Characterization of the intrahippocampal kainic acid model in female mice with a special focus on seizure suppression by antiseizure medications.

Despite special challenges in the medical treatment of women with epilepsy, in particular preclinical animal studies were focused on males for decades and females have only recently moved into the focus of scientific interest. The intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy (TLE) is one of the most studied models in males reproducing electroencephalographic (EEG) and histopathological features of human TLE. Hippocampal paroxysmal discharges (HPDs) were described as drug resistant focal seizures in males. Here, we investigated the IHKA model in female mice, in particular drug-resistance of HPDs and the influence of antiseizure medications (ASMs) on the power spectrum. After injecting kainic acid (KA) unilaterally into the hippocampus of female mice, we monitored the development of epileptiform activity by local field potential (LFP) recordings. Subsequently, we evaluated the effect of the commonly prescribed ASMs lamotrigine (LTG), oxcarbazepine (OXC) and levetiracetam (LEV), as well as the benzodiazepine diazepam (DZP) with a focus on HPDs and power spectral analysis and assessed neuropathological alterations of the hippocampus. In the IHKA model, female mice replicated key features of human TLE as previously described in males. Importantly, HPDs in female mice did not respond to commonly prescribed ASMs in line with the drug-resistance in males, thus representing a suitable model of drug-resistant seizures. Intriguingly, we observed an increased occurrence of generalized seizures after LTG. Power spectral analysis revealed a pronounced increase in the delta frequency range after the higher dose of 30 mg/kg LTG. DZP abolished HPDs and caused a marked reduction over a wide frequency range (delta, theta, and alpha) of the power spectrum. By characterizing the IHKA model of TLE in female mice we address an important gap in basic research. Considering the special challenges complicating the therapeutic management of epilepsy in women, inclusion of females in preclinical studies is imperative. A well-characterized female model is a prerequisite for the development of novel therapeutic strategies tailored to sex-specific needs and for studies on the effect of epilepsy and ASMs during pregnancy.

Dimethyl sulfoxide's impact on epileptiform activity in a mouse model of chronic temporal lobe epilepsy.

In the quest for novel treatments for patients with drug-resistant seizures, poor water solubility of potential drug candidates is a frequent obstacle. Literature indicated that the highly efficient solvent dimethyl sulfoxide (DMSO) may have a confounding influence in epilepsy research, reporting both pro- and antiepileptic effects. In this study, we aim to clarify the effects of DMSO on epileptiform activity in one of the most frequently studied models of chronic epilepsy, the intrahippocampal kainic acid (IHKA) mouse model, and in a model of acute seizures. We show that 100 % DMSO (in a volume of 1.5 µl/g corresponding to 1651 mg/kg) causes a significant short-term anti-seizure effect in epileptic IHKA mice of both sexes, but does not affect the threshold of acute seizures induced by pentylenetetrazol (PTZ). These findings highlight that the choice of solvent and appropriate vehicle control is crucial to minimize undesirable misleading effects and that drug candidates exclusively soluble in 100 % DMSO need to be modified for better solubility already at initial testing.

Dynorphin-based "release on demand" gene therapy for drug-resistant temporal lobe epilepsy.

Focal epilepsy represents one of the most common chronic CNS diseases. The high incidence of drug resistance, devastating comorbidities, and insufficient responsiveness to surgery pose unmet medical challenges. In the quest of novel, disease-modifying treatment strategies of neuropeptides represent promising candidates. Here, we provide the "proof of concept" that gene therapy by adeno-associated virus (AAV) vector transduction of preprodynorphin into the epileptogenic focus of well-accepted mouse and rat models for temporal lobe epilepsy leads to suppression of seizures over months. The debilitating long-term decline of spatial learning and memory is prevented. In human hippocampal slices obtained from epilepsy surgery, dynorphins suppressed seizure-like activity, suggestive of a high potential for clinical translation. AAV-delivered preprodynorphin expression is focally and neuronally restricted and release is dependent on high-frequency stimulation, as it occurs at the onset of seizures. The novel format of "release on demand" dynorphin delivery is viewed as a key to prevent habituation and to minimize the risk of adverse effects, leading to long-term suppression of seizures and of their devastating sequel.

Role for Chromatin Remodeling Factor Chd1 in Learning and Memory.

