RESUMO
The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K+ concentration ([K+]extra) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K+]extra that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride cotransporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), both in vitro and in vivo This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally.SIGNIFICANCE STATEMENT Halorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K+ ions into the cells; (2) the resultant drop in extracellular [K+] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K+] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K+]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.
Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Potássio , Masculino , Feminino , Camundongos , Animais , Potássio/metabolismo , Halorrodopsinas/farmacologia , Cloretos/metabolismo , Neurônios/metabolismo , Células Piramidais/metabolismo , Depressão Alastrante da Atividade Elétrica Cortical/fisiologiaRESUMO
The transcriptional coactivator, PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), plays a key role in coordinating energy requirement within cells. Its importance is reflected in the growing number of psychiatric and neurological conditions that have been associated with reduced PGC-1α levels. In cortical networks, PGC-1α is required for the induction of parvalbumin (PV) expression in interneurons, and PGC-1α deficiency affects synchronous GABAergic release. It is unknown, however, how this affects cortical excitability. We show here that knocking down PGC-1α specifically in the PV-expressing cells (PGC-1αPV-/-) blocks the activity-dependent regulation of the synaptic proteins, SYT2 and CPLX1. More surprisingly, this cell class-specific knockout of PGC-1α appears to have a novel antiepileptic effect, as assayed in brain slices bathed in 0 Mg2+ media. The rate of occurrence of preictal discharges developed approximately equivalently in wild-type and PGC-1αPV-/- brain slices, but the intensity of these discharges was lower in PGC-1αPV-/- slices, as evident from the reduced power in the γ range and reduced firing rates in both PV interneurons and pyramidal cells during these discharges. Reflecting this reduced intensity in the preictal discharges, the PGC-1αPV-/- brain slices experienced many more discharges before transitioning into a seizure-like event. Consequently, there was a large increase in the latency to the first seizure-like event in brain slices lacking PGC-1α in PV interneurons. We conclude that knocking down PGC-1α limits the range of PV interneuron firing and this slows the pathophysiological escalation during ictogenesis.NEW & NOTEWORTHY Parvalbumin expressing interneurons are considered to play an important role in regulating cortical activity. We were surprised, therefore, to find that knocking down the transcriptional coactivator, PGC-1α, specifically in this class of interneurons appears to slow ictogenesis. This anti-ictogenic effect is associated with reduced activity in preictal discharges, but with a far longer period of these discharges before the first seizure-like events finally start. Thus, PGC-1α knockdown may promote schizophrenia while reducing epileptic tendencies.
Assuntos
Excitabilidade Cortical/fisiologia , Interneurônios/metabolismo , Neocórtex/metabolismo , Parvalbuminas/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Células Piramidais/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/deficiênciaRESUMO
KEY POINTS: There is a rapid interneuronal response to focal activity in cortex, which restrains laterally propagating activity, including spreading epileptiform activity. The interneuronal response involves intense activation of both parvalbumin- and somatostatin-expressing interneurons. Interneuronal bursting is time-locked to glutamatergic barrages in the pre-ictal period. Ca2+ imaging using conditional expression of GCaMP6f provides an accurate readout of the evolving firing patterns in both types of interneuron. The activation profiles of the two interneuronal classes are temporally offset, with the parvalbumin population being activated first, and typically, at higher rates. ABSTRACT: Previous work has described powerful restraints on laterally spreading activity in cortical networks, arising from a rapid feedforward interneuronal response to focal activity. This response is particularly prominent ahead of an ictal wavefront. Parvalbumin-positive interneurons are considered to be critically involved in this feedforward inhibition, but it is not known what role, if any, is provided by somatostatin-expressing interneurons, which target the distal dendrites of pyramidal cells. We used a combination of electrophysiology and cell class-specific Ca2+ imaging in mouse brain slices bathed in 0 Mg2+ medium to characterize the activity profiles of pyramidal cells and parvalbumin- and somatostatin-expressing interneurons during epileptiform activation. The GCaMP6f signal strongly correlates with the level of activity for both interneuronal classes. Both interneuronal classes participate in the feedfoward inhibition. This contrasts starkly with the pattern of pyramidal recruitment, which is greatly delayed. During these barrages, both sets of interneurons show intense bursting, at rates up to 300Hz, which is time-locked to the glutamatergic barrages. The activity of parvalbumin-expressing interneurons appears to peak early in the pre-ictal period, and can display depolarizing block during the ictal event. In contrast, somatostatin-expressing interneuronal activity peaks significantly later, and firing persists throughout the ictal events. Interictal events appear to be very similar to the pre-ictal period, albeit with slightly lower firing rates. Thus, the inhibitory restraint arises from a coordinated pattern of activity in the two main classes of cortical interneurons.
