Search

Objective: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. Methods: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. Results: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. Significance: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine. Keywords: Dravet syndrome; epileptogenesis; fenfluramine; sodium channel; zebrafish. publishedVersion

Climate change is the biggest challenge facing humanity today. The associated global warming and humidification, increases in the severity and frequency of extreme climate events, extension of the ranges of vector‐borne diseases, and the consequent social and economic stresses and disruption will have major negative consequences on many aspects of health care. People whose resilience to change is already impaired may suffer disproportionately from these environmental changes, which are of unprecedented reach and magnitude. There has been little connection made so far between climate change and epilepsy. We briefly review the history of climate change science and the subsequent response of the global scientific community. We consider how climate change effects might in general affect health and disease. We consider some of the underlying complex interactions that, for example, favor the spread of vector‐borne diseases and how climate models operate and may help plan for global and local changes. We then speculate specifically on how these generic ideas may apply specifically to epilepsy. We consider these impacts at levels from molecular to the epidemiological. Data are sparse, and there is undoubtedly a need for more information to enable better estimation of possible effects of climate change on care in epilepsy. We also consider how the professional activities of those involved in epilepsy health care might contribute to global carbon emissions, for example, through flying for conference attendance. Healthcare organizations across the world are already considering, and responding to, many of these issues. We argue for more research in this area, but also for action today. Actions today are likely to generate cobenefits for health care, including care in epilepsy, resulting from efforts to decarbonize, mitigate effects of climate change that has already happened, and plan for adaptation to climate change.

In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZ-induced seizures in mice. Thus, AS-1 may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 μM, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 μM). Electronic supplementary material The online version of this article (10.1007/s13311-019-00773-w) contains supplementary material, which is available to authorized users.

Persons with certain single nucleotide polymorphisms (SNPs) in theCACNA1Dgene (encoding voltage-gated calcium channel subunit alpha 1-D) have increased risk of developing neuropsychiatric disorders such as bipolar, schizophrenia and autism. The molecular consequences of SNPs on gene expression and protein function are not well understood. Thus, the use of animal models to determine genotype-phenotype correlations is critical to understanding disease pathogenesis. Here, we describe the behavioural changes in larval zebrafish carrying an essential splice site mutation (sa17298) incacna1da. Heterozygous mutation resulted in 50% reduction of splice variants 201 and 202 (haploinsufficiency), while homozygosity increased transcript levels of variant 201 above wild type (WT; gain-of-function, GOF). Due to low homozygote viability, we focused primarily on performing the phenotypic analysis on heterozygotes. Indeed,cacna1dasa17298/WTlarvae displayed hyperlocomotion—a behaviour characterised in zebrafish as a surrogate phenotype for epilepsy, anxiety or psychosis-like behaviour. Follow-up tests ruled out anxiety or seizures, however, as neither thigmotaxis defects nor epileptiform-like discharges in larval brains were observed. We therefore focused on testing for potential “psychosis-like” behaviour by assayingcacna1dasa17298/WTlarval locomotor activity under constant light, during light-dark transition and in startle response to dark flashes. Furthermore, exposure of larvae to the antipsychotics, risperidone and haloperidol reversedcacna1da-induced hyperactivity to WT levels while valproate decreased but did not reverse hyperactivity. Together, these findings demonstrate thatcacna1dahaploinsufficiency induces behaviours in larval zebrafish analogous to those observed in rodent models of psychosis. Future studies on homozygous mutants will determine howcacna1dGOF alters behaviour in this context.

Palmatine (PALM) and berberine (BERB) are widely identified isoquinoline alkaloids among the representatives of the Berberidaceae botanical family. The antiseizure activity of BERB was shown previously in experimental epilepsy models. We assessed the effect of PALM in a pentylenetetrazole (PTZ)-induced seizure assay in zebrafish, with BERB as an active reference compound. Both alkaloids were isolated from the methanolic root extract of Berberis sibirica by counter-current chromatography, and their ability to cross the blood&ndash, brain barrier was determined via quantitative structure&ndash, activity relationship assay. PALM exerted antiseizure activity, as confirmed by electroencephalographic analysis, and decreased c-fos and bdnf levels in PTZ-treated larvae. In a behavioral assay, PALM dose-dependently decreased PTZ-induced hyperlocomotion. The combination of PALM and BERB in ED16 doses revealed hyperadditive activity towards PTZ-induced hyperlocomotion. Notably, we have indicated that both alkaloids may exert their anticonvulsant activity through different mechanisms of action. Additionally, the combination of both alkaloids in a 1:2.17 ratio (PALM: BERB) mimicked the activity of the pure extract, which indicates that these two active compounds are responsible for its anticonvulsive activity. In conclusion, our study reveals for the first time the anticonvulsant activity of PALM and suggests the combination of PALM and BERB may have higher therapeutic value than separate usage of these compounds.

