogen-activated protein kinase signal transduction pathway. Taken together, the results are consistent with a novel biologic role for the neurosteroid PregS that acts at picomolar concentrations to intensify the intracellular response to glutamatergic signaling at synaptic but not extrasynaptic, NMDARs by differentially augmenting CREB activation. This provides a genomic signal transduction mechanism by which PregS could participate in memory consolidation of relevance to cognitive function.
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Señalización del Calcio , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Pregnenolona/farmacología , Potenciales Sinápticos , Animales , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas , Masculino , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/fisiologíaRESUMEN
RATIONALE: The neurosteroid pregnenolone sulfate (PregS) acts as a cognitive enhancer and modulator of neurotransmission, yet aligning its pharmacological and physiological effects with reliable measurements of endogenous local concentrations and pharmacological and therapeutic targets has remained elusive for over 20 years. OBJECTIVES: New basic and clinical research concerning neurosteroid modulation of the central nervous system (CNS) function has emerged over the past 5 years, including important data involving pregnenolone and various neurosteroid precursors of PregS that point to a need for a critical status update. RESULTS: Highly specific actions of PregS affecting excitatory N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic transmission and the pharmacological effects of PregS on various receptors and ion channels are discussed. The discovery of a high potency (nanomolar) signal transduction pathway for PregS-induced NMDAR trafficking to the cell surface via a Ca(2+)- and G protein-coupled receptor (GPCR)-dependent mechanism and a potent (EC50 ~ 2 pM) direct enhancement of intracellular Ca(2+) levels is discussed in terms of its agonist effects on long-term potentiation (LTP) and memory. Lastly, preclinical and clinical studies assessing the promnestic effects of PregS and pregnenolone toward cognitive dysfunction in schizophrenia, and altered serum levels in epilepsy and alcohol dependence, are reviewed. CONCLUSIONS: PregS is present in human and rodent brain at physiologically relevant concentrations and meets most of the criteria for an endogenous neurotransmitter/neuromodulator. PregS likely plays a significant role in modulation of glutamatergic excitatory synaptic transmission underlying learning and memory, yet the molecular target(s) for its action awaits identification.
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Plasticidad Neuronal/efectos de los fármacos , Pregnanolona/farmacología , Sinapsis/efectos de los fármacos , Animales , Humanos , Neurotransmisores , Nootrópicos/farmacología , Pregnanolona/fisiología , Pregnanolona/uso terapéuticoRESUMEN
The use-dependent regulation of the GABAA receptor occurs under physiological, pathological, and pharmacological conditions. Tolerance induced by prolonged administration of benzodiazepines is associated with changes in GABAA receptor function. Chronic exposure of neurons to GABA for 48 hr induces a downregulation of the GABAA receptor number and an uncoupling of the GABA/benzodiazepine site interactions. A single brief exposure ((t1/2) = 3 min) of rat neocortical neurons to the neurotransmitter initiates a process that results in uncoupling hours later (t(1/2) = 12 hr) without alterations in the number of GABAA receptors and provides a paradigm to study the uncoupling mechanism selectively. Here we report that uncoupling induced by a brief GABAA receptor activation is blocked by the coincubation with inhibitors of protein kinases A and C, indicating that the uncoupling is mediated by the activation of a phosphorylation cascade. GABA-induced uncoupling is accompanied by subunit-selective changes in the GABAA receptor mRNA levels. However, the GABA-induced downregulation of the α3 subunit mRNA level is not altered by the kinase inhibitors, suggesting that the uncoupling is the result of a posttranscriptional regulatory process. GABA exposure also produces an increase in the serine phosphorylation on the GABAA receptor γ2 subunit. Taken together, our results suggest that the GABA-induced uncoupling is mediated by a posttranscriptional mechanism involving an increase in the phosphorylation of GABAA receptors. The uncoupling of the GABAA receptor may represent a compensatory mechanism to control GABAergic neurotransmission under conditions in which receptors are persistently activated.
