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OBJECTIVE. The purpose of this article is to summarize the role of molecular imaging of the brain by use of SPECT, FDG PET, and non-FDG PET radiotracers in epilepsy. CONCLUSION. Quantitative image analysis with PET and SPECT has increased the diagnostic utility of these modalities in localizing epileptogenic onset zones. A multi-modal platform approach integrating the functional imaging of PET and SPECT with the morphologic information from MRI in presurgical evaluation of epilepsy can greatly improve outcomes.
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Encéfalo/diagnóstico por imagem , Epilepsia/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada de Emissão de Fóton Único , Adolescente , Adulto , Criança , Pré-Escolar , Cisteína/análogos & derivados , Cisteína/farmacocinética , Feminino , Fluordesoxiglucose F18/farmacocinética , Humanos , Masculino , Pessoa de Meia-Idade , Compostos de Organotecnécio/farmacocinética , Oximas/farmacocinética , Compostos Radiofarmacêuticos/farmacocinéticaRESUMO
OBJECTIVE: Postictal generalized electroencephalographic suppression (PGES) is a pattern of low-voltage scalp electroencephalographic (EEG) activity following termination of generalized seizures. PGES has been associated with both sudden unexplained death in patients with epilepsy and therapeutic efficacy of electroconvulsive therapy (ECT). Automated detection of PGES epochs may aid in reliable quantification of this phenomenon. METHODS: We developed a voltage-based algorithm for detecting PGES. This algorithm applies existing criteria to simulate expert epileptologist readings. Validation relied on postictal EEG recording from patients undergoing ECT (NCT02761330), assessing concordance among the algorithm and four clinical epileptologists. RESULTS: We observed low-to-moderate concordance among epileptologist ratings of PGES. Despite this, the algorithm displayed high discriminability in comparison to individual epileptologists (C-statistic range: 0.86-0.92). The algorithm displayed high discrimination (C-statistic: 0.91) and substantial peak agreement (Cohen's Kappa: 0.65) in comparison to a consensus of clinical ratings. Interrater agreement between the algorithm and individual epileptologists was on par with that among expert epileptologists. CONCLUSIONS: An automated voltage-based algorithm can be used to detect PGES following ECT, with discriminability nearing that of experts. SIGNIFICANCE: Algorithmic detection may support clinical readings of PGES and improve precision when correlating this marker with clinical outcomes following generalized seizures.
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Algoritmos , Eletroencefalografia/normas , Epilepsia/epidemiologia , Epilepsia/fisiopatologia , Morte Súbita Inesperada na Epilepsia/epidemiologia , Eletroencefalografia/métodos , Epilepsia/diagnóstico , Humanos , Reprodutibilidade dos Testes , Morte Súbita Inesperada na Epilepsia/prevenção & controleRESUMO
OBJECTIVE: We devise a data-driven framework to assess the level of consciousness in etiologically heterogeneous comatose patients using intrinsic dynamical changes of resting-state Electroencephalogram (EEG) signals. METHODS: EEG signals were collected from 54 comatose patients (GCSâ¯â©½â¯8) and 20 control patients (GCSâ¯>â¯8). We analyzed the EEG signals using a new technique, termed Intrinsic Network Reactivity Index (INRI), that aims to assess the overall lability of brain dynamics without the use of extrinsic stimulation. The proposed technique uses three sigma EEG events as a trigger for ensuing changes to the directional derivative of signals across the EEG montage. RESULTS: The INRI had a positive relationship with GCS and was significantly different between various levels of consciousness. In comparison, classical band-limited power analysis did not show any specific patterns correlated to GCS. CONCLUSIONS: These findings suggest that reaching low variance EEG activation patterns becomes progressively harder as the level of consciousness of patients deteriorate, and provide a quantitative index based on passive measurements that characterize this change. SIGNIFICANCE: Our results emphasize the role of intrinsic brain dynamics in assessing the level of consciousness in coma patients and the possibility of employing simple electrophysiological measures to recognize the severity of disorders of consciousness (DOC).
