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PURPOSE: The P2X7 receptor, an adenosine triphosphate (ATP)-gated purinoreceptor, has emerged as one of the key players in neuroinflammatory processes. Therefore, developing a positron emission tomography (PET) tracer for imaging of P2X7 receptors in vivo presents a promising approach to diagnose, monitor, and study neuroinflammation in a variety of brain disorders. To fulfill the goal of developing a P2X7 PET ligand as a biomarker of neuroinflammation, [18F]JNJ-64413739 has been recently disclosed. PROCEDURES: We evaluated [18F]JNJ-64413739 in a rat model of neuroinflammation induced by an intracerebral injection of lipopolysaccharide (LPS). In vivo brain uptake was determined by PET imaging. Upregulation of neuroinflammatory biomarkers was determined by quantitative polymerase chain reaction (qPCR). Distribution of the tracer in the brain was determined by ex vivo autoradiography (ARG). The specificity of [18F]JNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446. RESULTS: Brain regions of rats injected with LPS had a significantly increased uptake (34 % ± 3 % s.e.m., p = 0.036, t test, standardized uptake value measured over the entire scanning period) of [18F]JNJ-64413739 relative to the corresponding brain regions of control animals injected with phosphate-buffered saline (PBS). The uptake in the contralateral regions and cerebellum was not significantly different between the groups of animals. The increase in uptake of [18F]JNJ-64413739 at the LPS-injected site observed by PET imaging was concordant with ex vivo ARG, upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. CONCLUSIONS: While further work is needed to study [18F]JNJ-64413739 in other types of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential PET tracer of central neuroinflammation.
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Encéfalo/diagnóstico por imagem , Inflamação/diagnóstico por imagem , Inflamação/patologia , Peptídeos/química , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Receptores Purinérgicos P2X7/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Lipopolissacarídeos , Peptídeos/farmacocinética , Projetos Piloto , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Piridinas/química , Ratos , Triazóis/químicaRESUMO
Imaging neuronal activity in awake behaving mice with miniature fluorescence microscopes requires the implementation of a variety of procedures. Surgeries are performed to gain access to the cell population of interest and to implant microscope components. After a recovery period, mice are trained to exhibit a desired behavior. Finally, neuronal activity is imaged and synchronized with that behavior. To take full advantage of the technology, selection of the calcium indicator and experimental design must be carefully considered. In this article, we explain the procedures and considerations that are critical for obtaining high-quality calcium imaging data. As an example, we describe how to utilize miniature fluorescence microscopy to image hippocampal place cell activity during linear track running in Thy1.GCaMP6f transgenic mice. © 2018 by John Wiley & Sons, Inc.
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Células de Lugar/fisiologia , Animais , Comportamento Animal , Cálcio/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Camundongos Transgênicos , Microscopia de Fluorescência , Atividade MotoraRESUMO
Prolonged exposure to abnormally high calcium concentrations is thought to be a core mechanism underlying hippocampal damage in epileptic patients; however, no prior study has characterized calcium activity during seizures in the live, intact hippocampus. We have directly investigated this possibility by combining whole-brain electroencephalographic (EEG) measurements with microendoscopic calcium imaging of pyramidal cells in the CA1 hippocampal region of freely behaving mice treated with the pro-convulsant kainic acid (KA). We observed that KA administration led to systematic patterns of epileptiform calcium activity: a series of large-scale, intensifying flashes of increased calcium fluorescence concurrent with a cluster of low-amplitude EEG waveforms. This was accompanied by a steady increase in cellular calcium levels (>5 fold increase relative to the baseline), followed by an intense spreading calcium wave characterized by a 218% increase in global mean intensity of calcium fluorescence (n = 8, range [114-349%], p < 10(-4); t-test). The wave had no consistent EEG phenotype and occurred before the onset of motor convulsions. Similar changes in calcium activity were also observed in animals treated with 2 different proconvulsant agents, N-methyl-D-aspartate (NMDA) and pentylenetetrazol (PTZ), suggesting the measured changes in calcium dynamics are a signature of seizure activity rather than a KA-specific pathology. Additionally, despite reducing the behavioral severity of KA-induced seizures, the anticonvulsant drug valproate (VA, 300 mg/kg) did not modify the observed abnormalities in calcium dynamics. These results confirm the presence of pathological calcium activity preceding convulsive motor seizures and support calcium as a candidate signaling molecule in a pathway connecting seizures to subsequent cellular damage. Integrating in vivo calcium imaging with traditional assessment of seizures could potentially increase translatability of pharmacological intervention, leading to novel drug screening paradigms and therapeutics designed to target and abolish abnormal patterns of both electrical and calcium excitation.
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Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal's state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (â¼ 65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders.
