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Try out PMC Labs and tell us what you think. Learn More. Recent work has shown that limbic seizures depress brainstem arousal systems, including reduced action potential firing in a key node: cholinergic neurons of the pedunculopontine tegmental nucleus PPT. In vivo whole-cell recordings have not ly been achieved in PPT, but are used here with the goal of elucidating the mechanisms of reduced PPT cholinergic neuronal activity.

An established model of focal limbic seizures was used in rats following brief hippocampal stimulation under light anesthesia. Whole-cell in vivo recordings were obtained from PPT neurons using custom-fabricated mm tapered patch pipettes, and cholinergic neurons were identified histologically.

Average membrane potential, input resistance, membrane potential fluctuations and variance were analyzed during seizures. A subset of PPT neurons exhibited reduced firing and hyperpolarization during seizures and stained positive for choline acetyltransferase. The combination of increased input resistance, decreased EPSP-like events and decreased variance weigh against active ictal inhibition and support withdrawal of excitatory input as the dominant mechanism of decreased activity of cholinergic neurons in the PPT.

Further identifying synaptic mechanisms of depressed arousal during seizures may lead to new treatments to improve ictal and postictal cognition. The mechanisms by which subcortical activating networks regulate states of arousal have long been a subject of debate and experimentation, with evidence pointing to the pedunculopontine tegmental nucleus PPT as a crucial node in this far-reaching system Mena-Segovia and Bolam, ; Mena-Segovia et al. The association of lesions in the brainstem tegmentum with the most extreme disturbance of arousal, coma, illustrates the critical role of brainstem structures in the regulation and maintenance of arousal Parvizi and Damasio, Early scientific probes into the location of brainstem arousal structures studied the effects of sequential brainstem transections on wakefulness, and isolated the mesopontine area as one crucial to maintaining an awake state Moruzzi and Magoun,with subsequent work identifying an important component of this system as cholinergic neurons in the PPT Mesulam et al.

A dominant target of PPT axonal fibers are thalamic nuclei Mena-Segovia and Bolam, known to play a central role in transitions between states of conscious arousal and their electrophysiological correlates McCormick and Bal, ; Steriade et al. Of particular interest is the intralaminar central lateral nucleus of the thalamus, which has been shown to have decreased firing during focal limbic seizures Feng et al. The strong thalamic projections of cholinergic neurons in the PPT and their increased activity preceding transitions from sleep to wake and rapid-eye-movement REM sleep, suggest a causal relationship between cholinergic PPT tone and transition to higher states of arousal Steriade et al.

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Stimulation of intralaminar thalamic nuclei that are downstream targets of the ascending reticular activating system has even been shown to reverse electrophysiological and behavioral measures of decreased arousal in animal seizure models Gummadavelli et al. Temporal lobe seizures are commonly associated with loss of consciousness despite the temporal lobes not being regarded as part of the canonical consciousness system Blumenfeld, It is not immediately clear why persons with focal temporal lobe seizures should lose consciousness Escueta et al.

Evidence is now growing in support of a network inhibition hypothesis to explain these seeming disparities, proposing that the loss of consciousness accompanying focal epilepsies is mediated through suppression of subcortical arousal circuits Blumenfeld, ; Norden and Blumenfeld, Recent work has shown that cholinergic PPT neuronal activity is decreased in an established model of focal temporal lobe seizures with decreased cortical arousal and that optogenetic stimulation of these neurons can reverse electrophysiological correlates of decreased arousal and increase gamma frequency cortical activity Furman et al.

On a brain-wide scale, prior work has dissected out areas of inhibitory and excitatory activity during focal limbic seizures Blumenfeld et al. At the cellular level, single-unit recordings have revealed that neurons of specific nuclei exhibit reduced neuronal firing during seizures Motelow et al.

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In the present study, we delve into the intracellular membrane potential V m level using in vivo whole-cell recordings from areas deep in the pedunculopontine brainstem to elucidate the synaptic mechanisms underlying ictal changes in neuronal activity of the PPT. A subset of PPT neurons, identified as cholinergic, exhibit reduced firing during focal limbic seizures, but the mechanism of reduced firing is unknown. The activity of these neurons is likely modified by contributions of both excitatory and inhibitory input Higley and Contreras, ; Isaacson and Scanziani, ; Wehr and Zador,however, the afferent synaptic changes resulting in decreased firing can be conceptually dichotomized into either an increase in inhibitory input, or a withdrawal of excitatory input.