TheCACNA1Agene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partialcacna1aaknockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression ofcacna1aain the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants ofcacna1aainto fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown ofcacna1aaon protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate thatcacna1aaloss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation ofCacna1a-mediated epileptogenesis and for in vivo drug screening.

Preclinical data indicated that the metabotropic glutamate receptors 5 (mGlu5) and glutamate receptors 2/3 (mGlu2/3) are involved in modulating morphine antinociception. However, little is known about the role of metabotropic glutamate receptors 7 (mGlu7) in this phenomenon. We compared the effects of AMN082 (0.1, 1 or 5mg/kg, ip), a selective mGlu7 allosteric agonist, LY354740 (0.1, 1 or 5mg/kg, ip), an mGlu2/3 agonist and MTEP (0.1, 1 or 5mg/kg, ip), a selective mGlu5 antagonist, on the acute antinociceptive effect of morphine (5mg/kg, sc) and also on the development and expression of tolerance to morphine analgesia in the tail-immersion test in mice. To determine the role of mGlu7 in morphine tolerance, and the association of the mGlu7 effect with the N-methyl-d-aspartate (NMDA) receptors regulation, we used MMPIP (10mg/kg, ip), a selective mGlu7 antagonist and MK-801, a NMDA antagonist. Herein, the acute administration of AMN082, MTEP or LY354740 alone failed to evoked antinociception, and did not affect morphine (5mg/kg, sc) antinociception. However, these ligands inhibited the development of morphine tolerance, and we indicated that MMPIP reversed the inhibitory effect of AMN082. When given together, the non-effective doses of AMN082 and MK-801 did not alter the tolerance to morphine. Thus, mGlu7, similarly to mGlu2/3 and mGlu5, are involved in the development of tolerance to the antinociceptive effects of morphine, but not in the acute morphine antinociception. Furthermore, while mGlu7 are engaged in the development of morphine tolerance, no interaction exists between mGlu7 and NMDA receptors in this phenomenon.

Repeated exposure to and withdrawal from ethanol induces deficits in spatial reversal learning. Data indicate that metabotropic glutamate 5 (mGlu5) receptors are implicated in synaptic plasticity and learning and memory. These receptors functionally interact with N-methyl- d -aspartate (NMDA) receptors, and activation of one type results in the activation of the other. We examined whether ( S )-(4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)-piperidin-1-yl (ADX-47273), a positive allosteric modulator (PAM) of mGlu5 receptor, attenuates deficits in reversal learning induced by withdrawal (11-13 days) from ‘binge-like’ ethanol input (5.0 g/kg, i.g. for 5 days) in the Barnes maze (a spatial learning) task in rats. We additionally examined the effects of ADX-47273 on the expression of the NMDA receptors subunit, GluN2B, in the hippocampus and prefrontal cortex, on the 13th day of ethanol withdrawal. Herein, withdrawal from repeated ethanol administration impaired reversal learning, but not the probe trial. Moreover, ADX-47273 (30 mg/kg, i.p.) given prior to the first reversal learning trial for 3 days in the Barnes maze, significantly enhanced performance in the ethanol-treated group. The 13th day of ethanol abstinence decreased the expression of the GluN2 B subunit in the selected brain regions, but ADX-47273 administration increased it. In conclusion, positive allosteric modulation of mGlu5 receptors recovered spatial reversal learning impairment induced by withdrawal from ‘binge-like’ ethanol exposure. Such effect seems to be correlated with the mGlu5 receptors mediated potentiation of GluN2B-NMDA receptor mediated responses in the hippocampus and prefrontal cortex. Thus, our results emphasize the role of mGlu5 receptor PAM in the adaptive learning impaired by ethanol exposure.