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Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Subunidades de Proteína/metabolismo , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/farmacología , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
An earlier age at onset of Parkinson's disease (PD) has been reported to be associated with occupational exposures to manganese and hydrocarbon solvents suggesting that exposure to neurotoxic chemicals may hasten the progression of idiopathic PD. In this study the role of occupational exposure to metals and pesticides in the progression of idiopathic PD was assessed by looking at age at disease onset. The effects of heritable genetic risk factors, which may also influence age at onset, was minimized by including only sporadic cases of PD with no family history of the disease (n=58). Independent samples Student t-test revealed that subjects with occupational exposure to metals and/or pesticides (n=36) were significantly (p=0.013) younger than unexposed controls (n=22). These subjects were then divided into three groups [high (n=18), low (n=18), and unexposed (n=22)] to ascertain if duration of exposure further influenced age at onset of PD. One-way ANOVA revealed that subjects in the high exposure group were significantly (p=0.0121) younger (mean age: 50.33 years) than unexposed subjects (mean age: 60.45 years). Subjects were also stratified by exposure type (metals vs. pesticides). These results suggest that chronic exposure to metals and pesticides is associated with a younger age at onset of PD among patients with no family history of the disease and that duration of exposure is a factor in the magnitude of this effect.
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BACKGROUND: The γ-aminobutyric acid (GABA) type A receptor (GABA(A)R) contains the recognition sites for a variety of agents used in the treatment of brain disorders, including anxiety and epilepsy. A better understanding of how receptor expression is regulated in individual neurons may provide novel opportunities for therapeutic intervention. Towards this goal we have studied transcription of a GABA(A)R subunit gene (GABRB1) whose activity is autologously regulated by GABA via a 10 base pair initiator-like element (ß(1)-INR). METHODS: By screening a human cDNA brain library with a yeast one-hybrid assay, the Polycomblike (PCL) gene product PHD finger protein transcript b (PHF1b) was identified as a ß(1)-INR associated protein. Promoter/reporter assays in primary rat cortical cells demonstrate that PHF1b is an activator at GABRB1, and chromatin immunoprecipitation assays reveal that presence of PHF1 at endogenous Gabrb1 is regulated by GABA(A)R activation. RESULTS: PCL is a member of the Polycomb group required for correct spatial expression of homeotic genes in Drosophila. We now show that PHF1b recognition of ß(1)-INR is dependent on a plant homeodomain, an adjacent helix-loop-helix, and short glycine rich motif. In neurons, it co-immunoprecipitates with SUZ12, a key component of the Polycomb Repressive Complex 2 (PRC2) that regulates a number of important cellular processes, including gene silencing via histone H3 lysine 27 trimethylation (H3K27me3). CONCLUSIONS: The observation that chronic exposure to GABA reduces PHF1 binding and H3K27 monomethylation, which is associated with transcriptional activation, strongly suggests that PHF1b may be a molecular transducer of GABA(A)R function and thus GABA-mediated neurotransmission in the central nervous system.
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Proteínas de Unión al ADN/metabolismo , Neuronas/efectos de los fármacos , Subunidades de Proteína/metabolismo , Receptores de GABA-A/metabolismo , Factores de Transcripción/metabolismo , Ácido gamma-Aminobutírico/farmacología , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Hipocampo/citología , Humanos , Masculino , Neocórtex/citología , Neuronas/metabolismo , Proteínas del Grupo Polycomb , Subunidades de Proteína/genética , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Factores de Transcripción/genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60-100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin-sensitive, G protein-coupled, and Ca(2+)-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.
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Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Pregnenolona/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células Cultivadas , Exocitosis/efectos de los fármacos , N-Metilaspartato/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Receptores de GABA-A/metabolismo , Receptores sigma/metabolismo , Xenopus laevis/metabolismoRESUMEN
Regulation of gene expression via brain-derived neurotrophic factor (BDNF) is critical to the development of the nervous system and may well underlie cognitive performance throughout life. We now describe a mechanism by which BDNF can exert its effects on postsynaptic receptor populations that may have relevance to both the normal and diseased brain where BDNF levels either rise or fall in association with changes in excitatory neurotransmission. Increased levels of NMDA receptors (NMDARs) occur in rat cortical neurons via synthesis of new NMDA receptor 1 (NR1) subunits. The majority of synthesis is controlled by binding of cAMP response element binding protein (CREB) and early growth response factor 3 (Egr3) to the core NR1 promoter (NR1-p) region. BDNF-mediated NR1 transcription depends upon induction of the mitogen-activated protein kinase (MAPK) pathway through activation of the TrK-B receptor. Taken together with the fact that NMDAR activation stimulates BDNF synthesis, our results uncover a feed-forward gene regulatory network that may enhance excitatory neurotransmission to change neuronal behavior over time.
