Direct Imaging of Hippocampal Epileptiform Calcium Motifs Following Kainic Acid Administration in Freely Behaving Mice

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

Relation of ordinal position signals to the expectation of reward and passage of time in four areas of the macaque frontal cortex

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

Rank Signals in Four Areas of Macaque Frontal Cortex During Selection of Actions and Objects in Serial Order

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

Zolpidem Reduces Hippocampal Neuronal Activity in Freely Behaving Mice: A Large Scale Calcium Imaging Study with Miniaturized Fluorescence Microscope

<div><p>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 (∼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.</p></div

Zolpidem decreased frequencies of calcium transients in CA1.

<p><b>A</b>: Calcium transients (indicated by tick marks) detected in individual cells (vertical axis) are plotted across time following vehicle (water, left) and Zolpidem (10 mg/kg, right) administration in a representative animal. <b>B</b>: Histogram of calcium transients (“Event Rate”) in the representative animal. <b>C</b>: Average rate of calcium transients (s.e.m. error bar) in all animals used in the study. Zolpidem decreased the frequency of calcium transients by 71% (from 0.7120 to 0.2087 events/min/cell, p<0.0001, Wilcoxon Signed Rank Test).</p

Similar sleep-promoting effects of Zolpidem in “Telemetry+Imaging” and “Telemetry only” mice.

<p>Duration of Wake, NREM and REM sleep, and Activity counts were measured during the first 2 hour period after oral dosing of Zolpidem (10 mg/kg). Latency to NREM and REM sleep were measured during the first 3 hour period. Measurements are expressed in minutes and represented as means ± s.e.m. (n = 5 animals per condition).</p><p>*p<0.05 and **p<0.01; comparison assessed by two-way mixed-design ANOVA.</p><p>Similar sleep-promoting effects of Zolpidem in “Telemetry+Imaging” and “Telemetry only” mice.</p

Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem.

<p><b>A</b>: Representative raster plot of calcium transients in individual cells (n = 195) that are color-coded depending on their response to Zolpidem (Mann-Whitney-Wilcoxon test, p<0.05 criterion of significance): significant decrease (blue); significant increase (red), non-significant change (black). <b>B:</b> Locations of individual cells identified in the same representative imaging session, layered atop a mean fluorescent image. <b>C</b>: Rate of calcium transients post-Zolpidem (“Zolpidem Event Rate”) vs post-vehicle (“Vehicle Event Rate”); each dot is an individual cell (n = 1275). The majority of individual neurons (65%) significantly lowered neuronal activity following Zolpidem administration; 32% of neurons did not show a significant change; and a small neuronal subset (∼3%) showed a significant increase.</p

Decrease in locomotion was not sufficient to explain Zolpidem-induced decrease in neuronal activity.

<p><b>A</b>: Raster plot of calcium transients in individual cells (vertical axis) following vehicle (left) and Zolpidem (10 mg/kg, right) administration in an example animal with identified inactive (displacements <−.2 cm/min) periods (green shading). The corresponding speed (in cm/min) trace is plotted below the raster plot. <b>B</b>: Comparison of average frequencies of calcium transients (number of events/minute/cell) during active periods (black bars) and inactive periods (green bars) in the representative animal (s.e.m. error bar). <b>C</b>: Average rate of calcium transients (s.e.m. error bar) in all animals with identified inactive periods following both vehicle (left) and Zolpidem (right) administration.</p