Assuntos
Interneurônios/fisiologia , Parvalbuminas/fisiologia , Somatostatina/fisiologia , Animais , Encéfalo/fisiologia , Feminino , Masculino , Camundongos TransgênicosRESUMO
Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we used a combination of Drosophila and mouse, and, in the latter, both a pentylenetetrazole (PTZ)-induced seizure model and an electrically induced seizure model for refractory seizures to evaluate the anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode of action included increased expression of the firing rate homeostatic regulator Pumilio (PUM). Knockdown of pum expression, in Drosophila, blocked anticonvulsive effects of 4-TBB, while analysis of validated PUM targets in mouse brain revealed significant reductions following exposure to this compound. A structure-activity study identified the active parts of the molecule and, further, showed that the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-induced and electrically induced seizures. This study provides a proof of principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development.
Assuntos
Anticonvulsivantes , Pentilenotetrazol , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Benzaldeídos/efeitos adversos , Drosophila , Homeostase , Camundongos , Neurônios , Pentilenotetrazol/efeitos adversos , Pirazóis/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/prevenção & controleRESUMO
Recent studies have implicated impaired Parvalbumin Fast-Spiking Interneuron (PVIN) function as a precipitating factor underlying abnormalities in network synchrony, oscillatory rhythms, and cognition associated with Alzheimer's disease (AD). However, a complete developmental investigation of potential gamma deficits, induced by commonly used carbachol or kainate in ex vivo slice preparations, within AD model mice is lacking. We examined gamma oscillations using field recordings in acute hippocampal slices from 5xFAD and control mice, through the period of developing pathology, starting at 3 months of age, when there is minimal plaque presence in the hippocampus, through to 12+ months of age, when plaque burden is high. In addition, we examined PVIN participation in gamma rhythms using targeted cell-attached recordings of genetically-reported PVINs, in both wild type and mutant mice. In parallel, a developmental immunohistochemical characterisation probing the PVIN-associated expression of PV and perineuronal nets (PNNs) was compared between control and 5xFAD mice. Remarkably, this comprehensive longitudinal evaluation failed to reveal any obvious correlations between PVIN deficits (electrical and molecular), circuit rhythmogenesis (gamma frequency and power), and Aß deposits/plaque formation. By 6-12 months, 5xFAD animals have extensive plaque formation throughout the hippocampus. However, a deficit in gamma oscillatory power was only evident in the oldest 5xFAD animals (12+ months), and only when using kainate, and not carbachol, to induce the oscillations. We found no difference in PV firing or phase preference during kainate-induced oscillations in younger or older 5xFAD mice compared to control, and a reduction of PV and PNNs only in the oldest 5xFAD mice. The lack of a clear relationship between PVIN function, network rhythmicity, and plaque formation in our study highlights an unexpected resilience in PVIN function in the face of extensive plaque pathology associated with this model, calling into question the presumptive link between PVIN pathology and Alzheimer's progression.