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Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteína de Unión a CREB/metabolismo , Corteza Cerebral/citología , Canales de Potasio Éter-A-Go-Go/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Canales de Potasio Éter-A-Go-Go/genética , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas Luminiscentes/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Receptor trkB/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Transfección , Dedos de Zinc/genéticaRESUMEN
NMDA receptor (NMDAR)-mediated excitatory synaptic transmission plays a critical role in synaptic plasticity and memory formation, whereas its dysfunction may underlie neuropsychiatric and neurodegenerative diseases. The neuroactive steroid pregnenolone sulfate (PS) acts as a cognitive enhancer in impaired animals, augments LTP in hippocampal slices by enhancing NMDAR activity, and may participate in the reduction of schizophrenia's negative symptoms by systemic pregnenolone. We report that the effects of PS on NMDAR function are diverse, varying with subunit composition and NR1 splice variant. While PS potentiates NR1-1a/NR2B receptors through a critical steroid modulatory domain in NR2B that also modulates tonic proton inhibition, potentiation of the NMDA response is not dependent upon relief of such inhibition, a finding that distinguishes it from spermine. In contrast, the presence of an NR2A subunit confers enhanced PS-potentiation at reduced pH, suggesting that it may indeed act like spermine does at NR2B-containing receptors. Additional tuning of the NMDAR response by PS comes via the N-terminal exon-5 splicing insert of NR1-1b, which regulates the magnitude of proton-dependent PS potentiation. For NR2C- and NR2D-containing receptors, negative modulation at NR2C receptors is pH-independent (like NR2B) while negative modulation at NR2D receptors is pH-dependent (like NR2A). Taken together, PS displays a rich modulatory repertoire that takes advantage of the structural diversity of NMDARs in the CNS. The differential pH sensitivity of NMDAR isoforms to PS modulation may be especially important given the emerging role of proton sensors to both learning and memory, as well as brain injury.
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Exones , Pregnenolona/fisiología , Protones , Receptores de N-Metil-D-Aspartato/fisiología , Empalme Alternativo/genética , Animales , Cristalografía por Rayos X , Exones/genética , Femenino , Inhibición Neural/genética , Mutación Puntual , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevisRESUMEN
Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.
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Cuerpo Estriado/ultraestructura , Dopamina/metabolismo , Pregnenolona/farmacología , Terminales Presinápticos/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Glicina/farmacología , Masculino , Microscopía Electrónica de Rastreo/métodos , N-Metilaspartato/farmacología , Cloruro de Potasio/farmacología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Tritio/metabolismo , Valina/análogos & derivados , Valina/farmacologíaRESUMEN
BACKGROUND: Compounds targeting the benzodiazepine binding site of the GABAA-R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for pre-anesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABAA-R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABAA-R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between alpha1/alpha2-containing receptors for an anxioselective that is predicted by studies using transgenic mice. RESULTS: We hypothesized that the pharmacological properties of ocinaplon in vivo might be influenced by an active biotransformation product with greater selectivity for the alpha2 subunit relative to alpha1. One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABAA-Rs, but that its selectivity profile is similar to that of ocinaplon. CONCLUSION: These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABAA-R modulators.
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Ansiolíticos/farmacología , Óxidos N-Cíclicos/farmacología , Diazepam/farmacología , Pirimidinas/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Ansiolíticos/metabolismo , Línea Celular , Óxidos N-Cíclicos/metabolismo , Diazepam/metabolismo , Humanos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Pirimidinas/metabolismo , Receptores de GABA-A/fisiología , Xenopus laevisRESUMEN
The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.
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Proteína de Unión a CREB/metabolismo , Corteza Cerebral/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/biosíntesis , Elementos de Respuesta , Transcripción Genética , Alcoholismo/metabolismo , Alcoholismo/patología , Animales , Proteína de Unión a CREB/genética , Células Cultivadas , Corteza Cerebral/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Activación Enzimática , Epilepsia/metabolismo , Epilepsia/patología , Neuronas/patología , Proteína Quinasa C/metabolismo , ARN Mensajero/biosíntesis , Ratas , Sinaptosomas/metabolismoRESUMEN
Central nervous system function is critically dependent upon an exquisitely tuned balance between excitatory synaptic transmission, mediated primarily by glutamate, and inhibitory synaptic transmission, mediated primarily by GABA. Modulation of either excitation or inhibition would be expected to result in altered functionality of finely tuned synaptic pathways and global neural systems, leading to altered nervous system function. Administration of positive or negative modulators of ligand-gated ion channels has been used extensively and successfully in CNS therapeutics, particularly for the induction of sedation and treatment of anxiety, seizures, insomnia, and pain. Excessive activation of excitatory glutamate receptors, such as in cerebral ischemia, can result in neuronal damage via excitotoxic mechanisms. The discovery that neuroactive steroids exert rapid, direct effects upon the function of both excitatory and inhibitory neurotransmitter receptors has raised the possibility that endogenous neurosteroids may play a regulatory role in synaptic transmission by modulating the balance between excitatory and inhibitory neurotransmission. The sites to which neuroactive steroids bind may also serve as targets for the discovery of therapeutic neuromodulators.
