Evoked responses to single pulse electrical stimulation reveal impaired striatal excitability in a rat model of Parkinson's disease.

BACKGROUND: Sensorimotor beta oscillations are increased in Parkinson's disease (PD) due to the alteration of dopaminergic transmission. This electrophysiological read-out is reported both in patients and in animal models such as the 6-OHDA rat model obtained with unilateral nigral injection of 6-hydroxydopamine (6-OHDA). Current treatments, based on dopaminergic replacement, transiently normalize this pathological beta activity and improve patients' quality of life. OBJECTIVES: We wanted to assess in vivo whether the abnormal beta oscillations can be correlated with impaired striatal or cortical excitability of the sensorimotor system and modulated by the pharmacological manipulation of the dopaminergic system. METHODS: In the unilateral 6-OHDA rat model and control animals, we used intra-striatal and intra-cortical single-pulse electrical stimulation (SPES) and concurrent local field potentials (LFP) recordings. In the two groups, we quantified basal cortico-striatal excitability from time-resolved spectral analyses of LFP evoked responses induced remotely by intracerebral stimulations. The temporal dependance of cortico-striatal excitability to dopaminergic transmission was further tested using electrophysiological recordings combined with levodopa injection. RESULTS: LFP evoked responses after striatal stimulation showed a transient reduction of power in a large time-frequency domain in the 6-OHDA group compared to the sham group. This result was specific to the striatum, as no significant difference was observed in cortical LFP evoked responses between the two groups. This impaired striatal excitability in the 6-OHDA group was observed in the striatum at least during the first 3 months after the initial lesion. In addition, the striatum responses to SPES during a levodopa challenge showed a transient potentiation of the decrease of responsiveness in frequencies below 40 Hz. CONCLUSION: The spectral properties of striatal responses to SPES show high sensitivity to dopaminergic transmission in the unilateral 6-OHDA rat model. We thus propose that this approach could be used in preclinical models as a time-resolved biomarker of impaired dopaminergic transmission capable of monitoring progressive neurodegeneration and/or challenges to drug intake.

Pronounced antiseizure activity of the subtype-selective GABAA positive allosteric modulator darigabat in a mouse model of drug-resistant focal epilepsy.

AIM: Darigabat is an α2/3/5 subunit-selective positive allosteric modulator of GABAA receptors that has demonstrated broad-spectrum activity in several preclinical models of epilepsy as well as in a clinical photoepilepsy trial. The objective here was to assess the acute antiseizure effect of darigabat in the mesial temporal lobe epilepsy (MTLE) mouse model of drug-resistant focal seizures. METHODS: The MTLE model is generated by single unilateral intrahippocampal injection of low dose (1 nmole) kainic acid in adult mice, and subsequent epileptiform activity is recorded following implantation of a bipolar electrode under general anesthesia. After a period of epileptogenesis (~4 weeks), spontaneous and recurrent hippocampal paroxysmal discharges (HPD; focal seizures) are recorded using intracerebral electroencephalography. The number and cumulated duration of HPDs were recorded following administration of vehicle (PO), darigabat (0.3-10 mg kg-1 , PO), and positive control diazepam (2 mg kg-1 , IP). RESULTS: Darigabat dose-dependently reduced the expression of HPDs, demonstrating comparable efficacy profile to diazepam at doses of 3 and 10 mg kg-1 . CONCLUSIONS: Darigabat exhibited a robust efficacy profile in the MTLE model, a preclinical model of drug-resistant focal epilepsy. A Phase II proof-of-concept placebo-controlled, adjunctive-therapy trial (NCT04244175) is ongoing to evaluate efficacy and safety of darigabat in patients with drug-resistant focal seizures.

Single-pulse electrical stimulation methodology in freely moving rat.

