Increased Dentate Gyrus Excitability in the Intrahippocampal Kainic Acid Mouse Model for Temporal Lobe Epilepsy.

The intrahippocampal kainic acid (IHKA) mouse model is an extensively used in vivo model to investigate the pathophysiology of mesial temporal lobe epilepsy (mTLE) and to develop novel therapies for drug-resistant epilepsy. It is characterized by profound hippocampal sclerosis and spontaneously occurring seizures with a major role for the injected damaged hippocampus, but little is known about the excitability of specific subregions. The purpose of this study was to electrophysiologically characterize the excitability of hippocampal subregions in the chronic phase of the induced epilepsy in the IHKA mouse model. We recorded field postsynaptic potentials (fPSPs) after electrical stimulation in the CA1 region and in the dentate gyrus (DG) of hippocampal slices of IHKA and healthy mice using a multielectrode array (MEA). In the DG, a significantly steeper fPSP slope was found, reflecting higher synaptic strength. Population spikes were more prevalent with a larger spatial distribution in the IHKA group, reflecting a higher degree of granule cell output. Only minor differences were found in the CA1 region. These results point to increased neuronal excitability in the DG but not in the CA1 region of the hippocampus of IHKA mice. This method, in which the excitability of hippocampal slices from IHKA mice is investigated using a MEA, can now be further explored as a potential new model to screen for new interventions that can restore DG function and potentially lead to novel therapies for mTLE.

Investigating the Effect of Transcutaneous Auricular Vagus Nerve Stimulation on Cortical Excitability in Healthy Males.

OBJECTIVES: As a potential treatment for epilepsy, transcutaneous auricular vagus nerve stimulation (taVNS) has yielded inconsistent results. Combining transcranial magnetic stimulation with electromyography (TMS-EMG) and electroencephalography (TMS-EEG) can be used to investigate the effect of interventions on cortical excitability by evaluating changes in motor evoked potentials (MEPs) and TMS-evoked potentials (TEPs). The goal of this study is to objectively evaluate the effect of taVNS on cortical excitability with TMS-EMG and TMS-EEG. These findings are expected to provide insight in the mechanism of action and help identify more optimal stimulation paradigms. MATERIALS AND METHODS: In this prospective single-blind cross-over study, 15 healthy male subjects underwent active and sham taVNS for 60 min, using a maximum tolerated stimulation current. Single and paired pulse TMS was delivered over the right-sided motor hotspot to evaluate MEPs and TEPs before and after the intervention. MEP statistical analysis was conducted with a two-way repeated measures ANOVA. TEPs were analyzed with a cluster-based permutation analysis. Linear regression analysis was implemented to investigate an association with stimulation current. RESULTS: MEP and TEP measurements were not affected by taVNS in this study. An association was found between taVNS stimulation current and MEP outcome measures indicating a decrease in cortical excitability in participants who tolerated higher taVNS currents. A subanalysis of participants (n = 8) who tolerated a taVNS current ≥2.5 mA showed a significant increase in the resting motor threshold, decrease in MEP amplitude and modulation of the P60 and P180 TEP components. CONCLUSIONS: taVNS did not affect cortical excitability measurements in the overall population in this study. However, taVNS has the potential to modulate specific markers of cortical excitability in participants who tolerate higher stimulation levels. These findings indicate the need for adequate stimulation protocols based on the recording of objective outcome parameters.

Attenuation of Hippocampal Evoked Potentials in vivo by Activation of GtACR2, an Optogenetic Chloride Channel.

AIM: GtACR2, a light-activated chloride channel, is an attractive tool for neural inhibition as it can shunt membrane depolarizations. In this study, we assessed the effect of activating GtACR2 on in vivo hippocampal CA1 activity evoked by Schaffer collateral (SC) stimulation. METHODS: Adult male Wistar rats were unilaterally injected with 0.5 µL of adeno associated viral vector for induction of GtACR2-mCherry (n = 10, GtACR2 group) or mCherry (n = 4, Sham group) expression in CA1 pyramidal neurons of the hippocampus. Three weeks later, evoked potentials (EPs) were recorded from the CA1 subfield placing an optrode (bipolar recording electrode attached to an optic fiber) at the injection site and a stimulation electrode targeting SCs. Effects of illumination parameters required to activate GtACR2 such as light power densities (LPDs), illumination delays, and light-pulse durations were tested on CA1 EP parameters [population spike (PS) amplitude and field excitatory postsynaptic potential (fEPSP) slope]. RESULTS: In the GtACR2 group, delivery of a 10 ms light-pulse induced a negative deflection in the local field potential which increased with increasing LPD. When combined with electrical stimulation of the SCs, light-induced activation of GtACR2 had potent inhibitory effects on CA1 EPs. An LPD of 160 mW/mm2 was sufficient to obtain maximal inhibition CA1 EPs. To quantify the duration of the inhibitory effect, a 10 ms light-pulse of 160 mW/mm2 was delivered at increasing delays before the CA1 EPs. Inhibition of EPs was found to last up to 9 ms after the cessation of the light-pulse. Increasing light-pulse durations beyond 10 ms did not result in larger inhibitory effects. CONCLUSION: Precisely timed activation of GtACR2 potently blocks evoked activity of CA1 neurons. The strength of inhibition depends on LPD, lasts up to 9 ms after a light-pulse of 10 ms, and is independent of the duration of the light-pulse given.