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Coma/diagnóstico , Estado de Consciência , Eletroencefalografia/métodos , Adulto , Idoso , Algoritmos , Encéfalo/fisiopatologia , Coma/classificação , Eletroencefalografia/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Rapidly determining the causes of a depressed level of consciousness (DLOC) including coma is a common clinical challenge. Quantitative analysis of the electroencephalogram (EEG) has the potential to improve DLOC assessment by providing readily deployable, temporally detailed characterization of brain activity in such patients. While used commonly for seizure detection, EEG-based assessment of DLOC etiology is less well-established. As a first step towards etiological diagnosis, we sought to distinguish focal and diffuse causes of DLOC through assessment of temporal dynamics within EEG signals. METHODS: We retrospectively analyzed EEG recordings from 40 patients with DLOC with consensus focal or diffuse culprit pathology. For each recording, we performed a suite of time-series analyses, then used a statistical framework to identify which analyses (features) could be used to distinguish between focal and diffuse cases. RESULTS: Using cross-validation approaches, we identified several spectral and non-spectral EEG features that were significantly different between DLOC patients with focal vs. diffuse etiologies, enabling EEG-based classification with an accuracy of 76%. CONCLUSIONS: Our findings suggest that DLOC due to focal vs. diffuse injuries differ along several electrophysiological parameters. These results may form the basis of future classification strategies for DLOC and coma that are more etiologically-specific and therefore therapeutically-relevant.
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Coma/etiologia , Transtornos da Consciência/etiologia , Eletroencefalografia/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
OBJECTIVES: Specific changes in the functional connectivity of brain networks occur in patients with epilepsy. Yet whether such changes reflect a stable disease effect or one that is a function of active seizure burden remains unclear. Here, we longitudinally assessed the connectivity of canonical cognitive functional networks in patients with intractable temporal lobe epilepsy (TLE), both before and after patients underwent epilepsy surgery and achieved seizure freedom. METHODS: Seventeen patients with intractable TLE who underwent epilepsy surgery with Engel class I outcome and 17 matched healthy controls took part in the study. The functional connectivity of a set of cognitive functional networks derived from typical cognitive tasks was assessed in patients, preoperatively and postoperatively, as well as in controls, using stringent methods of artifact reduction. RESULTS: Preoperatively, functional networks in TLE patients differed significantly from healthy controls, with differences that largely, but not exclusively, involved the default mode and temporal/auditory subnetworks. However, undergoing epilepsy surgery and achieving seizure freedom did not lead to significant changes in network connectivity, with postoperative functional network abnormalities closely mirroring the preoperative state. SIGNIFICANCE: This result argues for a stable chronic effect of the disease on brain connectivity, with changes that are largely "burned in" by the time a patient with intractable TLE undergoes epilepsy surgery, which typically occurs years after the initial diagnosis. The result has potential implications for the treatment of intractable epilepsy, suggesting that delaying surgical intervention that may achieve seizure freedom may lead to functional network changes that are no longer reversible by the time of epilepsy surgery.