Precise temporal and spatial regulation of gene expression in the brain is a prerequisite for cognitive processes such as learning and memory. Epigenetic mechanisms that modulate the chromatin structure have emerged as important regulators in this context. While posttranslational modification of histones or the modification of DNA bases have been examined in detail in many studies, the role of ATP-dependent chromatin remodeling factors (ChRFs) in learning- and memory-associated gene regulation has largely remained obscure. Here we present data that implicate the highly conserved chromatin assembly and remodeling factor Chd1 in memory formation and the control of immediate early gene (IEG) response in the hippocampus. We used various paradigms to assess short-and long-term memory in mice bearing a mutated Chd1 gene that gives rise to an N-terminally truncated protein. Our data demonstrate that the Chd1 mutation negatively affects long-term object recognition and short- and long-term spatial memory. We found that Chd1 regulates hippocampal expression of the IEG early growth response 1 (Egr1) and activity-regulated cytoskeleton-associated (Arc) but not cFos and brain derived neurotrophic factor (Bdnf), because the Chd1-mutation led to dysregulation of Egr1 and Arc expression in naive mice and in mice analyzed at different stages of object location memory (OLM) testing. Of note, Chd1 likely regulates Egr1 in a direct manner, because chromatin immunoprecipitation (ChIP) assays revealed enrichment of Chd1 upon stimulation at the Egr1 genomic locus in the hippocampus and in cultured cells. Together these data support a role for Chd1 as a critical regulator of molecular mechanisms governing memory-related processes, and they show that this function involves the N-terminal serine-rich region of the protein.

The ketamine-like compound methoxetamine substitutes for ketamine in the self-administration paradigm and enhances mesolimbic dopaminergic transmission.

RATIONALE: Recently, an increasing number of emergency cases due to a novel ketamine-like drug, methoxetamine (MXE), were reported in several countries. However, very little is known about the neuropsychopharmacological and reinforcing profile of this compound. OBJECTIVES: Our study aims to investigate the effects of MXE on self-administration (SA) behaviour in comparison to ketamine and on dopaminergic transmission. METHODS: A SA substitution study was performed in male rats trained to intravenously (IV) self-administer ketamine. At responding stability, rats were exposed to sequential phases of MXE substitution at different dosages (starting from 0.5 and then decreasing to 0.25 and 0.125 mg/kg). Standard electrophysiological techniques were used to record changes in firing activities of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (NAc) shell after acute injection of cumulative doses of MXE (0.031-0.5 mg/kg IV). Finally, in vivo microdialysis was performed in freely moving rats to evaluate the effect of acute MXE administration (0.125, 0.25 and 0.5 mg/kg IV) on dopamine release in the NAc shell. RESULTS: MXE 0.125 and 0.25 mg/kg, but not 0.5 mg/kg, substituted for ketamine SA. MXE also induced a dose-dependent stimulation of firing rate (p < 0.0001) and burst firing (p < 0.05) of NAc-projecting VTA dopamine neurons. Consistently, MXE significantly (p < 0.05) increased dopamine extracellular levels in the NAc shell at 0.5 and 0.25 mg/kg with different time onsets, i.e. at 40 and 100 min, respectively. CONCLUSIONS: This study, while confirming the reinforcing effects of MXE, highlights an electrophysiological and neurochemical profile predictive of its addictive properties.

The ketamine analogue methoxetamine generalizes to ketamine discriminative stimulus in rats.

Methoxetamine (MXE) is a chemical analogue of ketamine. Originally proposed as a ketamine-like fast-acting antidepressant, owing to similar N-methyl-D-aspartate blocker properties, it is now scheduled for reports of hallucinations and psychosis similar to ketamine and lysergic acid. As little is known about the addictive properties of MXE, the aim of this study was to investigate the similarity between discriminative stimuli of MXE and ketamine, as well as to provide data and protocols that could be used in the future for the characterization of novel ketamine-like drugs. The paradigm used was a two-lever operant conditioning paradigm in which rats were trained to discriminate ketamine (7.5 mg/kg/ml, intraperitoneal) from vehicle. Generalization tests were performed with MXE (0.0625, 0.125, 0.25, 0.5, or 1.0). We also tested the N-methyl-D-aspartate channel blocker MK-801 (0.005-0.1), lysergic acid (0.025-0.30), a serotonergic drug that had similar hallucinogenic effects as ketamine and methamphetamine (0.15-0.60) a drug with no generalization with ketamine, injected intraperitoneally presession (mg/kg). MXE and MK-801 fully generalized to ketamine. Lysergic acid and methamphetamine partially substituted for the ketamine stimulus, although the highest lysergic acid dose showed a 77.7% generalization. The present findings suggest that investigation of 'ketamine-like compounds' should explore not only substances with chemical analogy and common molecular mechanisms with ketamine, but also with similar psychopharmacological effects.