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Região CA1 Hipocampal , Cálcio/metabolismo , Agonistas de Receptores de GABA-A/farmacologia , Imagem Molecular/métodos , Imagem Óptica/métodos , Células Piramidais , Piridinas/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Microscopia de Fluorescência/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , ZolpidemRESUMO
The orexin/hypocretin neuropeptides are produced by a cluster of neurons within the lateral posterior hypothalamus and participate in neuronal regulation by activating their receptors (OX1 and OX2 receptors). The orexin system projects widely through the brain and functions as an interface between multiple regulatory systems including wakefulness, energy balance, stress, reward, and emotion. Recent studies have demonstrated that orexins and glutamate interact at the synaptic level and that orexins facilitate glutamate actions. We tested the hypothesis that orexins modulate glutamate signaling via OX1 receptors by monitoring levels of glutamate in frontal cortex of freely moving mice using enzyme coated biosensors under inhibited OX1 receptor conditions. MK-801, an NMDA receptor antagonist, was administered subcutaneously (0.178 mg/kg) to indirectly disinhibit pyramidal neurons and therefore increase cortical glutamate release. In wild-type mice, pretreatment with the OX1 receptor antagonist GSK-1059865 (10 mg/kg S.C.) which had no effect by itself, significantly attenuated the cortical glutamate release elicited by MK-801. OX1 receptor knockout mice had a blunted glutamate release response to MK-801 and exhibited about half of the glutamate release observed in wild-type mice in agreement with the data obtained with transient blockade of OX1 receptors. These results indicate that pharmacological (transient) or genetic (permanent) inhibition of the OX1 receptor similarly interfere with glutamatergic function in the cortex. Selectively targeting the OX1 receptor with an antagonist may normalize hyperglutamatergic states and thus may represent a novel therapeutic strategy for the treatment of various psychiatric disorders associated with hyperactive states.
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Neurons in the supplementary eye field (SEF) of the macaque monkey exhibit rank selectivity, firing differentially as a function of the phase attained during the performance of a task requiring the execution of saccades to a series of objects in fixed order. The activity of these neurons is commonly thought to represent ordinal position in the service of serial-order performance. However, there is little evidence causally linking neuronal activity in the SEF to sequential behavior. To explore the role of the SEF in serial-order performance, we delivered intracortical microstimulation while monkeys performed a task requiring them to make saccades to three objects in a fixed order on each trial. Microstimulation, considered on average across all SEF sites and all phases of the trial, affected saccadic kinematics. In particular, it prolonged the reaction time, increased the peak velocity, and slightly increased the amplitude of saccades. In addition, it interfered with the monkeys' ability to select the target appropriate to a given phase of the trial. The pattern of the errors was such as would be expected if microstimulation shifted the neural representation of ordinal position toward a later phase of the trial.
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Lobo Frontal/fisiologia , Movimentos Sacádicos/fisiologia , Animais , Estimulação Elétrica , Macaca mulatta , MasculinoRESUMO
Neurons in several areas of the monkey frontal cortex exhibit rank selectivity, firing differentially as a function of the stage attained during the performance of a serial order task. The activity of these neurons is commonly thought to represent ordinal position within the trial. However, they might also be sensitive to factors correlated with ordinal position including time elapsed during the trial (which is greater for each successive stage) and the degree of anticipation of reward (which probably increases at each successive stage). To compare the influences of these factors, we monitored neuronal activity in the supplementary motor area (SMA), presupplementary motor area (pre-SMA), supplementary eye field (SEF), and dorsolateral prefrontal cortex during the performance of a serial order task (requiring a series of saccades in three specified directions), a variable reward task (in which a cue displayed early in the trial indicated whether the reward received at the end of the trial would be large or small), and a long delay task (in which the monkey had simply to maintain fixation during a period of time approximating the duration of an average trial in the serial order task). We found that rank signals were partially correlated with sensitivity to elapsed time and anticipated reward. The connection to elapsed time was strongest in the pre-SMA. The connection to anticipated reward was most pronounced in the SMA and SEF. However, critically, these factors could not fully explain rank selectivity in any of the areas tested.
