Exploration of Theta Burst-Induced Modulation of Transcranial Magnetic Stimulation-Evoked Potentials Over the Motor Cortex.

OBJECTIVES: This study investigates the way theta burst stimulation (TBS) applied to the motor cortex (M1) affects TMS-evoked potentials (TEPs). There have been few direct comparisons of continuous TBS (cTBS) and intermittent TBS (iTBS), and there is a lack of consensus from existing literature on the induced effects. We performed an exploratory trial to assess the effect of M1-cTBS and M1-iTBS on TEP components. MATERIALS AND METHODS: In a cross-over design, 15 participants each completed three experimental sessions with ≥one week in between sessions. The effect of a single TBS train administered over M1 was investigated using TEPs recorded at the same location, 20 to 30 minutes before and in the first 10 minutes after the intervention. In each session, a different type of TBS (cTBS, iTBS, or active control cTBS) was administered in a single-blinded randomized order. For six different TEP components (N15, P30, N45, P60, N100, and P180), amplitude was compared before and after the intervention using cluster-based permutation (CBP) analysis. RESULTS: We were unable to identify a significant modulation of any of the six predefined M1 TEP components after a single train of TBS. When waiving statistical correction for multiple testing in view of the exploratory nature of the study, the CBP analysis supports a reduction of the P180 amplitude after iTBS (p = 0.015), whereas no effect was observed after cTBS or in the active control condition. The reduction occurred in ten of 15 subjects, showing intersubject variability. CONCLUSIONS: The observed decrease in the P180 amplitude after iTBS may suggest a neuromodulatory effect of iTBS. Despite methodologic issues related to our study and the potential sensory contamination within this latency range of the TEP, we believe that our finding deserves further investigation in hypothesis-driven trials of adequate power and proper design, focusing on disentanglement between TEPs and peripherally evoked potentials, in addition to indicating reproducibility across sessions and subjects. CLINICAL TRIAL REGISTRATION: The Clinicaltrials.gov registration number for the study is NCT05206162.

Investigating the Working Mechanism of Transcranial Direct Current Stimulation.

BACKGROUND: Transcranial direct current stimulation (tDCS) is used to modulate neuronal activity, but the exact mechanism of action (MOA) is unclear. This study investigates tDCS-induced modulation of the corticospinal excitability and the underlying MOA. By anesthetizing the scalp before applying tDCS and by stimulating the cheeks, we investigated whether stimulation of peripheral and/or cranial nerves contributes to the effects of tDCS on corticospinal excitability. MATERIALS AND METHODS: In a randomized cross-over study, four experimental conditions with anodal direct current stimulation were compared in 19 healthy volunteers: 1) tDCS over the motor cortex (tDCS-MI), 2) tDCS over the motor cortex with a locally applied topical anesthetic (TA) on the scalp (tDCS-MI + TA), 3) DCS over the cheek region (DCS-C), and 4) sham tDCS over the motor cortex(sham). tDCS was applied for 20 minutes at 1 mA. Motor evoked potentials (MEPs) were measured before tDCS and immediately, 15, 30, 45, and 60 minutes after tDCS. A questionnaire was used to assess the tolerability of tDCS. RESULTS: A significant MEP amplitude increase compared with baseline was found 30 minutes after tDCS-MI, an effect still observed 60 minutes later; no time∗condition interaction effect was detected. In the other three conditions (tDCS-MI + TA, DCS-C, sham), no significant MEP modulation was found. The questionnaire indicated that side effects are significantly lower when the local anesthetic was applied before stimulation than in the other three conditions. CONCLUSIONS: The significant MEP amplitude increase observed from 30 minutes on after tDCS-MI supports the modulatory effect of tDCS on corticospinal neurotransmission. This effect lasted one hour after stimulation. The absence of a significant modulation when a local anesthetic was applied suggests that effects of tDCS are not solely established through direct cortical stimulation but that stimulation of peripheral and/or cranial nerves also might contribute to tDCS-induced modulation.

Glioblastoma-Associated Mesenchymal Stem/Stromal Cells and Cancer-Associated Fibroblasts: Partners in Crime?