Identification of vagus nerve stimulation parameters affecting rat hippocampal electrophysiology without temperature effects.

BACKGROUND: Recent experiments in rats have demonstrated significant effects of VNS on hippocampal excitability but were partially attributed to hypothermia, induced by the applied VNS parameters. OBJECTIVE: To allow meaningful preclinical research on the mechanisms of VNS and translation of rodent results to clinical VNS trials, we aimed to identify non-hypothermia inducing VNS parameters that significantly affect hippocampal excitability. METHODS: VNS was administered in cycles of 30 s including either 0.1, 0.16, 0.25, 0.5, 1.5, 3 or 7 s of VNS ON time (biphasic pulses, 250µs/phase, 1 mA, 30 Hz) and the effect of different VNS ON times on brain temperature was evaluated. VNS paradigms with and without hypothermia were compared for their effects on hippocampal neurophysiology in freely moving rats. RESULTS: Using VNS parameters with an ON time/OFF time of up to 0.5 s/30 s did not cause hypothermia, while clear hypothermia was detected with ON times of 1.5, 3 and 7 s/30 s. Relative to SHAM VNS, the normothermic 0.5 s VNS condition significantly decreased hippocampal EEG power and changed dentate gyrus evoked potentials with an increased field excitatory postsynaptic potential slope and a decreased population spike amplitude. CONCLUSION: VNS can be administered in freely moving rats without causing hypothermia, while profoundly affecting hippocampal neurophysiology suggestive of reduced excitability of hippocampal neurons despite increased synaptic transmission efficiency.

Deep brain stimulation reduces evoked potentials with a dual time course in freely moving rats: Potential neurophysiological basis for intermittent as an alternative to continuous stimulation.

OBJECTIVE: Deep brain stimulation (DBS) is an increasingly applied treatment for various neuropsychiatric disorders including drug-resistant epilepsy, and it may be optimized by rationalizing the stimulation protocol based on increased knowledge of its mechanism of action. We evaluated the effects of minutes to hours of hippocampal DBS on hippocampal evoked potentials (EPs) and local field potentials (LFPs) in freely moving male rats to further investigate some of the previously proposed mechanisms of action. METHODS: Hippocampal high-frequency (130 Hz) DBS was administered for 0, 1, or 6 min every 10 min for 160 min. Stimulation parameter settings were similar to those that had previously been shown to reduce seizures in epileptic rats. EPs and LFPs were recorded in the stimulation-free intervals. We investigated both the immediate temporary effects of 1 or 6 min of DBS and the effects of 160 min of intermittent DBS. Input specificity was investigated by using two different stimulation electrodes. RESULTS: Relatively low DBS intensities corresponding to only 1.8% of the intensity evoking a maximum EP were required to prevent unintended seizure occurrence in healthy rats. Both 1 and 6 min of DBS caused input-specific short-lasting (<60 s) reductions (5%-7%) of the field excitatory postsynaptic potential (fEPSP) slope (P = .005). We observed longer-lasting, input-specific EP reductions during the 160 min intermittent DBS, with statistically significant reductions (3%-4%) of the fEPSP slope (P = .009-.018). The LFP spectrogram remained unaltered. SIGNIFICANCE: Deep brain stimulation induced both acute temporary effects compatible with axonal block and/or synaptic depression, and longer-lasting potentially cumulative EP reductions, suggesting the involvement of homeostatic plasticity or long-term depression. This dual time course may parallel the different temporal patterns of improvement observed in clinical trials. The longer-lasting reductions provide a potential neurophysiological basis for the use of intermittent DBS-as typically used in epilepsy patients-as an alternative to continuous DBS.

A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity.