Kynurenine (KYN), a main metabolite of tryptophan in mammals, is a direct precursor of kynurenic acid, anthranilic acid and 3-hydroxykynurenine (3-HK). Under physiological conditions, KYN is produced endogenously mainly in the liver by tryptophan 2,3-dioxygenase (TDO). Tumorigenesis and inflammatory conditions increase the activity of another KYN synthetizing enzyme, indoleamine 2,3-dioxygenase (IDO). However, knowledge about the exogenous sources and the fate of KYN in mammals is still limited. While most papers deal with the contribution of KYN to pathologies of the central nervous system, its role in the periphery has almost been ignored. KYN is a ligand for the aryl hydrocarbon receptor (AhR). As a receptor for KYN and its downstream metabolites, AhR is involved in several physiological and pathological conditions, including inflammation and carcinogenesis. Recent studies have shown that KYN suppresses immune response and is strongly involved in the process of carcinogenesis and tumour metastasis. Thus, inhibition of activity of the enzymes responsible for KYN synthesis, TDO, IDO or genetic manipulation leading to reduction of KYN synthesis, could be considered as innovative strategies for improving the efficacy of immunotherapy. Surprisingly, however, genetic or pharmacological approaches for reducing tryptophan catabolism to KYN do not necessarily result in decrease of KYN level in the main circulation. This review aims to summarize the current knowledge of KYN fate and function and to emphasize its importance for vital physiological and pathological processes.

Zingiber officinale is one of the most frequently used medicinal herbs in Asia. Using rodent seizure models, it was previously shown that Zingiber officinale hydroethanolic extract exerts antiseizure activity, but the active constituents responsible for this effect have not been determined. In this paper, we demonstrated that Zingiber officinale methanolic extract exerts anticonvulsant activity in the pentylenetetrazole (PTZ)-induced hyperlocomotion assay in larval zebrafish. Next, we isolated 6-gingerol (6-GIN) – a major constituent of Zingiber officinale rhizoma. We observed that 6-GIN exerted potent dose-dependent anticonvulsant activity in the PTZ-induced hyperlocomotion seizure assay in zebrafish, which was confirmed electroencephalographically. To obtain further insight into the molecular mechanisms of 6-GIN antiseizure activity, we assessed the concentration of two neurotransmitters in zebrafish, i.e., inhibitory γ-aminobutyric acid (GABA) and excitatory glutamic acid (GLU), and their ratio after exposure to acute PTZ dose. Here, 6-GIN decreased GLU level and reduced the GLU/GABA ratio in PTZ-treated fish compared with only PTZ-bathed fish. This activity was associated with the decrease in grin2b, but not gabra1a, grin1a, gria1a, gria2a, and gria3b expression in PTZ-treated fish. Molecular docking to the human NR2B-containing N-methyl-D-aspartate (NMDA) receptor suggests that 6-GIN might act as an inhibitor and interact with the amino terminal domain, the glutamate-binding site, as well as within the ion channel of the NR2B-containing NMDA receptor. In summary, our study reveals, for the first time, the anticonvulsant activity of 6-GIN. We suggest that this effect might at least be partially mediated by restoring the balance between GABA and GLU in the epileptic brain, however, more studies are needed to prove our hypothesis.

Schizophrenia is a mental disorder that affects 1% of the population worldwide and is manifested as a broad spectrum of symptoms, from hallucinations to memory impairment. It is believed that genetic and/or environmental factors may contribute to the occurrence of this disease. Recently, the zebrafish has emerged as a valuable and attractive model for various neurological disorders including schizophrenia. In this review, we describe current pharmacological models of schizophrenia with special emphasis on providing insights into the pros and cons of using zebrafish as a behavioural model of this disease. Moreover, we highlight the advantages and utility of using zebrafish for elucidating the genetic mechanisms underlying this psychiatric disorder. We believe that the zebrafish has high potential also in the area of precision medicine and may complement the development of therapeutics, especially for pharmacoresistant patients.