BACKGROUND: Cortico-cortical evoked potentials (CCEP) are becoming popular to infer brain connectivity and cortical excitability in implanted refractory epilepsy patients. Our goal was to transfer this methodology to the freely moving rodent. NEW METHOD: CCEP were recorded on freely moving Sprague-Dawley rats, from cortical and subcortical areas using depth electrodes. Electrical stimulation was applied using 1 ms biphasic current pulse, cathodic first, at a frequency of 0.5 Hz, with intensities ranging from 0.2 to 0.8 mA. Data were then processed in a similar fashion to human clinical studies, which included epoch selection, artefact correction and smart averaging. RESULTS: For a large range of tested intensities, we recorded CCEPs with very good signal to noise ratio and reproducibility between animals, without any behavioral modification. The CCEP were composed of different components according to recorded and stimulated sites, similarly to human recordings. COMPARISON WITH EXISTING METHODS: We minimally adapted a clinically-motivated methodology to a freely moving rodent model to achieve high translational relevance of future preclinical studies. CONCLUSIONS: Our results indicate that the CCEP methodology can be applied to freely moving rodents and transferred to preclinical research. This will be of interest to address various neuroscientific questions, in physiological and pathological conditions.

Differential effects of traxoprodil and S-ketamine on quantitative EEG and auditory event-related potentials as translational biomarkers in preclinical trials in rats and mice.

Quantitative Electroencephalography (qEEG) and event-related potential (ERP) assessment have emerged as powerful tools to unravel translational biomarkers in preclinical and clinical psychiatric drug discovery trials. The aim of the present study was to compare the GluN2B negative allosteric modulator (NAM) traxoprodil (CP-101,606) with the unselective NMDA receptor channel blocker S-ketamine to give insight into central target engagement and differentiation on multiple EEG readouts. For qEEG recordings telemetric transmitters were implanted in male Wistar rats. Recorded EEG data were analyzed using fast Fourier transformation to determine power spectra and vigilance states. Additionally, body temperature and locomotor activity were assessed via telemetry. For recordings of auditory event-related potentials (AERP) male C57Bl/6J mice were chronically implanted with deep electrodes using a tethered system. Power spectral analysis revealed a significant increase in gamma power following ketamine treatment, whereas traxoprodil (6&18 mg/kg) induced an overall decrease primarily within alpha and beta bands. Additionally, ketamine disrupted sleep and enhanced time spent in wake vigilance states, whereas traxoprodil did not alter sleep-wake architecture. AERP and mismatch negativity (MMN) revealed that ketamine (10 mg/kg) selectively disrupts auditory deviance detection, whereas traxoprodil (6 mg/kg) did not alter MMN at clinically relevant doses. In contrast to ketamine treatment, traxoprodil did not produce hyperactivity and hypothermia. In conclusion, ketamine and traxoprodil showed very different effects on diverse EEG readouts differentiating selective GluN2B antagonism from non-selective pan-NMDA-R antagonists like ketamine. These readouts are thus perfectly suited to support drug discovery efforts on NMDA-R and understanding the different functions of NMDA-R subtypes.

Pronounced antiepileptic activity of the subtype-selective GABAA -positive allosteric modulator PF-06372865 in the GAERS absence epilepsy model.

AIM: Antiepileptic drugs that modulate GABA have the potential to aggravate or improve the symptoms of absence epilepsy. PF-06372865 is a positive allosteric modulator (PAM) of α2/3/5 subunit-containing GABAA receptors with minimal activity at α1-containing receptors, which are believed to mediate many of the adverse events associated with benzodiazepines. The aim of this study was to assess the antiepileptic effect of PF-06372865 in a preclinical model of absence seizures. METHODS: Genetic absence epilepsy rats from Strasbourg (GAERS) was implanted with four cortical electrodes over the frontoparietal cortex, and the number and cumulated duration of spike-and-wave discharges (SWDs) were recorded for 10-90 minutes following administration of vehicle, PF-06372865, and positive controls diazepam and valproate. RESULTS: PF-06372865 (0.3, 1, 2, 10 mg kg-1 ) dose-dependently reduced the expression of SWDs, including full suppression at the highest doses by 30 minutes after administration. CONCLUSIONS: PF-06372865 demonstrated robust efficacy in suppressing SWDs in the GAERS model of absence epilepsy. To our knowledge, this is the first demonstration of antiepileptic activity of an α2/3/5-subtype-selective GABAA PAM in a model of absence epilepsy. Further study of the antiepileptic properties of PF-06372865 is warranted in patients with absence seizures.

