Optogenetic determination of dynamic and cell-type-specific inhibitory reversal potentials.

The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo, and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically-defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials, and thereby expand the repertoire of optogenetics.Significance statement The strength of synaptic inhibition in the brain is determined, in part, by the reversal potential of the ionic currents that flow through inhibitory ligand-gated ion channels (EInh). Estimates of EInh have traditionally used agonists to activate receptors on the cell surface, but this has limitations. Our study presents an optogenetic strategy for performing agonist-independent measurements of EInh in the brain. We demonstrate the effectiveness of the approach in vitro, in vivo, and across different neuronal subtypes. Its excellent temporal control allows for measurements of EInh dynamics, which reveal differences between genetically-defined neuronal subpopulations. This expands the application of optogenetics and affords new opportunities to study synaptic inhibition.

Research on the Control of Gastrointestinal Strongyles in Sheep by Using Lotus corniculatus or Cichorium intybus in Feed.

The general practice of sheep farmers in gastrointestinal helminth control is based on the use of commercial drugs, making chemoresistance very common. Considering this, our study focused on the biological control of gastrointestinal parasitism using high-tannin plant hay. Three groups of 30 animals each were formed. The control group was additionally fed meadow hay, while the other two groups received chicory (group 2) and bird's foot trefoil hay (group 3). The number of gastrointestinal strongyle eggs, shed through faeces (EPG), was surveyed for 28 days for all animals. The amounts of total tannins for meadow, chicory, and Lotus corniculatus hay supplements were 13.92 mg/g, 78.59 mg/g, and 94.43 mg/g, while their condensed tannin contents were 2.58 mg/g, 29.84 mg/g, and 15.94 mg/g, respectively. Compared to experimental day 0, there was an increase in EPG of 80.83% in the control group, a decrease of 24.72% in group 2, and a 20% decrease in group 3, by day 28. The p-value was <0.05 between group 1 and the other groups, showing significant differences between the control and experimental groups. The decrease in EPG rates in the experimental groups compared to the control group demonstrates an antiparasitic effect of Lotus corniculatus and chicory.

A genetically targeted ion sensor reveals distinct seizure-related chloride and pH dynamics in GABAergic interneuron populations.

Intracellular chloride and pH play fundamental roles in determining a neuron's synaptic inhibition and excitability. Yet it has been difficult to measure changes in these ions during periods of heightened network activity, such as occur in epilepsy. Here we develop a version of the fluorescent reporter, ClopHensorN, to enable simultaneous quantification of chloride and pH in genetically defined neurons during epileptiform activity. We compare pyramidal neurons to the major GABAergic interneuron subtypes in the mouse hippocampus, which express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal polypeptide (VIP). Interneuron populations exhibit higher baseline chloride, with PV interneurons exhibiting the highest levels. During an epileptiform discharge, however, all subtypes converge upon a common elevated chloride level. Concurrent with these dynamics, epileptiform activity leads to different degrees of intracellular acidification, which reflect baseline pH. Thus, a new optical tool for dissociating chloride and pH reveals neuron-specific ion dynamics during heightened network activity.

Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization.

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.

Disrupting Epileptiform Activity by Preventing Parvalbumin Interneuron Depolarization Block.

Inhibitory synaptic mechanisms oppose epileptic network activity in the brain. The breakdown in this inhibitory restraint and propagation of seizure activity has been linked to the overwhelming of feedforward inhibition, which is provided in large part by parvalbumin-expressing (PV) interneurons in the cortex. The underlying cellular processes therefore represent potential targets for understanding and preventing the propagation of seizure activity. Here we use an optogenetic strategy to test the hypothesis that depolarization block in PV interneurons is a significant factor during the loss of inhibitory restraint. Depolarization block results from the inactivation of voltage-gated sodium channels and leads to impaired action potential firing. We used focal NMDA stimulation to elicit reproducible epileptiform discharges in hippocampal organotypic brain slices from male and female mice and combined this with targeted recordings from defined neuronal populations. Simultaneous patch-clamp recordings from PV interneurons and pyramidal neurons revealed epileptiform activity that was associated with an overwhelming of inhibitory synaptic mechanisms and the emergence of a partial, and then complete, depolarization block in PV interneurons. To counteract this depolarization block, we developed protocols for eliciting pulsed membrane hyperpolarization via the inhibitory opsin, archaerhodopsin. This optical approach was effective in counteracting cumulative inactivation of voltage-gated channels, maintaining PV interneuron action potential firing properties during the inhibitory restraint period, and reducing the probability of initiating epileptiform activity. These experiments support the idea that depolarization block is a point of weakness in feedforward inhibitory synaptic mechanisms and represents a target for preventing the initiation and spread of seizure activity.SIGNIFICANCE STATEMENT GABAA receptor-mediated synaptic transmission opposes seizure activity by establishing an inhibitory restraint against spreading excitation. Parvalbumin-expressing (PV) interneurons contribute significantly to this inhibitory restraint, but it has been suggested that these cells are overwhelmed as they enter a state of "depolarization block." Here we test the importance of this process by devising an optogenetic strategy to selectively relieve depolarization block in PV interneurons. By inducing brief membrane hyperpolarization, we show that it is possible to reduce depolarization block in PV interneurons, maintain their action potential firing in the face of strong excitation, and disrupt epileptiform activity in an in vitro model. This represents a proof of principle that targeting rate-limiting processes can strengthen the inhibitory restraint of epileptiform activity.