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Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/cirurgia , Rede Nervosa/fisiopatologia , Cuidados Pós-Operatórios/tendências , Convulsões/fisiopatologia , Convulsões/cirurgia , Adulto , Eletroencefalografia/tendências , Epilepsia do Lobo Temporal/diagnóstico por imagem , Feminino , Humanos , Imageamento por Ressonância Magnética/tendências , Masculino , Pessoa de Meia-Idade , Rede Nervosa/diagnóstico por imagem , Convulsões/diagnóstico por imagem , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/fisiopatologia , Lobo Temporal/cirurgia , Adulto JovemRESUMO
N-methyl-d-aspartate receptors (NMDARs) govern synaptic plasticity, development, and neuronal response to insult. Prolonged activation of NMDARs such as during an insult may activate secondary currents or modulate Mg2+ sensitivity, but the conditions under which these occur are not fully defined. We reexamined the effect of prolonged NMDAR activation in juvenile mouse hippocampal slices. NMDA (10 µM) elicited current with the expected negative-slope conductance in the presence of 1.2 mM Mg2+ However, several minutes of continued NMDA exposure elicited additional inward current at -70 mV. A higher concentration of NMDA (100 µM) elicited the current more rapidly. The additional current was not dependent on Ca2+, network activity, or metabotropic NMDAR function and did not persist on agonist removal. Voltage ramps revealed no alteration of either reversal potential or NMDA-elicited conductance between -30 mV and +50 mV. The result was a more linear NMDA current-voltage relationship. The current linearization was also induced in interneurons and in mature dentate granule neurons but not immature dentate granule cells, dissociated cultured hippocampal neurons, or nucleated patches excised from CA1 pyramidal neurons. Comparative simulations of NMDA application to a CA1 pyramidal neuron and to a cultured neuron revealed that linearization can be explained by space-clamp errors arising from gradual recruitment of distal dendritic NMDARs. We conclude that persistent secondary currents do not strongly contribute to NMDAR responses in juvenile mouse hippocampus and careful discernment is needed to exclude contributions of clamp artifacts to apparent secondary currents.NEW & NOTEWORTHY We report that upon sustained activation of NMDARs in juvenile mouse hippocampal neurons there is apparent loss of Mg2+ block at negative membrane potentials. However, the phenomenon is explained by loss of dendritic voltage clamp, leading to a linear current-voltage relationship. Our results give a specific example of how spatial voltage errors in voltage-clamp recordings can readily be misinterpreted as biological modulation.
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Magnésio/metabolismo , Potenciais da Membrana/fisiologia , N-Metilaspartato/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Artefatos , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Células Cultivadas , Simulação por Computador , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Técnicas de Cultura de TecidosRESUMO
The major cholesterol metabolite in brain, 24(S)-hydroxycholesterol (24S-HC), serves as a vehicle for cholesterol removal. Its effects on neuronal function, however, have only recently begun to be investigated. Here, we review that nascent work. Our own studies have demonstrated that 24S-HC has potent positive modulatory effects on N-methyl-d-aspartate (NMDA) receptor (NMDAR) function. This could have implications not only for brain plasticity but also for pathological NMDAR overuse. Other work has demonstrated effects of 24S-HC on neuronal survival and as a possible biomarker of neurodegenerative disease. Depending on circumstances, both upregulation/mimicry of 24S-HC signaling and down-regulation/antagonism may have therapeutic potential. We are interested in the possibility that synthetic analogues of 24S-HC with positive effects at NMDARs may hold neurotherapeutic promise, given the role of NMDA receptor hypofunction in certain neuropsychiatric disorders.
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Hidroxicolesteróis/metabolismo , Hidroxicolesteróis/farmacologia , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Encefalopatias/tratamento farmacológico , Encefalopatias/genética , Encefalopatias/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colesterol 24-Hidroxilase/genética , Humanos , Camundongos , Neurônios/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
It is widely appreciated that neuronal networks exhibit patterns of bursting and synchrony that are not captured by simple measures such as average spike rate. These patterns can encode information or represent pathological behavior such as seizures. However, methods for quantifying bursting and synchrony are not agreed upon and can be confounded with spike rate measures. Previous validation has largely relied on in silico networks and single experimental conditions. How published measures of bursting and synchrony perform when applied to biological networks of varied average spike rate and subjected to varied experimental challenges is unclear. In multielectrode array recordings of network activity, we found that two mechanistically distinct drugs, cyclothiazide and bicuculline, produced equivalent increases in average spike rate but differed in bursting and synchrony. We applied several measures of bursting to the recordings (2 threshold interval methods and a surprise-based method) and found that a measure based on an average critical interval, adjusted for the array-wide spike rate, performed best in quantifying differential drug effects. To quantify synchrony, we compared a coefficient of variation-based measure, the recently proposed spike time tiling coefficient, the SPIKE-distance measure, and a global synchrony index. The spike time tiling coefficient, the SPIKE-distance measure, and the global synchrony index all captured a difference between drugs with the best performance exhibited by the global synchrony index. In summary, our exploration should aid other investigators by highlighting strengths and limitations of current methods.