Methodological standards and interpretation of video-electroencephalography in adult control rodents. A TASK1-WG1 report of the AES/ILAE Translational Task Force of the ILAE.

In vivo electrophysiological recordings are widely used in neuroscience research, and video-electroencephalography (vEEG) has become a mainstay of preclinical neuroscience research, including studies of epilepsy and cognition. Studies utilizing vEEG typically involve comparison of measurements obtained from different experimental groups, or from the same experimental group at different times, in which one set of measurements serves as "control" and the others as "test" of the variables of interest. Thus, controls provide mainly a reference measurement for the experimental test. Control rodents represent an undiagnosed population, and cannot be assumed to be "normal" in the sense of being "healthy." Certain physiological EEG patterns seen in humans are also seen in control rodents. However, interpretation of rodent vEEG studies relies on documented differences in frequency, morphology, type, location, behavioral state dependence, reactivity, and functional or structural correlates of specific EEG patterns and features between control and test groups. This paper will focus on the vEEG of standard laboratory rodent strains with the aim of developing a small set of practical guidelines that can assist researchers in the design, reporting, and interpretation of future vEEG studies. To this end, we will: (1) discuss advantages and pitfalls of common vEEG techniques in rodents and propose a set of recommended practices and (2) present EEG patterns and associated behaviors recorded from adult rats of a variety of strains. We will describe the defining features of selected vEEG patterns (brain-generated or artifactual) and note similarities to vEEG patterns seen in adult humans. We will note similarities to normal variants or pathological human EEG patterns and defer their interpretation to a future report focusing on rodent seizure patterns.

NADPH oxidases as drug targets and biomarkers in neurodegenerative diseases: What is the evidence?

Neurodegenerative disease are frequently characterized by microglia activation and/or leukocyte infiltration in the parenchyma of the central nervous system and at the molecular level by increased oxidative modifications of proteins, lipids and nucleic acids. NADPH oxidases (NOX) emerged as a novel promising class of pharmacological targets for the treatment of neurodegeneration due to their role in oxidant generation and presumably in regulating microglia activation. The unique function of NOX is the generation of superoxide anion (O2•-) and hydrogen peroxide (H2O2). However in the context of neuroinflammation, they present paradoxical features since O2•-/H2O2 generated by NOX and/or secondary reactive oxygen species (ROS) derived from O2•-/H2O2 can either lead to neuronal oxidative damage or resolution of inflammation. The role of NOX enzymes has been investigated in many models of neurodegenerative diseases by using either genetic or pharmacological approaches. In the present review we provide a critical assessment of recent findings related to the role of NOX in the CNS as well as how the field has advanced over the last 5 years. In particular, we focus on the data derived from the work of a consortium (Neurinox) funded by the European Commission's Programme 7 (FP7). We discuss the evidence gathered from animal models and human samples linking NOX expression/activity with neuroinflammation in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease as well as autoimmune demyelinating diseases like multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy (CIDP). We address the possibility to use measurement of the activity of the NOX2 isoform in blood samples as biomarker of disease severity and treatment efficacy in neurodegenerative disease. Finally we clarify key controversial aspects in the field of NOX, such as NOX cellular expression in the brain, measurement of NOX activity, impact of genetic deletion of NOX in animal models of neurodegeneration and specificity of NOX inhibitors.

Activation of GABAA receptors controls mesiotemporal lobe epilepsy despite changes in chloride transporters expression: In vivo and in silico approach.