AIM: Selective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine. METHODS: The LC of male Sprague-Dawley rats was injected with 10 nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n = 19, DREADD group) or AAV2/7-PRSx8-eGFP (n = 13, Controls). Three weeks later, LC unit recordings were performed in anesthetized rats. We investigated whether clozapine, a drug known to bind to modified neurons expressing hM3Dq receptors, was able to increase the LC firing rate. Baseline unit activity was recorded followed by subsequent administration of 0.01 and 0.1 mg/kg of clozapine in all rats. hM3Dq-mcherry expression levels were investigated using immunofluorescence staining of brainstem slices at the end of the experiment. RESULTS: Unit recordings could be performed in 12 rats and in a total of 12 neurons (DREADDs: n = 7, controls: n = 5). Clozapine 0.01 mg/kg did not affect the mean firing rate of recorded LC-neurons; 0.1 mg/kg induced an increased firing rate, irrespective whether neurons were recorded from DREADD or control rats (p = 0.006). Co-labeling of LC neurons and mCherry-tag showed that 20.6 ± 2.3% LC neurons expressed the hM3Dq receptor. Aspecific expression of hM3Dq-mCherry was also observed in non-LC neurons (26.0 ± 4.1%). CONCLUSION: LC unit recording is feasible in an experimental set-up following manipulations for DREADD induction. A relatively low transduction efficiency of the used AAV was found. In view of this finding, the effect of injected clozapine on LC-NA could not be investigated as a reliable outcome parameter for activation of chemogenetically modified LC neurons. The use of AAV2/7, a vector previously applied successfully to target dopaminergic neurons in the substantia nigra, leads to insufficient chemogenetic modification of the LC compared to transduction with AAV2/9.

Clinical Vagus Nerve Stimulation Paradigms Induce Pronounced Brain and Body Hypothermia in Rats.

Vagus nerve stimulation (VNS) is a widely used neuromodulation technique that is currently used or being investigated as therapy for a wide array of human diseases such as epilepsy, depression, Alzheimer's disease, tinnitus, inflammatory diseases, pain, heart failure and many others. Here, we report a pronounced decrease in brain and core temperature during VNS in freely moving rats. Two hours of rapid cycle VNS (7s on/18s off) decreased brain temperature by around [Formula: see text]C, while standard cycle VNS (30[Formula: see text]s on/300[Formula: see text]s off) was associated with a decrease of around [Formula: see text]C. Rectal temperature similarly decreased by more than [Formula: see text]C during rapid cycle VNS. The hypothermic effect triggered by VNS was further associated with a vasodilation response in the tail, which reflects an active heat release mechanism. Despite previous evidence indicating an important role of the locus coeruleus-noradrenergic system in therapeutic effects of VNS, lesioning this system with the noradrenergic neurotoxin DSP-4 did not attenuate the hypothermic effect. Since body and brain temperature affect most physiological processes, this finding is of substantial importance for interpretation of several previously published VNS studies and for the future direction of research in the field.

Modulation of Hippocampal Activity by Vagus Nerve Stimulation in Freely Moving Rats.

BACKGROUND: Vagus Nerve Stimulation (VNS) has seizure-suppressing effects but the underlying mechanism is not fully understood. To further elucidate the mechanisms underlying VNS-induced seizure suppression at a neurophysiological level, the present study examined effects of VNS on hippocampal excitability using dentate gyrus evoked potentials (EPs) and hippocampal electroencephalography (EEG). METHODS: Male Sprague-Dawley rats were implanted with a VNS electrode around the left vagus nerve. A bipolar stimulation electrode was implanted in the left perforant path and a bipolar recording electrode was implanted in the left dentate gyrus for EEG and dentate field EP recording. Following recovery, VNS was applied in freely moving animals, using a duty cycle of 7 s on/18 s off, 30 Hz frequency, 250 µs pulse width, and an intensity of either 0 (SHAM), 25 µA or 1000 µA, while continuously monitoring EEG and dentate field EPs. RESULTS: VNS at 1000 µA modulated dentate field EPs by decreasing the field excitatory post-synaptic potential (fEPSP) slope and increasing the latency and amplitude of the population spike. It additionally influenced hippocampal EEG by slowing theta rhythm from 7 Hz to 5 Hz and reducing theta peak and gamma band power. No effects were observed in the SHAM or 25 µA VNS conditions. CONCLUSION: VNS modulated hippocampal excitability of freely moving rats in a complex way. It decreased synaptic efficacy, reflected by decreased fEPSP slope and EEG power, but it simultaneously facilitated dentate granule cell discharge indicating depolarization of dentate granule cells.