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Potenciais de Ação/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Benzotiadiazinas/farmacologia , Bicuculina/farmacologia , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Hipocampo/efeitos dos fármacos , Microeletrodos , Neurônios/efeitos dos fármacos , Periodicidade , Ratos , Processamento de Sinais Assistido por Computador , SoftwareRESUMO
Preclinical and clinical studies have demonstrated the significance of inflammation and autoantibodies in epilepsy, and the use of immunotherapies in certain situations has become an established practice. Temporal lobe epilepsy can follow paraneoplastic or nonparaneoplastic limbic encephalitis associated with antibodies directed against brain antigens. Here, we focus on a patient with worsening confusion and temporal lobe seizures despite treatment with antiepileptic medications. Serial brain MRIs did not conclusively reveal structural abnormalities, so the patient underwent brain PET/MRI to simultaneously evaluate brain structure and function, revealing bitemporal abnormalities. The patient was diagnosed with voltage-gated potassium channel antibody-related limbic encephalitis based on clinical presentation, imaging findings, and antibody testing. Treatment included the addition of a second antiepileptic agent and oral steroids. His seizures and cognitive deficits improved and stabilized.
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BACKGROUND AND PURPOSE: Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers. EXPERIMENTAL APPROACH: We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. KEY RESULTS: SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks - measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. CONCLUSIONS AND IMPLICATIONS: Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour.
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Regulação Alostérica/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Noresteroides/farmacologia , Pregnenolona/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hidroxicolesteróis/química , Noresteroides/química , Pregnenolona/química , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Relação Estrutura-AtividadeRESUMO
Memantine and ketamine, voltage- and activation-dependent channel blockers of N-methyl-d-aspartate (NMDA) receptors (NMDARs), have enjoyed a recent resurgence in clinical interest. Steady-state pharmacodynamic differences between these blockers have been reported, but it is unclear whether the compounds differentially affect dynamic physiologic signaling. In this study, we explored nonequilibrium conditions relevant to synaptic transmission in hippocampal networks in dissociated culture and hippocampal slices. Equimolar memantine and ketamine had indistinguishable effects on the following measures: steady-state NMDA currents, NMDAR excitatory postsynaptic current (EPSC) decay kinetics, progressive EPSC inhibition during repetitive stimulation, and extrasynaptic NMDAR inhibition. Therapeutic drug efficacy and tolerability of memantine have been attributed to fast kinetics and strong voltage dependence. However, pulse depolarization in drug presence revealed a surprisingly slow and similar time course of equilibration for the two compounds, although memantine produced a more prominent fast component (62% versus 48%) of re-equilibration. Simulations predicted that low gating efficacy underlies the slow voltage-dependent relief from block. This prediction was empirically supported by faster voltage-dependent blocker re-equilibration with several experimental manipulations of gating efficacy. Excitatory postsynaptic potential-like voltage commands produced drug differences only with large, prolonged depolarizations unlikely to be attained physiologically. In fact, we found no difference between drugs on measures of spontaneous network activity or acute effects on plasticity in hippocampal slices. Despite indistinguishable synaptic pharmacodynamics, ketamine provided significantly greater neuroprotection from damage induced by oxygen glucose deprivation, consistent with the idea that under extreme depolarizing conditions, the biophysical difference between drugs becomes detectable. We conclude that despite subtle differences in voltage dependence, during physiologic activity, blocker pharmacodynamics are largely indistinguishable and largely voltage independent.