Excitatory GABAergic signalling is associated with benzodiazepine resistance in status epilepticus.

Status epilepticus is defined as a state of unrelenting seizure activity. Generalized convulsive status epilepticus is associated with a rapidly rising mortality rate, and thus constitutes a medical emergency. Benzodiazepines, which act as positive modulators of chloride (Cl-) permeable GABAA receptors, are indicated as first-line treatment, but this is ineffective in many cases. We found that 48% of children presenting with status epilepticus were unresponsive to benzodiazepine treatment, and critically, that the duration of status epilepticus at the time of treatment is an important predictor of non-responsiveness. We therefore investigated the cellular mechanisms that underlie acquired benzodiazepine resistance, using rodent organotypic and acute brain slices. Removing Mg2+ ions leads to an evolving pattern of epileptiform activity, and eventually to a persistent state of repetitive discharges that strongly resembles clinical EEG recordings of status epilepticus. We found that diazepam loses its antiseizure efficacy and conversely exacerbates epileptiform activity during this stage of status epilepticus-like activity. Interestingly, a low concentration of the barbiturate phenobarbital had a similar exacerbating effect on status epilepticus-like activity, while a high concentration of phenobarbital was effective at reducing or preventing epileptiform discharges. We then show that the persistent status epilepticus-like activity is associated with a reduction in GABAA receptor conductance and Cl- extrusion capability. We explored the effect on intraneuronal Cl- using both gramicidin, perforated-patch clamp recordings and Cl- imaging. This showed that during status epilepticus-like activity, reduced Cl- extrusion capacity was further exacerbated by activity-dependent Cl- loading, resulting in a persistently high intraneuronal Cl-. Consistent with these results, we found that optogenetic stimulation of GABAergic interneurons in the status epilepticus-like state, actually enhanced epileptiform activity in a GABAAR dependent manner. Together our findings describe a novel potential mechanism underlying benzodiazepine-resistant status epilepticus, with relevance to how this life-threatening condition should be managed in the clinic.

Burst-Suppression Ratio on Electrocorticography Depends on Interelectrode Distance.

INTRODUCTION: With deepening of anesthesia-induced comatose states, the EEG becomes fragmented by increasing periods of suppression. When measured from conventional EEG recordings, the binary burst-suppression signal (BS) appears similar across the scalp. As such, the BS ratio (BSR), quantifying the fraction of time spent in suppression, is clinically considered a global index of brain function in sedation monitoring. Recent studies indicate that BS may be considerably asynchronous when measured with higher spatial resolution such as on electrocorticography. The authors investigated the magnitude of BSR changes with cortical recording interelectrode distance. METHODS: The authors selected fronto-parietal electrocorticography recordings showing propofol-induced BS recorded via 8-electrode strips (1-cm interelectrode distance) during cortical motor mapping in 31 patients. For 1-minute epochs, bipolar recordings were computed between each electrode pair. The median BSR, burst duration (BD), and bursting frequency were derived for each interelectrode distance. RESULTS: At 1-cm interelectrode distance, with increasing BSR, BD decreased exponentially. For a BSR between 50% and 80%, BD reached a plateau of 2.1 seconds while the bursting frequency decreased from 14 to 6 bursts per minute. With increasing interelectrode distance, BD increased at a rate of 0.2 seconds per cm. This correlated with a decrease in BSR with distance that reached the rate of -4.4 percentage per centimeters during deepest anesthesia. CONCLUSIONS: With increasing cortical interelectrode recording distance, burst summation leads to an increasing BD associated with a reduction in BSR. Standardization of interelectrode distance is important for cortical BSR measurements.

Intraoperative somatosensory evoked potential monitoring decreases EEG burst suppression ratio during deep general anesthesia.

PURPOSE: The burst suppression (BS) EEG patterns induced by general anesthesia can react to somatosensory stimuli. We investigated this reactivity by studying the effect of peripheral nerve stimulation used for routine intraoperative spinal cord monitoring by somatosensory evoked potentials on BS patterns. METHODS: The relative time spent in suppression expressed as BS ratio (BSR) and mean burst duration were measured before (BSR(Pre)), during (BSR(Stim)), and after (BSR(Post)) a 60-second repetitive electrical ulnar nerve stimulation in nine patients under total intravenous general anesthesia with propofol. The BS reactivity was measured as BSR(Pre)-BSR(Stim). RESULTS: Overall, 27 trials were included with BSR(Pre) up to 77%, indistinguishable from BSR(Post). During stimulation, the mean BSR transiently decreased from 42% to 35%. For each 1% increase in BSR(Pre), the BS reactivity increased with 0.6%, whereas the burst duration remained approximately 3 seconds. For BSR(Pre) below 30%, the BS reactivity was negligible. CONCLUSIONS: Data from this study show that somatosensory input can evoke bursts, altering the "spontaneous" deep BS patterns (BSR(Pre) >30%). Further studies are necessary to objectively assess the clinical relevance of stimulus-induced BS reactivity during deep general anesthesia.