Mesiotemporal lobe Epilepsy (MTLE), the most frequent form of focal epilepsy, is often drug-resistant. Enriching the epileptic focus with GABA-releasing engineered cells has been proposed as a strategy to prevent seizures. However, ex vivo data from animal models and MTLE patients suggest that, due to changes in chloride homeostasis, GABAA receptor activation is depolarizing and partly responsible for focal interictal discharges and seizure initiation. To understand how these two contradictory aspects of GABAergic neurotransmission coexist in MTLE, we used an established mouse model of MTLE presenting hippocampal sclerosis and recurrent hippocampal paroxysmal discharges (HPDs) 30-40days after a unilateral injection of kainate in the dorsal hippocampus. We first showed that injections of GABAA receptor agonists either systemically or directly into hippocampus suppressed HPDs. Western-blotting and immunostaining revealed that levels of α1, α3 and γ2 GABAA receptor subunits were increased in epileptic mice, compared to saline controls, while levels of R1 and R2 GABAB receptor subunits but also NR1, NR2A and NR2B NMDA receptor subunits and GluR1 and GluR2 AMPA receptor subunits were decreased. In addition, we showed that the expression of the transporter NKCC1, which load neurons with chloride, was increased, whereas KCC2, a chloride extruder, was decreased and that HPDs were suppressed by injection of blockers of NKCC1. These different changes were integrated in a numerical model, and in silico simulations supported the notion that chloride imbalance impair local inhibitory control of pyramidal neurons' activity in this model of MTLE. However, our numerical model also suggested that lasting activation of these receptors restore physiological intracellular chloride concentrations and suppress HPDs. Overall, our study suggests that activation of GABAA receptor remains an effective antiepileptic strategy to suppress focal seizures in MTLE, and demonstrates that modeling and simulation studies provide new insights about the cellular and synaptic mechanisms of this disease.

High-Throughput LC-MS/MS Proteomic Analysis of a Mouse Model of Mesiotemporal Lobe Epilepsy Predicts Microglial Activation Underlying Disease Development.

Uncovering the molecular mechanisms of mesiotemporal lobe epilepsy (MTLE) is critical to identify therapeutic targets. In this study, we performed global protein expression analysis of a kainic acid (KA) MTLE mouse model at various time-points (1, 3, and 30 days post-KA injection -dpi), representing specific stages of the syndrome. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination with label-free protein quantification using three processing approaches for quantification, was applied. Following comparison of KA versus NaCl-injected mice, 22, 53, and 175 proteins were differentially (statistically significant) expressed at 1, 3 and 30dpi, respectively, according to all three quantification approaches. Selected findings were confirmed by multiple reaction monitoring LC-MS/MS. As a positive control, the astrocyte marker GFAP was found to be upregulated (3dpi: 1.9 fold; 30dpi: 12.5 fold), also verified by IHC. The results collectively suggest that impairment in synaptic transmission occurs even right after initial status epilepticus (1dpi), with neurodegeneration becoming more extensive during epileptogenesis (3dpi) and sustained at the chronic phase (30dpi), where also extensive glial- and astrocyte-mediated inflammation is evident. This molecular profile is in line with observed phenotypic changes in human MTLE, providing the basis for future studies on new molecular targets for the disease.

Differential Effects of Antiepileptic Drugs on Focal Seizures in the Intrahippocampal Kainate Mouse Model of Mesial Temporal Lobe Epilepsy.