Assuntos
Ketamina/farmacologia , Memantina/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Glucose/deficiência , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Masculino , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/fisiologia , Oxigênio/metabolismo , Ratos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
How epilepsy affects brain functional networks remains poorly understood. Here we investigated resting state functional connectivity of the temporal region in temporal lobe epilepsy. Thirty-two patients with unilateral temporal lobe epilepsy underwent resting state blood-oxygenation level dependent functional magnetic resonance imaging. We defined regions of interest a priori focusing on structures involved, either structurally or metabolically, in temporal lobe epilepsy. These structures were identified in each patient based on their individual anatomy. Our principal findings are decreased local and inter-hemispheric functional connectivity and increased intra-hemispheric functional connectivity ipsilateral to the seizure focus compared to normal controls. Specifically, several regions in the affected temporal lobe showed increased functional coupling with the ipsilateral insula and immediately neighboring subcortical regions. Additionally there was significantly decreased functional connectivity between regions in the affected temporal lobe and their contralateral homologous counterparts. Intriguingly, decreased local and inter-hemispheric connectivity was not limited or even maximal for the hippocampus or medial temporal region, which is the typical seizure onset region. Rather it also involved several regions in temporal neo-cortex, while also retaining specificity, with neighboring regions such as the amygdala remaining unaffected. These findings support a view of temporal lobe epilepsy as a disease of a complex functional network, with alterations that extend well beyond the seizure onset area, and the specificity of the observed connectivity changes suggests the possibility of a functional imaging biomarker for temporal lobe epilepsy.
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OBJECTIVE: Selective amygdalohippocampectomy (AHC) has evolved to encompass a variety of techniques to resect the mesial temporal lobe. To date, there have been few large-scale evaluations of trans-middle temporal gyrus selective AHC. The authors examine a large series of patients who have undergone the trans-middle temporal gyrus AHC and assess its clinical and neuropsychological impact. METHODS: A series of 76 adult patients underwent selective AHC via the trans-middle temporal gyrus approach over a 10-year period, 19 of whom underwent pre- and postoperative neuropsychological evaluations. RESULTS: Favorable seizure response rates were achieved (92% Engel class I or II), with very low surgical morbidity and no mortality. Postoperative neuropsychological assessment revealed a decline in verbal memory for the left AHC group. No postoperative memory decline was identified for the right AHC group, but rather some improvements were noted within this group. CONCLUSIONS: The trans-middle temporal gyrus selective AHC is a safe and effective choice for management of medically refractory epilepsy in adults.
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Tonsila do Cerebelo/cirurgia , Epilepsia do Lobo Temporal/cirurgia , Hipocampo/cirurgia , Procedimentos Neurocirúrgicos/métodos , Resultado do Tratamento , Adulto , Idoso , Análise de Variância , Eletroencefalografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Gravação em Vídeo , Adulto JovemRESUMO
BACKGROUND: The emerging insight into resting-state cortical networks has been important in our understanding of the fundamental architecture of brain organization. These networks, which were originally identified with functional magnetic resonance imaging, are also seen in the correlation topography of the infraslow rhythms of local field potentials. Because of the fundamental nature of these networks and their independence from task-related activations, we posit that, in addition to their neuroscientific relevance, these slow cortical potential networks could play an important role in clinical brain mapping. OBJECTIVE: To assess whether these networks would be useful in identifying eloquent cortex such as sensorimotor cortex in patients both awake and under anesthesia. METHODS: This study included 9 subjects undergoing surgical treatment for intractable epilepsy. Slow cortical potentials were recorded from the cortical surface in patients while awake and under propofol anesthesia. To test brain-mapping utility, slow cortical potential networks were identified with data-driven (seed-independent) and anatomy-driven (seed-based) approaches. With electrocortical stimulation used as the gold standard for comparison, the sensitivity and specificity of these networks for identifying sensorimotor cortex were calculated. RESULTS: Networks identified with a data-driven approach in patients under anesthesia and awake were 90% and 93% sensitive and 58% and 55% specific for sensorimotor cortex, respectively. Networks identified with systematic seed selection in patients under anesthesia and awake were 78% and 83% sensitive and 67% and 60% specific, respectively. CONCLUSION: Resting-state networks may be useful for tailoring stimulation mapping and could provide a means of identifying eloquent regions in patients while under anesthesia.