Tight-seal whole-cell intracellular recording is a powerful tool for investigating membrane properties and synaptic input on individual neurons Neher and Sakmann, ; Sakmann, The targeting of deep brain structures in vivohowever, has been limited by technical complications of their access, with prior reports removing large sections of brain tissue e. The network of brain circuitry involved in seizures likely extends far beyond areas of canonical seizure activity Blumenfeld, ; Englot et al.

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In the present study, a minimally invasive technique was used to access deep brainstem nuclei of the rat midbrain tegmentum, with negligible disturbance of brain architecture. Using this technique, we attained stable, in vivo whole-cell recordings from neurons in the PPT while triggering focal limbic seizures.

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The contributions of excitatory versus inhibitory input as regulators of neuronal activity are integral to answering questions regarding mechanisms by which neurons process information Holt and Koch, ; Isaacson and Scanziani, ; Mitchell and Silver, ; Wehr and Zador, We identified a subset of reduced-firing hyperpolarizing RfHp neurons in the PPT, putatively cholinergic by histology. The technical challenges associated with blind-patch recording from individual neurons and attaining whole cell configurations using a pipette embedded in 7mm of living neural tissue resulted in a low sample-size of neurons in this study.

As such, firm conclusions from these data may be premature. The pattern of characteristics described here, however, could be explained by a mechanism of reduced excitatory synaptic input on cholinergic neurons in the PPT during seizures.

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For complete methods, see Supplemental Methods online. All procedures were conducted in compliance with approved institutional animal care and use protocols. A total of 54 female Sprague Dawley rats Charles River Laboratories age 6 — 10 weeks, weighing - g were used in these experiments.

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Female rats were used because seizures have been shown less likely to secondarily generalize in female rats compared to males. Janszky et al. Continuous recordings were made using Spike2 v8. Seizures were induced during a lightly anesthetized state as described ly see also Supplemental Methods Englot et al. Bipolar stimulating-recording electrodes were placed in the Hc bilaterally. Hc was stimulated, using a 2s, 60Hz square biphasic wave 1ms per phase. Hc stimulus current amplitude was titrated to produce focal hippocampal seizure activity based on polyspike discharges without propagation to frontal cortex.

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Propogation of polyspike activity to the orbitofrontal electrode was considered secondary generalization and such seizures were excluded from analyses. Because the reduced-firing hyperpolarizing phenotype Figure 1 was observed in comparatively short seizures as well as longer ones, no lower threshold on duration of seizures was stipulated for inclusion in analyses range of duration for included seizures was 6 to 72 seconds.

A Whole-cell current clamp recording of membrane potential Vm in PPT neuron shows reduced firing and hyperpolarization see inset during a focal seizure induced in the hippocampus by a 2s, 60 Hz stimulus gray bar. Concomitant recording of local field potential LFP shows polyspike seizure activity in the hippocampus Hc. B Histology demonstrates co-localization of biocytin from intracellular electrode solution staining and ChAT immunohistochemistry for neuron recorded in A.

Scale bar is 20 microns. For whole-cell recordings from PPT, procedures used ly for more superficial targets were modified See also Supplemental Methods McGinley et al. A potassium-gluconate-based intracellular solution with 0.

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Borosilicate glass pipettes 1BF-4, World Precision instruments were fabricated using a multi-line program to have a long mm taper to allow recordings deep below the cortical surface, and a resistance of 3. The neuron-searching step of acquiring whole cell recordings was undertaken in voltage-clamp mode, with current response visualized in real-time both in Spike2 software and on an external oscilloscope for blind-patching. To confirm locations of neurons, slices containing labelled neurons were compared to anatomical diagrams of rat brains Paxinos and Watson, supplemental figure S3. Data analysis was performed using Spike2 CED, v5.