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Antecipação Psicológica/fisiologia , Lobo Frontal/fisiologia , Estimulação Luminosa/métodos , Tempo de Reação/fisiologia , Recompensa , Animais , Macaca , Macaca mulatta , Masculino , Desempenho Psicomotor/fisiologiaRESUMO
Neurons in several areas of monkey frontal cortex exhibit ordinal position (rank) selectivity during the performance of serial order tasks. It has been unclear whether rank selectivity or the dependence of rank selectivity on task context varies across the areas of frontal cortex. To resolve this issue, we recorded from neurons in the supplementary motor area (SMA), presupplementary motor area (pre-SMA), supplementary eye field (SEF), and dorsolateral prefrontal cortex (dlPFC) as monkeys performed two oculomotor tasks, one requiring the selection of three actions in sequence and the other requiring the selection of three objects in sequence. We found that neurons representing all ranks were present in all areas. Only to a moderate degree did the prevalence and nature of rank selectivity vary from area to area. The two most prominent inter-area differences involved a lower prevalence of rank selectivity in the dlPFC than in the other areas and a higher proportion of neurons preferring late ranks in the SMA and SEF than in the other areas. Neurons in all four areas are rank generalists in the sense of favoring the same rank in both the serial action task and the serial object task.
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Lobo Frontal/fisiologia , Movimentos Sacádicos/fisiologia , Animais , Contagem de Células , Bases de Dados Factuais , Fenômenos Eletrofisiológicos , Fixação Ocular/fisiologia , Lobo Frontal/citologia , Macaca mulatta , Imageamento por Ressonância Magnética , Masculino , Córtex Motor/fisiologia , Neurônios/fisiologia , Estimulação Luminosa , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Desempenho Psicomotor/fisiologia , Aprendizagem Seriada/fisiologia , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Campos Visuais/fisiologiaRESUMO
In this issue of Neuron, Han and colleagues bring optogenetic methods into use in nonhuman primates by demonstrating the feasibility of achieving cell-type-specific photoactivation of macaque neocortical neurons. The use of optogenetic approaches in nonhuman primates promises to revolutionize our understanding of the neural circuitry that mediates complex cognitive functions.
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Macaca mulatta/fisiologia , Modelos Animais , Rodopsina/genética , Células Receptoras Sensoriais/fisiologia , Vias Visuais/fisiologia , Animais , Neocórtex/citologia , Estimulação Luminosa/métodos , Rodopsina/metabolismoRESUMO
When correlation is measured in the presence of noise, its value is decreased. In single-neuron recording experiments, for example, the correlation of selectivity indices in a pair of tasks may be assessed across neurons, but, because the number of trials is limited, the measured index values for each neuron will be noisy. This attenuates the correlation. A correction for such attenuation was proposed by Spearman more than 100 yr ago, and more recent work has shown how confidence intervals may be constructed to supplement the correction. In this paper, we propose an alternative Bayesian correction. A simulation study shows that this approach can be far superior to Spearman's, both in accuracy of the correction and in coverage of the resulting confidence intervals. We demonstrate the usefulness of this technology by applying it to a set of data obtained from the frontal cortex of a macaque monkey while performing serial order and variable reward saccade tasks. There the correction results in a substantial increase in the correlation across neurons in the two tasks.
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Potenciais de Ação/fisiologia , Teorema de Bayes , Neurônios/fisiologia , Animais , Simulação por Computador , Intervalos de Confiança , Lobo Frontal/citologia , Macaca mulatta , Modelos Neurológicos , Recompensa , Movimentos Sacádicos/fisiologia , Estatísticas não ParamétricasRESUMO
When a monkey executes a learned series of eye movements (for example, rightward followed by upward followed by leftward), neurons in the supplementary eye field (SEF) fire differentially in conjunction with the first, second, and third movements. It has not been clear whether such ordinal position signals are truly general, accompanying all forms of sequential behavior, or accompany only learned sequences of movements. To resolve this issue, we trained monkeys to perform both a serial action task (making saccades in a fixed sequence of directions) and a serial object task (making saccades to a fixed sequence of objects). We found concordant ordinal position selectivity in the two tasks. Neuronal selectivity for the passage of time and expectation of reward could not explain such concordance. We conclude that SEF neurons signal ordinal position consistently across different task contexts. These signals presumably underlie the ability of primates including humans to perform a broad range of serial order tasks.
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Movimentos Oculares/fisiologia , Olho , Neurônios/fisiologia , Córtex Visual/citologia , Campos Visuais/fisiologia , Percepção Visual/fisiologia , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Sinais (Psicologia) , Macaca mulatta , Masculino , Estimulação Luminosa , Tempo de Reação/fisiologia , Recompensa , Fatores de TempoRESUMO
The decrease in elasticity of epithelial tissues with ageing contributes to many human diseases. This change was previously attributed to increased crosslinking of extracellular matrix proteins. Here we show that individual human epithelial cells also become significantly more rigid during ageing in vitro. Using atomic force microscopy (AFM), we found that the Young's modulus of viable cells was consistently increased two- to four-fold in older versus younger cells. Direct visualization of the cytoskeleton using a novel method involving the AFM suggested that increased rigidity of ageing cells was due to a higher density of cytoskeletal fibres. Our results identify a unique mechanism that might contribute to the age-related loss of elasticity in epithelial tissues.