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Mapeamento Encefálico/métodos , Epilepsia/fisiopatologia , Córtex Motor/fisiopatologia , Rede Nervosa/fisiopatologia , Propofol/uso terapêutico , Córtex Somatossensorial/fisiopatologia , Vigília/efeitos dos fármacos , Adulto , Anestésicos Intravenosos/uso terapêutico , Feminino , Humanos , Masculino , Córtex Motor/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Descanso , Córtex Somatossensorial/efeitos dos fármacosRESUMO
GABA(A) receptors are found on the somatodendritic compartment and on the axon initial segment of many principal neurons. The function of axonal receptors remains obscure, although it is widely assumed that axonal receptors must have a strong effect on excitability. We found that activation of GABA(A) receptors on the dentate granule neuron axon initial segment altered excitability by depolarizing the voltage threshold for action potential initiation under conditions that minimally affected overall cell input resistance. In contrast, activation of somatic GABA(A) receptors strongly depressed the input resistance of granule neurons without affecting the voltage threshold of action potential initiation. Although these effects were observed over a range of intracellular chloride concentrations, average voltage threshold was unaffected when E(Cl) rendered GABA(A) axon initial segment responses explicitly excitatory. A compartment model of a granule neuron confirmed these experimental observations. Low ambient agonist concentrations designed to activate granule neuron tonic currents did not stimulate axonal receptors sufficiently to raise voltage threshold. Using excitatory postsynaptic current (EPSC)-like depolarizations, we show physiological consequences of axonal versus somatic GABA(A) receptor activation. With axonal inhibition, individual excitatory postsynaptic potentials (EPSPs) largely retained their amplitude and time course, but EPSPs that were suprathreshold under basal conditions failed to reach threshold with GABA(A) activation. By contrast, somatic inhibition depressed individual EPSPs because of strong shunting. Our results suggest that axonal GABA(A) receptors have a privileged effect on voltage threshold and that two major measures of neuronal excitability, voltage threshold and rheobase, are differentially affected by axonal and somatic GABA(A) receptor activation.
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Axônios/metabolismo , Dendritos/metabolismo , Giro Denteado/fisiologia , Neurônios/fisiologia , Receptores de GABA-A/metabolismo , Potenciais de Ação/fisiologia , Animais , Giro Denteado/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Modelos Animais , Neurônios/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologiaRESUMO
Endogenous neurosteroids and their synthetic analogs (neuroactive steroids) are potent modulators of GABA(A) receptors. Thus, they are of physiological and clinical relevance for their ability to modulate inhibitory function in the CNS. Despite their importance, fundamental issues of neurosteroid actions remain unresolved. Recent evidence suggests that glutamatergic principal neurons, rather than glia, are the major sources of neurosteroid synthesis. Other recent studies have identified putative neurosteroid binding sites on GABA(A) receptors. In this Opinion, we argue that neurosteroids require a membranous route of access to transmembrane-domain binding sites within GABA(A) receptors. This has implications for the design of future neuroactive steroids because the lipid solubility and related accessibility properties of the ligand are likely to be key determinants of receptor modulation.
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Neurotransmissores/metabolismo , Receptores de GABA-A/metabolismo , Animais , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/fisiologia , Modelos Moleculares , Neurônios/citologia , Neurônios/metabolismo , Conformação Proteica , Receptores de GABA-A/química , Transmissão Sináptica/fisiologiaRESUMO
Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABA(A) receptor with maximum efficacy and potency similar to clinically used GABA(A) receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABA(A) receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABA(A) receptor function. Even dyes with no activity on GABA(A) receptors at baseline induced photodynamic enhancement of GABA(A) receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.