Analysis epoch were defined as follows: 1. Baseline: 20—0 s before stimulus; 2. Ictal: the first 20 s of hippocampal seizure activity or the entire period of seizure activity where indicatedbased on large amplitude, polyspike activity in the hippocampal LFP recordings; 3. Postictal: 0—20 s after seizure; 4. Recovery: 20 s following the postictal period. All included neurons had RC charging curve compatible with transition to intracellular recordings, exhibited reproducible action potentials, and stable resting potential see Supplemental Methods for details. Spike sorting on the whole cell recordings was performed in Spike 2 using template-matching based on waveform shapes to identify single units.

Raster plots of neuronal firing were generated for each neuron, and then histograms of mean firing rate were calculated across neurons in 1s non-overlapping bins for each epoch Figures 2 and supplemental figure S3. A Raster plots five cells from five animals show decreased firing during the Ictal period compared with Baseline. Boxes in ictal panel indicate duration of seizures up to the first 20 s. B Histograms of firing rate in 1 s bins across neurons.

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Baseline is defined as the 20 s preceding seizure onset. Ictal is defined as up to the first 20 s following seizure onset. Postictal is the first 20 s following seizure end. Recovery is the 20 s following the Postictal period. Membrane potential in baseline and ictal epochs were averaged in 3s bins after excluding the 20ms period surrounding each action potential 5ms prior, 15ms following.

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The bin just prior to seizure stimulation within the baseline period was compared to the bin of most negative membrane potential during the ictal period to determine the amplitude of ictal hyperpolarization. Time course graphs of mean change in membrane potential, V m mV were constructed using 3s bins of V m data Figure 3 C.

Absolute change from baseline V m was calculated by taking the mean baseline V m for the 20s prior to seizure onset, and subtracting that mean baseline V m from all plotted 3s V m bins.

Action potentials are truncated. The trace shows reduced firing and hyperpolarization in the ictal period blueas well as a larger magnitude of V m response to constant current steps, indicating an increase in ictal input resistance. Inset on right shows expanded view of baseline black and ictal blue V m responses to current pulses, with resting potentials aligned vertically to enable differences in response amplitude to be compared more easily.

RfHp neurons D show consistent increases in R in during the ictal period compared to baseline. None of the non-RfHp neurons E show ificant increase in R in during seizures. Top black trace shows the baseline V m of an RfHp neuron, while the lower blue trace shows ictal V m of the same RfHp neuron.

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Orange markings overly EPSP-like events, with each event also marked as a black baseline or blue ictal tick-mark for easier comparison of relative frequency. In group analysis, frequency of EPSP-like events was ificantly decreased during the ictal periods of RfHp neurons compared to baseline see text.

Square hyperpolarizing pulses were delivered at a constant frequency and amplitude during the recording of each neuron Figure 3 A. Absolute change from baseline R in was calculated by taking the mean baseline R in for the 20s prior to seizure onset, and subtracting that mean baseline R in from all plotted 3s R in bins. As an additional measure of synaptic activity, we investigated the variance of voltage before, during and after seizures.

Variance in these epochs was compared using paired two-tailed t-tests. To graphically depict membrane potential distribution, we created voltage histograms centered to the mean of a fitted Gaussian.

Our goal was to find the mechanism for reduced firing of cholinergic neurons in PPT. To this end, whole-cell configuration was attained from 36 neurons in the stereotactic area of the PPT, after a total of pipette passes into the brains of 54 rats. Electrophysiologic definition of RfHp as used in this paper is as follows: 1.

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Most neurons that were recorded did not show the RfHp phenotype and are therefore referred to in this study as non-RfHp neurons supplemental figure S1. The locations of all histologically recovered neurons are shown in supplemental figure S2. Among the 5 neurons observed to show the RfHp phenotype on electrophysiology and included in firing rate analysis Figure 24 were recovered histologically. The action potential firing of RfHp neurons before, during and after focal seizures is depicted in the raster plots in Figure 2 A.

Histograms in Figure 2 B present the average firing rate in 1s bins over the course of each 20s epoch.

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For the purposes of directly comparing seizures of differing lengths, only up to the first 20s of the ictal epoch are depicted in Figure 2 and used in statistical analysis.

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