AIMS: Mesial temporal lobe epilepsy (MTLE) is the most common form of drug-refractory epilepsy. Most of the morphological and electrophysiological features of human MTLE can be reproduced in a mouse by a unilateral intrahippocampal injection of kainate (MTLE mouse model). The effects of antiepileptic drugs (AEDs) on the occurrence of recurrent focal hippocampal seizures in this model remain to be specified. Here, we addressed the pharmacological reactivity of this model to the most commonly used AEDs. METHODS: Using depth electroencephalographical (EEG) recordings, we tested the dose-response effects of acute injection of nine AEDs on the occurrence of hippocampal paroxysmal discharges (HPDs) as well as on ictal and interictal power spectra in the MTLE mouse model. RESULTS: Valproate, carbamazepine, and lamotrigine dose dependently suppressed HPDs and modified the general behavior and/or EEG activity. Levetiracetam and pregabalin suppressed HPDs at high doses but without any behavioral nor interictal EEG changes. Finally, phenobarbital, tiagabine, vigabatrin, and diazepam suppressed HPDs in a dose-dependent manner at doses devoid of obvious behavioral effects. CONCLUSION: The MTLE mouse model displays a differential sensitivity to AEDs with a greater efficacy of drug that facilitates GABAergic transmission. This model provides an efficient tool to identify new treatment for drug-resistant forms of focal epilepsies.

Spatiotemporal specificity of GABAA receptor-mediated regulation of adult hippocampal neurogenesis.

GABAergic transmission regulates adult neurogenesis by exerting negative feedback on cell proliferation and enabling dendrite formation and outgrowth. Further, GABAergic synapses target differentiating dentate gyrus granule cells prior to formation of glutamatergic connections. GABA(A) receptors (GABA(A) Rs) mediating tonic (extrasynaptic) and phasic (synaptic) transmission are molecularly and functionally distinct, but their specific role in regulating adult neurogenesis is unknown. Using global and single-cell targeted gene deletion of subunits contributing to the assembly of GABA(A) Rs mediating tonic (α4, δ) or phasic (α2) GABAergic transmission, we demonstrate here in the dentate gyrus of adult mice that GABA(A) Rs containing α4, but not δ, subunits mediate GABAergic effects on cell proliferation, initial migration and early dendritic development. In contrast, α2-GABA(A) Rs cell-autonomously signal to control positioning of newborn neurons and regulate late maturation of their dendritic tree. In particular, we observed pruning of distal dendrites in immature granule cells lacking the α2 subunit. This alteration could be prevented by pharmacological inhibition of thrombospondin signaling with chronic gabapentin treatment, shown previously to reduce glutamatergic synaptogenesis. These observations point to homeostatic regulation of inhibitory and excitatory inputs onto newborn granule cells under the control of α2-GABA(A) Rs. Taken together, the availability of distinct GABA(A) R subtypes provides a molecular mechanism endowing spatiotemporal specificity to GABAergic control of neuronal maturation in adult brain.

Impaired reelin processing and secretion by Cajal-Retzius cells contributes to granule cell dispersion in a mouse model of temporal lobe epilepsy.

Cajal-Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin-positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal-Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal-Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal-Retzius cells in reeler Orleans (reln (orl/orl) ) mice, which express a secretion-deficient 310-kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal-Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln (orl/+) mice, which induced severe GCD. Furthermore, Western blot analysis in KA-treated wildtype mice revealed increased production of ∼300-kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA-lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal-Retzius cells, suggesting that production of truncated reelin in KA-treated hippocampus is activity-dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal-Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal-Retzius cells for hippocampal neuronal reorganization in TLE.

PSA-NCAM-dependent GDNF signaling limits neurodegeneration and epileptogenesis in temporal lobe epilepsy.