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Corantes Fluorescentes/farmacologia , GABAérgicos/farmacologia , Hipocampo/metabolismo , Receptores de GABA-A/metabolismo , Coloração e Rotulagem/métodos , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Corantes Fluorescentes/química , GABAérgicos/química , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Hipocampo/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Microscopia Confocal , Microscopia de Fluorescência , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos , Técnicas de Patch-Clamp , Ratos , Receptores de GABA-A/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Xenopus laevisRESUMO
Intrinsic excitability is a key feature dictating neuronal response to synaptic input. Here we investigate the recent observation that dentate granule neurons exhibit a more depolarized voltage threshold for action potential initiation than CA3 pyramidal neurons. We find no evidence that tonic GABA currents, leak or voltage-gated potassium conductances, or the expression of sodium channel isoform differences can explain this depolarized threshold. Axonal initial segment voltage-gated sodium channels, which are dominated by the Na(V)1.6 isoform in both cell types, distribute more proximally and exhibit lower overall density in granule neurons than in CA3 neurons. To test possible contributions of sodium channel distributions to voltage threshold and to test whether morphological differences participate, we performed simulations of dentate granule neurons and of CA3 pyramidal neurons. These simulations revealed that cell morphology and sodium channel distribution combine to yield the characteristic granule neuron action potential upswing and voltage threshold. Proximal axon sodium channel distribution strongly contributes to the higher voltage threshold of dentate granule neurons for two reasons. First, action potential initiation closer to the somatodendritic current sink causes the threshold of the initiating axon compartment to rise. Second, the proximity of the action potential initiation site to the recording site causes somatic recordings to more faithfully reflect the depolarized threshold of the axon than in cells like CA3 neurons, with distally initiating action potentials. Our results suggest that the proximal location of axon sodium channels in dentate granule neurons contributes to the intrinsic excitability differences between DG and CA3 neurons and may participate in the low-pass filtering function of dentate granule neurons.
Assuntos
Potenciais de Ação/fisiologia , Giro Denteado/fisiologia , Neurônios/fisiologia , Canais de Sódio/fisiologia , Animais , Axônios/fisiologia , Região CA3 Hipocampal/fisiologia , Simulação por Computador , Eletrofisiologia , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Condução Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Análise de Regressão , Processamento de Sinais Assistido por Computador , Transmissão Sináptica/fisiologiaRESUMO
We have shown that fluorescent, 7-nitro-2,1,3-benzoxadiazol-4-yl amino (NBD)-conjugated neurosteroid analogs photopotentiate GABA(A) receptor function. These compounds seem to photosensitize a modification of receptor function, resulting in long-lived increases in responses to exogenous or synaptic GABA. Here we extend this work to examine the effectiveness of different fluorophore positions, conjugations, steroid structures, and fluorophores. Our results are generally in agreement with the idea that steroids with activity at GABA(A) receptors are the most potent photopotentiators. In particular, we find that an unnatural enantiomer of an effective photopotentiating steroid is relatively weak, excluding the idea that membrane solubility alone, which is identical for enantiomer pairs, is solely responsible for potent photopotentiation. Furthermore, there is a significant correlation between baseline GABA(A) receptor activity and photopotentiation. Curiously, both sulfated steroids, which bind a presumed external neurosteroid antagonist site, and hydroxysteroids, which bind an independent site, are effective. We also find that a rhodamine dye conjugated to a 5beta-reduced 3alpha-hydroxy steroid is a particularly potent and effective photopotentiator, with minimal baseline receptor activity up to 10 muM. Steroid conjugated fluorescein and Alexa Fluor 546 also supported photopotentiation, although the Alexa Fluor conjugate was weaker and required 10-fold higher concentration to achieve similar potentiation to the best NBD and rhodamine conjugates. Filling cells with steroid-conjugated or free fluorophores via whole-cell patch pipette did not support photopotentiation. FM1-43, another membrane-targeted, structurally unrelated fluorophore, also produced photopotentiation at micromolar concentrations. We conclude that further optimization of fluorophore and carrier could produce an effective, selective, light-sensitive GABA(A) receptor modulator.
Assuntos
Corantes Fluorescentes/química , Receptores de GABA-A/química , Animais , Feminino , Técnicas de Patch-Clamp , Ratos , Xenopus laevisRESUMO
Anesthetic steroids with actions at gamma-aminobutyric acid type A receptors (GABA(A)Rs) may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA(A)Rs. The hydrophobicity ranking predicted relative potency of GABA(A)R potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by gamma-cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of nonspecific accumulation. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster nonspecific accumulation and departitioning than those of a fluorescent analogue of the high-lipophilicity (3alpha,5alpha)-3-hydroxypregnan-20-one (C17-NBD-3alpha5alphaA). These differences were paralleled by differences in potentiation of GABA(A)R function. The enantiomer of C17-NBD-3alpha5alphaA, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-3alpha5alphaA, but was inactive at GABA(A)Rs. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore both specific (e.g., fulfilling pharmacophore requirements) and nonspecific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.