Tumor-associated mesenchymal stem/stromal cells (TA-MSCs) have been recognized as attractive therapeutic targets in several cancer types, due to their ability to enhance tumor growth and angiogenesis and their contribution to an immunosuppressive tumor microenvironment (TME). In glioblastoma (GB), mesenchymal stem cells (MSCs) seem to be recruited to the tumor site, where they differentiate into glioblastoma-associated mesenchymal stem/stromal cells (GA-MSCs) under the influence of tumor cells and the TME. GA-MSCs are reported to exert important protumoral functions, such as promoting tumor growth and invasion, increasing angiogenesis, stimulating glioblastoma stem cell (GSC) proliferation and stemness, mediating resistance to therapy and contributing to an immunosuppressive TME. Moreover, they could act as precursor cells for cancer-associated fibroblasts (CAFs), which have recently been identified in GB. In this review, we provide an overview of the different functions exerted by GA-MSCs and CAFs and the current knowledge on the relationship between these cell types. Increasing our understanding of the interactions and signaling pathways in relevant models might contribute to future regimens targeting GA-MSCs and GB-associated CAFs to inhibit tumor growth and render the TME less immunosuppressive.

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.

Hippocampal seizures differentially modulate locus coeruleus activity and result in consistent time-locked release of noradrenaline in rat hippocampus.

The locus coeruleus (LC) is a small brainstem nucleus and is the sole source of noradrenaline in the neocortex, hippocampus and cerebellum. Noradrenaline is a powerful neuromodulator involved in the regulation of excitability and plasticity of large-scale brain networks. In this study, we performed a detailed assessment of the activity of locus coeruleus neurons and changes in noradrenergic transmission during acute hippocampal seizures evoked with perforant path stimulation, using state-of-the-art methodology. Action potentials of LC neurons, of which some were identified by means of optogenetics, were recorded in anesthetized rats using a multichannel high-density electrophysiology probe. The seizure-induced change in firing rate differed between LC neurons: 55% of neurons decreased in firing rate during seizures, while 28% increased their firing rate. Topographic analysis of multi-unit activity over the electrophysiology probe showed a topographic clustering of neurons that were inhibited or excited during seizures. Changes in hippocampal noradrenaline transmission during seizures were assessed using a fluorescent biosensor for noradrenaline, GRABNE2m, in combination with fiber photometry, in both anesthetized and awake rats. Although our neuronal recordings indicated both inhibition and excitation of LC neurons during seizures, a consistent release of noradrenaline was observed. Concentrations of noradrenaline increased at seizure onset and decreased during or shortly after the seizure. In conclusion, this study showed consistent but heterogeneous modulation of LC neurons and a consistent time-locked release of hippocampal noradrenaline during acute hippocampal seizures.

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.

Laryngeal Muscle-Evoked Potential Recording as an Indicator of Vagal Nerve Fiber Activation.

BACKGROUND: Vagus nerve stimulation (VNS) is an adjunctive therapy for drug-resistant epilepsy. Noninvasive evoked potential recordings in laryngeal muscles (LMEPs) innervated by vagal branches may provide a marker to assess effective vagal nerve fiber activation. We investigated VNS-induced LMEPs in patients with epilepsy in acute and chronic settings. MATERIALS AND METHODS: A total of 17 of 25 patients underwent LMEP recordings at initiation of therapy (acute group); 15 of 25 patients after one year of VNS (chronic group); and 7 of 25 patients were tested at both time points (acute + chronic group). VNS-induced LMEPs were recorded following different pulse widths and output currents using six surface laryngeal EMG electrodes to calculate input/output curves and estimate LMEP latency, threshold current for minimal (Ithreshold), half-maximal (I50), and 95% of maximal (I95) response induction and amplitude of maximal response (Vmax). These were compared with the acute + chronic group and between responders and nonresponders in the acute and chronic group. RESULTS: VNS-induced LMEPs were present in all patients. Ithreshold and I95 values ranged from 0.25 to 1.00 mA and from 0.42 to 1.77 mA, respectively. Estimated mean LMEP latencies were 10 ± 0.1 milliseconds. No significant differences between responders and nonresponders were observed. In the acute + chronic group, Ithreshold values remained stable over time. However, at the individual level in this group, Vmax was lower in all patients after one year compared with baseline. CONCLUSIONS: Noninvasive VNS-induced LMEP recording is feasible both at initiation of VNS therapy and after one year. Low output currents (0.25-1.00 mA) may be sufficient to activate vagal Aα-motor fibers. Maximal LMEP amplitudes seemed to decrease after chronic VNS therapy in patients.

Description of Osmolyte Pathways in Maturing Mdx Mice Reveals Altered Levels of Taurine and Sodium/Myo-Inositol Co-Transporters.