The modulation of BDNF expression and signalling dissects the antidepressant from the reinforcing properties of ketamine: Effects of single infusion vs. chronic self-administration in rats.

Ketamine is a drug of abuse with a unique profile, which besides its inherent mechanism of action as a non-competitive antagonist of the NMDA glutamate receptor, displays both antidepressant and reinforcing properties. The major aim of our study was to find a molecular signature of ketamine that may help in discriminating between its reinforcing and antidepressant effects. To this end, we focused our attention on BDNF, a neurotrophin that has been shown to play a role in both antidepressant and reinforcing properties of several drugs. Rats were exposed to self-administer intravenous (IV) ketamine (S/A) for 43 days or to receive a single IV ketamine 0.5mg/kg, or vehicle infusion. Although the dose we employed is lower than that reported by the literature, it however yields Cmax values that correspond to those achieved in humans after antidepressant treatment. Our results show that while the single infusion of ketamine increased the neurotrophin expression in the hippocampus while reducing it in the ventral striatum, a feature shared with other antidepressants, the repeated self-administration reduced mBDNF expression and its downstream signalling in both ventral striatum and hippocampus. Further, we here show that phosphorylation of Akt is oppositely regulated by ketamine, pointing to this pathway as central to the different actions of the drug. Taken together, we here point to BDNF and its downstream signalling pathway as a finely tuned mechanism whose modulation might subserve the different features of ketamine.

Pharmacological and non-pharmacological factors that regulate the acquisition of ketamine self-administration in rats.

BACKGROUND: Recreational ketamine use may be modulated by factors such as ketamine infusion patterns, associated conditioned stimuli and spatial-temporal contexts. Our aim was to study the pharmacological and non-pharmacological factors that regulate the acquisition of ketamine use. METHODS: In experiment 1, four groups of male rats were trained to self-administer ketamine during nine 1-h daily sessions, under four reinforcement schedules: i) pre-session ketamine priming (Priming-[KET]), ii) conditioned stimulus (CS) paired to the ketamine infusions ([KET + CS]), iii) neither priming nor CS ([KET]), iv) combination of both (Priming-[KET + CS]). In experiment 2, two groups of male rats were trained to self-administer ketamine during nine 1-h daily or weekly sessions, under the Priming-[KET + CS] schedule. Lever pressing was then extinguished by saline substitution for ketamine infusion. Afterwards, ketamine was made available again upon responding under the same schedule. RESULTS: The Priming-[KET + CS] schedule of reinforcement showed a significant increase in the number of ketamine reinforcements and a significant discrimination between active vs. inactive levers. The same schedule allowed the establishment of ketamine self-administration on a weekly basis. During the extinction phase, rate of responding significantly dropped in both weekly and daily groups although it was twofold longer in the former, which showed a lack of reacquisition. CONCLUSIONS: Both pre-session ketamine priming and a conditioned stimulus paired to the ketamine infusions are required for the acquisition of ketamine self-administration. The longer extinction and the lack of reacquisition in the weekly group could be due to changes in temporal context that might affect the conditioning process.

Nicotine-seeking reinstatement is reduced by inhibition of instrumental memory reconsolidation.

The reinforcing properties of nicotine play a major role in instrumental conditioning to nicotine taking in smokers. Retrieval of nicotine-related memories may promote relapse to nicotine seeking after prolonged abstinence. Once consolidated, memories are stable, but they return to a labile phase, called reconsolidation, after their retrieval. The aim of our study was to investigate whether it was possible to interfere with the reconsolidation of instrumental nicotine-related memories by acting at glutamatergic receptors [N-methyl-D-aspartate receptors (NMDARs)] to prevent relapse to nicotine-seeking behaviour in the rat. We assessed whether the NMDAR antagonist MK-801, administered before or after nicotine-related instrumental memory retrieval, can reduce reinstatement of nicotine-seeking behaviour in rats previously trained to nicotine self-administration. Following a period of forced abstinence, MK-801 (0.1 mg/kg intraperitoneally) was administered 30 min before or 1 h after the re-exposure to 20 lever presses without any contingency in the training context to retrieve instrumental memory. MK-801 administered after, but not before, retrieval inhibited reinstatement compared with vehicle controls and groups without retrieval of instrumental memory. Interestingly, a retrieval factor effect was observed as an increase of reinstatement in vehicle-treated groups, suggesting a behavioural outcome of the occurrence of instrumental memory reconsolidation. Our findings suggest that, by acting on NMDARs, it is possible to reduce the reinstatement of nicotine-seeking behaviour through inhibition of instrumental nicotine-related memory reconsolidation.