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Neurotransmisores/fisiología , Esteroides/fisiología , Animales , Química Encefálica/fisiología , Sulfato de Deshidroepiandrosterona/farmacología , Humanos , Modelos Moleculares , Neurotransmisores/metabolismo , Pregnenolona/farmacología , Receptores de Glutamato/efectos de los fármacos , Receptores de Neurotransmisores/efectos de los fármacos , Esteroides/metabolismo , Sulfatasas/metabolismo , Sulfatos , Sulfotransferasas/metabolismoRESUMEN
Studies using mice with point mutations of GABA(A) receptor alpha subunits suggest that the sedative and anxiolytic properties of 1,4-benzodiazepines are mediated, respectively, by GABA(A) receptors bearing the alpha(1) and alpha(2) subunits. This hypothesis predicts that a compound with high efficacy at GABA(A) receptors containing the alpha(1) subunit would produce sedation, whereas an agonist acting at alpha(2) subunit-containing receptors (with low or null efficacy at alpha(1)-containing receptors) would be anxioselective. Electrophysiological studies using recombinant GABA(A) receptors expressed in Xenopus oocytes indicate that maximal potentiation of GABA-stimulated currents by the pyrazolo-[1,5-a]-pyrimidine, DOV 51892, at alpha(1)beta(2)gamma(2S) constructs of the GABA(A) receptor was significantly higher (148%) than diazepam. In contrast, DOV 51892 was considerably less efficacious and/or potent than diazepam in enhancing GABA-stimulated currents mediated by constructs containing alpha(2), alpha(3), or alpha(5) subunits. In vivo, DOV 51892 increased punished responding in the Vogel conflict test, an effect blocked by flumazenil, and increased the percentage of time spent in the open arms of the elevated plus-maze. However, DOV 51892 had no consistent effects on motor function or muscle relaxation at doses more than 1 order of magnitude greater than the minimal effective anxiolytic dose. Although the mutant mouse data predict that the high-efficacy potentiation of GABA(A1a) receptor-mediated currents by DOV 51892 would be sedating, behavioral studies demonstrate that DOV 51892 is anxioselective, indicating that GABA potentiation mediated by alpha(1) subunit-containing GABA(A) receptors may be neither the sole mechanism nor highly predictive of the sedative properties of benzodiazepine recognition site modulators.
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Ansiolíticos/farmacología , Canales de Cloruro/metabolismo , Diazepam/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/fisiología , Animales , Ansiolíticos/síntesis química , Ataxia/inducido químicamente , Ataxia/patología , Canales de Cloruro/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Conducta Exploratoria/efectos de los fármacos , Fuerza de la Mano/fisiología , Hipnóticos y Sedantes/farmacología , Masculino , Actividad Motora/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Oocitos/metabolismo , Equilibrio Postural/efectos de los fármacos , Pirazoles/síntesis química , Piridinas/síntesis química , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Xenopus laevisRESUMEN
Benzodiazepines (BZDs) have been used extensively for more than 40 years because of their high therapeutic index and low toxicity. Although BZDs are understood to act primarily as allosteric modulators of GABA(A) receptors, the mechanism of modulation is not well understood. The applicability of an allosteric model with two binding sites for gamma-aminobutyric acid (GABA) and one for a BZD-like modulator was investigated. This model predicts that BZDs should enhance the efficacy of partial agonists. Consistent with this prediction, diazepam increased the efficacy of the GABA(A) receptor partial agonist kojic amine in chick spinal cord neurons. To further test the validity of the model, the effects of diazepam, flurazepam, and zolpidem were examined using wild-type and spontaneously active mutant alpha1(L263S)beta3gamma2 GABA(A) receptors expressed in HEK-293 cells. In agreement with the predictions of the allosteric model, all three modulators acted as direct agonists for the spontaneously active receptors. The results indicate that BZD-like modulators enhance the amplitude of the GABA response by stabilizing the open channel active state relative to the inactive state by less than 1 kcal, which is similar to the energy of stabilization conferred by a single hydrogen bond.