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration. Osmotic stress participates to DMD pathology and altered levels of osmolyte pathway members have been reported. The goal of this study was to gain insight in osmoregulatory changes in the mdx mouse model by examining the expression of osmolyte pathway members, including taurine transporter (TauT), sodium myo-inositol co-transporter (SMIT), betaine GABA transporter (BGT), and aldose reductase (AR) in the skeletal muscles and diaphragm of mdx mice aged 4, 8, 12, and 26 weeks. Necrosis was most prominent in 12 week-old mdx mice, whereas the amount of regenerated fibers increased until week 26 in the tibialis anterior. TauT protein levels were downregulated in the tibialis anterior and gastrocnemius of 4 to 12 week-old mdx mice, but not in 26 week-old mice, whereas TauT levels in the diaphragm remained significantly lower in 26 week-old mdx mice. In contrast, SMIT protein levels were significantly higher in the muscles of mdx mice when compared to controls. Our study revealed differential regulation of osmolyte pathway members in mdx muscle, which points to their complex involvement in DMD pathogenesis going beyond general osmotic stress responses. These results highlight the potential of osmolyte pathway members as a research interest and future therapeutic target in dystrophinopathy.

Exploring the Therapeutic Potential of Ectoine in Duchenne Muscular Dystrophy: Comparison with Taurine, a Supplement with Known Beneficial Effects in the mdx Mouse.

Duchenne Muscular Dystrophy (DMD) is a debilitating muscle disorder that condemns patients to year-long dependency on glucocorticoids. Chronic glucocorticoid use elicits many unfavourable side-effects without offering satisfying clinical improvement, thus, the search for alternative treatments to alleviate muscle inflammation persists. Taurine, an osmolyte with anti-inflammatory effects, mitigated pathological features in the mdx mouse model for DMD but interfered with murine development. In this study, ectoine is evaluated as an alternative for taurine in vitro in CCL-136 cells and in vivo in the mdx mouse. Pre-treating CCL-136 cells with 0.1 mM taurine and 0.1 mM ectoine prior to exposure with 300 U/mL IFN-γ and 20 ng/mL IL-1ß partially attenuated cell death, whilst 100 mM taurine reduced MHC-I protein levels. In vivo, histopathological features of the tibialis anterior in mdx mice were mitigated by ectoine, but not by taurine. Osmolyte treatment significantly reduced mRNA levels of inflammatory disease biomarkers, respectively, CCL2 and SPP1 in ectoine-treated mdx mice, and CCL2, HSPA1A, TNF-α and IL-1ß in taurine-treated mdx mice. Functional performance was not improved by osmolyte treatment. Furthermore, ectoine-treated mdx mice exhibited reduced body weight. Our results confirmed beneficial effects of taurine in mdx mice and, for the first time, demonstrated similar and differential effects of ectoine.

Ex Vivo Feedback Control of Neurotransmission Using a Photocaged Adenosine A1 Receptor Agonist.

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability.

Long-term chemogenetic suppression of seizures in a multifocal rat model of temporal lobe epilepsy.

OBJECTIVE: One third of epilepsy patients do not become seizure-free using conventional medication. Therefore, there is a need for alternative treatments. Preclinical research using designer receptors exclusively activated by designer drugs (DREADDs) has demonstrated initial success in suppressing epileptic activity. Here, we evaluated whether long-term chemogenetic seizure suppression could be obtained in the intraperitoneal kainic acid rat model of temporal lobe epilepsy, when DREADDs were selectively expressed in excitatory hippocampal neurons. METHODS: Epileptic male Sprague Dawley rats received unilateral hippocampal injections of adeno-associated viral vector encoding the inhibitory DREADD hM4D(Gi), preceded by a cell-specific promotor targeting excitatory neurons. The effect of clozapine-mediated DREADD activation on dentate gyrus evoked potentials and spontaneous electrographic seizures was evaluated. Animals were systemically treated with single (.1 mg/kg/24 h) or repeated (.1 mg/kg/6 h) injections of clozapine. In addition, long-term continuous release of clozapine and olanzapine (2.8 mg/kg/7 days) using implantable minipumps was evaluated. All treatments were administered during the chronic epileptic phase and between 1.5 and 13.5 months after viral transduction. RESULTS: In the DREADD group, dentate gyrus evoked potentials were inhibited after clozapine treatment. Only in DREADD-expressing animals, clozapine reduced seizure frequency during the first 6 h postinjection. When administered repeatedly, seizures were suppressed during the entire day. Long-term treatment with clozapine and olanzapine both resulted in significant seizure-suppressing effects for multiple days. Histological analysis revealed DREADD expression in both hippocampi and some cortical regions. However, lesions were also detected at the site of vector injection. SIGNIFICANCE: This study shows that inhibition of the hippocampus using chemogenetics results in potent seizure-suppressing effects in the intraperitoneal kainic acid rat model, even 1 year after viral transduction. Despite a need for further optimization, chemogenetic neuromodulation represents a promising treatment prospect for temporal lobe epilepsy.