RESUMO
In violation of Dale's principle several neuronal subtypes utilize more than one classical neurotransmitter. Molecular identification of vesicular glutamate transporter three and cholecystokinin expressing cortical interneurons (CCK+VGluT3+INTs) has prompted speculation of GABA/glutamate corelease from these cells for almost two decades despite a lack of direct evidence. We unequivocally demonstrate CCK+VGluT3+INT-mediated GABA/glutamate cotransmission onto principal cells in adult mice using paired recording and optogenetic approaches. Although under normal conditions, GABAergic inhibition dominates CCK+VGluT3+INT signaling, glutamatergic signaling becomes predominant when glutamate decarboxylase (GAD) function is compromised. CCK+VGluT3+INTs exhibit surprising anatomical diversity comprising subsets of all known dendrite targeting CCK+ interneurons in addition to the expected basket cells, and their extensive circuit innervation profoundly dampens circuit excitability under normal conditions. However, in contexts where the glutamatergic phenotype of CCK+VGluT3+INTs is amplified, they promote paradoxical network hyperexcitability which may be relevant to disorders involving GAD dysfunction such as schizophrenia or vitamin B6 deficiency.
Assuntos
Ácido Glutâmico/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Interneurônios/metabolismo , CamundongosRESUMO
BACKGROUND: In June 2006, the Advisory Committee on Immunization Practices (ACIP) expanded its June 2005 recommendation for a second dose of varicella vaccine during outbreaks to a recommendation for routine school entry second dose varicella vaccination. In October 2006, the Arkansas Department of Health was notified of a varicella outbreak among students where some received a second dose during an outbreak-related vaccination campaign in February 2006. METHODS: The outbreak was investigated using a school-wide parental survey with a follow-up survey of identified case patients. Vaccination status was verified using state and local immunization records. Limited laboratory testing confirmed circulation of wild-type varicella, including varicella in 2-dose vaccine recipients. RESULTS: Vaccination information was available for 871 (99%) of the 880 children. Varicella vaccination coverage was 97% (2-dose, 39%; 1-dose, 58%). A review of the February vaccination clinic found no deficiencies; lot numbers did not differ between cases and noncases. Varicella was confirmed by PCR in 5 (42%) of 12 lesion specimens and by IgM in 1 (6%) of 16 serum specimens. Varicella was reported in 84 children, including 25 (30%) two-dose and 53 (63%) one-dose recipients. Attack rates among 2-dose recipients (10.4%) and 1-dose recipients (14.6%) were not significantly different (RR: 0.72, 95% CI: 0.44-1.15). All 2-dose recipients and 80% of 1-dose recipients reported having 50 or fewer skin lesions. CONCLUSION: This outbreak is the first to document varicella in both 1- and 2-dose vaccine recipients; both groups had mild disease. The vaccine effectiveness of 1 and 2 doses were similar.
Assuntos
Vacina contra Varicela/administração & dosagem , Varicela/epidemiologia , Surtos de Doenças , Arkansas , Distribuição de Qui-Quadrado , Varicela/imunologia , Varicela/prevenção & controle , Vacina contra Varicela/imunologia , Criança , Pré-Escolar , Humanos , Vacinação em Massa , Reação em Cadeia da Polimerase , Instituições Acadêmicas , Estatísticas não Paramétricas , Inquéritos e QuestionáriosRESUMO
Hyperprolinaemia is characterized by increased tissue accumulation of proline (Pro) and is known to exert serious cognitive and/or neuropsychiatric symptomatology as a direct result of Pro accumulation in the brain. The aim of this study was to explore a putative link between experimentally-simulated hyperprolinaemia and the activity of acetylcholinesterase (AChE); a crucial neurotoxicity marker. In vitro experiments were undertaken on purified eel-derived AChE, as well as on adult mouse brain homogenates, in order to examine the effect of a spectrum of Pro concentrations (3, 30, 500, and 1000 µM) on this marker. Our data showed that although Pro exerted a significant inhibitory effect on pure AChE activity, mouse brain-derived membrane-bound AChE activity was found either unaltered or significantly increased following incubation with Pro. The use of AChE activity as a neurotoxicity marker within the context of experimentally-simulated hyperprolinaemia should be considered with caution and in parallel with a number of other experimental parameters.