Polysialylated neuronal cell adhesion molecule (PSA-NCAM), a polysialylated protein constitutively expressed in the hippocampus, is involved in neuronal growth, synaptic plasticity and neurotrophin signaling. In particular, PSA-NCAM mediates Ret-independent glial-derived neurotrophic factor (GDNF) signaling, leading to downstream FAK activation. GDNF has potent seizure-suppressant action, whereas PSA-NCAM is upregulated by seizure activity. However, the involvement of Ret-independent GDNF signaling in temporal lobe epilepsy (TLE) is not established. We tested the effects of PSA-NCAM inactivation on neurodegeneration and epileptogenesis in a mouse model of TLE. In this model, unilateral intrahippocampal kainic acid (KA) injection induced degeneration of CA1, CA3c and hilar neurons, followed by spontaneous recurrent focal seizures. In the contralateral, morphologically preserved hippocampus, a long-lasting increase of PSA-NCAM immunoreactivity was observed. Inactivation of PSA-NCAM by endoneuraminidase (EndoN) administration into the contralateral ventricle of KA-treated mice caused severe degeneration of CA3a,b neurons and dentate gyrus granule cells in the epileptic focus, and led to early onset of focal seizures. This striking trans-hemispheric alteration suggested that PSA-NCAM mediates GDNF signaling, leading to transport of neuroprotective signals into the lesioned hippocampus. This hypothesis was confirmed by injecting GDNF antibodies into the contralateral hippocampus of KA-treated mice, thereby reproducing the enhanced neurodegeneration seen after PSA-NCAM inactivation. Furthermore, contralateral EndoN and anti-GDNF treatment decreased GDNF family receptor alpha1 immunoreactivity and FAK phosphorylation in the epileptic focus. Thus, Ret-independent GDNF signaling across the commissural projection might protect CA3a,b neurons and delay seizure onset. These findings implicate GDNF in the control of epileptogenesis and offer a possible mechanism explaining lesion asymmetry in mesial TLE.

Reorganization of GABAergic circuits maintains GABAA receptor-mediated transmission onto CA1 interneurons in alpha1-subunit-null mice.

The majority of hippocampal interneurons strongly express GABA(A) receptors containing the alpha1 subunit, suggesting that inhibitory control of interneurons is important for proper function of hippocampal circuits. Here, we investigated with immunohistochemical and electrophysiological techniques how these GABA(A) receptors are replaced in mice carrying a targeted deletion of the alpha1-subunit gene (alpha1(0/0) mice). Using markers of five major populations of CA1 interneurons (parvalbumin, calretinin, calbindin, neuropeptide Y and somatostatin), we show that these interneurons remain unaffected in alpha1(0/0) mice. In triple immunofluorescence staining experiments combining these markers with the GABA(A) receptor alpha1, alpha2 or alpha3 subunit and gephyrin, we demonstrate a strong increase in alpha3- and alpha2-GABA(A) receptors clustered at postsynaptic sites along with gephyrin in most CA1 interneurons in alpha1(0/0) mice. The changes were cell type-specific and resulted in an increased number of GABAergic synapses on interneurons. These adjustments were mirrored functionally by retention of spontaneous IPSCs with prolonged decay kinetics, as shown by whole-cell patch-clamp recordings of CA1 interneurons. However, a significant decrease in frequency and amplitude of miniature IPSCs was evident, suggesting reduced affinity of postsynaptic receptors and/or impaired vesicular GABA release. Finally, to assess whether these compensatory changes are sufficient to protect against a pathological challenge, we tested the susceptibility of alpha1(0/0) mice against kainic acid-induced excitotoxicity. No genotype difference was observed in the effects of kainic acid, indicating that the absence of a major GABA(A) receptor subtype is functionally compensated for in hippocampal interneurons by a reorganization of inhibitory circuits.

Transient hyperthermia protects against subsequent seizures and epilepsy-induced cell damage in the rat.

Many mild preconditioning stress conditions, including physical and metabolic injuries, increase the resistance of neurons to subsequent more severe stresses of the same or different type. This "tolerance phenomenon" lasts one to several weeks, providing a unique opportunity to investigate endogenous neuroprotective mechanisms. The aim of this study was to find a physiological and easily applicable preconditioning stimulus able to confer protection against convulsant-induced neuronal damage and seizures. We found that moderate transient hyperthermic preconditioning markedly reduced kainic-acid-induced neuronal cell loss and attenuated susceptibility to bicuculline-induced seizures. Prevention of cell damage (approximately 50%) was efficient both in vitro in organotypic hippocampal slice cultures and in vivo in adult rats. This protection lasted about 1 week and peaked 3 to 5 days after pretreatment. Unraveling the mechanisms of heat shock preconditioning-induced protection against epilepsy should lead to the development of new therapeutic strategies.