Dynamic functional connectivity and graph theory metrics in a rat model of temporal lobe epilepsy reveal a preference for brain states with a lower functional connectivity, segregation and integration.

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. The involvement of abnormal functional brain networks in the development of epilepsy and its comorbidities has been demonstrated by electrophysiological and neuroimaging studies in patients with epilepsy. This longitudinal study investigated changes in dynamic functional connectivity (dFC) and network topology during the development of epilepsy using the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE). Resting state functional magnetic resonance images (rsfMRI) of 20 IPKA animals and 7 healthy control animals were acquired before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) under medetomidine anaesthesia using a 7 T MRI system. Starting from 17 weeks post-SE, hippocampal EEG was recorded to determine the mean daily seizure frequency of each animal. Dynamic FC was assessed by calculating the correlation matrices between fMRI time series of predefined regions of interest within a sliding window of 50 s using a step length of 2 s. The matrices were classified into 6 FC states, each characterized by a correlation matrix, using k-means clustering. In addition, several time-variable graph theoretical network metrics were calculated from the time-varying correlation matrices and classified into 6 states of functional network topology, each characterized by a combination of network metrics. Our results showed that FC states with a lower mean functional connectivity, lower segregation and integration occurred more often in IPKA animals compared to control animals. Functional connectivity also became less variable during epileptogenesis. In addition, average daily seizure frequency was positively correlated with percentage dwell time (i.e. how often a state occurs) in states with high mean functional connectivity, high segregation and integration, and with the number of transitions between states, while negatively correlated with percentage dwell time in states with a low mean functional connectivity, low segregation and low integration. This indicates that animals that dwell in states of higher functional connectivity, higher segregation and higher integration, and that switch more often between states, have more seizures.

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.

Ambulatory Electrocardiographic Monitoring and Ectopic Beat Detection in Conscious Mice.

Ambulatory electrocardiography (AECG) is a primary diagnostic tool in patients with potential arrhythmic disorders. To study the pathophysiological mechanisms of arrhythmic disorders, mouse models are widely implemented. The use of a technique similar to AECG for mice is thus of great relevance. We have optimized a protocol which allows qualitative, long-term ECG data recording in conscious, freely moving mice. Automated algorithms were developed to efficiently process the large amount of data and calculate the average heart rate (HR), the mean peak-to-peak interval and heart rate variability (HRV) based on peak detection. Ectopic beats are automatically detected based on aberrant peak intervals. As we have incorporated a multiple lead configuration in our ECG set-up, the nature and origin of the suggested ectopic beats can be analyzed in detail. The protocol and analysis tools presented here are promising tools for studies which require detailed, long-term ECG characterization in mouse models with potential arrhythmic disorders.

The Signaling Pathways Involved in the Anticonvulsive Effects of the Adenosine A1 Receptor.

Adenosine acts as an endogenous anticonvulsant and seizure terminator in the brain. Many of its anticonvulsive effects are mediated through the activation of the adenosine A1 receptor, a G protein-coupled receptor with a wide array of targets. Activating A1 receptors is an effective approach to suppress seizures. This review gives an overview of the neuronal targets of the adenosine A1 receptor focusing in particular on signaling pathways resulting in neuronal inhibition. These include direct interactions of G protein subunits, the adenyl cyclase pathway and the phospholipase C pathway, which all mediate neuronal hyperpolarization and suppression of synaptic transmission. Additionally, the contribution of the guanyl cyclase and mitogen-activated protein kinase cascades to the seizure-suppressing effects of A1 receptor activation are discussed. This review ends with the cautionary note that chronic activation of the A1 receptor might have detrimental effects, which will need to be avoided when pursuing A1 receptor-based epilepsy therapies.

Spontaneous Right Ventricular Pseudoaneurysms and Increased Arrhythmogenicity in a Mouse Model of Marfan Syndrome.

Patients with Marfan syndrome (MFS), a connective tissue disorder caused by pathogenic variants in the gene encoding the extracellular matrix protein fibrillin-1, have an increased prevalence of primary cardiomyopathy, arrhythmias, and sudden cardiac death. We have performed an in-depth in vivo and ex vivo study of the cardiac phenotype of Fbn1mgR/mgR mice, an established mouse model of MFS with a severely reduced expression of fibrillin-1. Using ultrasound measurements, we confirmed the presence of aortic dilatation and observed cardiac diastolic dysfunction in male Fbn1mgR/mgR mice. Upon post-mortem examination, we discovered that the mutant mice consistently presented myocardial lesions at the level of the right ventricular free wall, which we characterized as spontaneous pseudoaneurysms. Histological investigation demonstrated a decrease in myocardial compaction in the MFS mouse model. Furthermore, continuous 24 h electrocardiographic analysis showed a decreased heart rate variability and an increased prevalence of extrasystolic arrhythmic events in Fbn1mgR/mgR mice compared to wild-type littermates. Taken together, in this paper we document a previously unreported cardiac phenotype in the Fbn1mgR/mgR MFS mouse model and provide a detailed characterization of the cardiac dysfunction and rhythm disorders which are caused by fibrillin-1 deficiency. These findings highlight the wide spectrum of cardiac manifestations of MFS, which might have implications for patient care.

Development of a Rat Model for Glioma-Related Epilepsy.

Seizures are common in patients with high-grade gliomas (30-60%) and approximately 15-30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures.

Alterations in the functional brain network in a rat model of epileptogenesis: A longitudinal resting state fMRI study.

Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. Electrophysiological and neuroimaging studies in patients with epilepsy suggest that abnormal functional brain networks play a role in the development of epilepsy, i.e. epileptogenesis, resulting in the generation of spontaneous seizures and cognitive impairment. In this longitudinal study, we investigated changes in functional brain networks during epileptogenesis in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE) using resting state functional magnetic resonance imaging (rsfMRI) and graph theory. Additionally, we investigated whether these changes are related to the frequency of occurrence of spontaneous epileptic seizures in the chronic phase of epilepsy. Using a 7T MRI system, rsfMRI images were acquired under medetomidine anaesthesia before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) induction in 20 IPKA animals and 7 healthy control animals. To obtain a functional network, correlation between fMRI time series of 38 regions of interest (ROIs) was calculated. Then, several graph theoretical network measures were calculated to describe and quantify the network changes. At least 17 weeks post-SE, IPKA animals were implanted with electrodes in the left and right dorsal hippocampus, EEG was measured for 7 consecutive days and spontaneous seizures were counted. Our results show that correlation coefficients of fMRI time series shift to lower values during epileptogenesis, indicating weaker whole brain network connections. Segregation and integration in the functional brain network also decrease, indicating a lower local interconnectivity and a lower overall communication efficiency. Secondly, this study demonstrates that the largest decrease in functional connectivity is observed for the retrosplenial cortex. Finally, post-SE changes in functional connectivity, segregation and integration are correlated with seizure frequency in the IPKA rat model.

Long-term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy.

OBJECTIVE: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. METHODS: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. RESULTS: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. SIGNIFICANCE: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.

Involvement of fast-spiking cells in ictal sequences during spontaneous seizures in rats with chronic temporal lobe epilepsy.

See Lenck-Santini (doi:10.1093/awx205) for a scientific commentary on this article. Epileptic seizures represent altered neuronal network dynamics, but the temporal evolution and cellular substrates of the neuronal activity patterns associated with spontaneous seizures are not fully understood. We used simultaneous recordings from multiple neurons in the hippocampus and neocortex of rats with chronic temporal lobe epilepsy to demonstrate that subsets of cells discharge in a highly stereotypical sequential pattern during ictal events, and that these stereotypical patterns were reproducible across consecutive seizures. In contrast to the canonical view that principal cell discharges dominate ictal events, the ictal sequences were predominantly composed of fast-spiking, putative inhibitory neurons, which displayed unusually strong coupling to local field potential even before seizures. The temporal evolution of activity was characterized by unique dynamics where the most correlated neuronal pairs before seizure onset displayed the largest increases in correlation strength during the seizures. These results demonstrate the selective involvement of fast spiking interneurons in structured temporal sequences during spontaneous ictal events in hippocampal and neocortical circuits in experimental models of chronic temporal lobe epilepsy.