Pterostilbene (PTE), a natural dimethylated analog of resveratrol, possesses numerous health-beneficial properties. The ability of PTE to cross the blood–brain barrier raised the possibility that this compound may modulate central nervous system functions, including seizure activity. The aim of our study was to investigate the activity of PTE in the larval zebrafish pentylenetetrazole (PTZ) seizure assay and three acute seizure tests in mice, i.e., in the maximal electroshock seizure threshold (MEST), 6 Hz-induced psychomotor seizure threshold and intravenous (iv) PTZ tests. Additionally, potential antidepressant activity of PTE was estimated in the forced swim test in mice. The chimney test was used to determine the influence of PTE on motor coordination in mice, while its influence on neuromuscular strength was assessed in the grip strength test in mice. Locomotor activity was determined to verify the results from the forced swim test. PTE revealed an evident anticonvulsant effect both in zebrafish larvae (10 µM; 2 h-incubation) and mice (at doses of 100 and 200 mg/kg, intraperitoneally) but it did not exhibit antidepressant potential in the forced swim test. Furthermore, it did not cause any statistically significant changes in motor coordination, neuromuscular strength and locomotor activity in mice. In conclusion, our present findings demonstrate for the first time the anticonvulsant potential of PTE. The aforementioned results suggest that it might be employed in epilepsy treatment, however, further precise studies are required to verify its activity in other experimental seizure and epilepsy models and its precise mechanism of action should be determined.

Central cholinergic dysfunction contributes to acute spatial memory deficits produced by ethanol administration. Donepezil and rivastigmine elevate acetylcholine levels in the synaptic cleft through the inhibition of cholinesterases—enzymes involved in acetylcholine degradation. The aim of our study was to reveal whether donepezil (acetylcholinesterase inhibitor) and rivastigmine (also butyrylcholinesterase inhibitor) attenuate spatial memory impairment as induced by acute ethanol administration in the Barnes maze task (primary latency and number of errors in finding the escape box) in rats. Additionally, we compared the influence of these drugs on ethanol-disturbed memory. In the first experiment, the dose of ethanol (1.75 g/kg, i.p.) was selected that impaired spatial memory, but did not induce motor impairment. Next, we studied the influence of donepezil (1 and 3 mg/kg, i.p.), as well as rivastigmine (0.5 and 1 mg/kg, i.p.), given either before the probe trial or the reversal learning on ethanol-induced memory impairment. Our study demonstrated that these drugs, when given before the probe trial, were equally effective in attenuating ethanol-induced impairment in both test situations, whereas rivastigmine, at both doses (0.5 and 1 mg/kg, i.p.), and donepezil only at a higher dose (3 mg/kg, i.p.) given prior the reversal learning, attenuated the ethanol-induced impairment in cognitive flexibility. Thus, rivastigmine appears to exert more beneficial effect than donepezil in reversing ethanol-induced cognitive impairments—probably due to its wider spectrum of activity. In conclusion, the ethanol-induced spatial memory impairment may be attenuated by pharmacological manipulation of central cholinergic neurotransmission.

The present study examined the influence of the cholinesterase inhibitors donepezil (a selective inhibitor of acetylcholinesterase) and rivastigmine (also an inhibitor of butyrylcholinesterase) on the acquisition and reinstatement of ethanol-induced conditioned place preference (CPP) in rats. Before the CPP procedure, animals received a single injection of ethanol (0.5 g/kg, 10% w/v, intraperitoneally [i.p.]) for 15 days. The ethanol-induced CPP (biased method) was developed by four injections of ethanol (0.5 g/kg, 10% w/v, i.p.) every second day. Control rats received saline instead of ethanol. Donepezil (0.5, 1 or 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5 or 1 mg/kg, i.p.) were administered before ethanol during conditioning or before the reinstatement of ethanol-induced CPP. The cholinesterase inhibitors were equally effective in increasing (dose dependently) the acquisition of ethanol-induced CPP. Furthermore, priming injections of both inhibitors reinstated (cross-reinstatement) the ethanol-induced CPP with similar efficacy. These effects of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist, but not by scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. Thus, our results show that the cholinergic system is involved in the reinforcing properties of ethanol, and nicotinic acetylcholine receptors play an important role in the relapse to ethanol-seeking behaviour.

Metabotropic glutamate subtype 5 (mGlu5) receptors are implicated in various forms of synaptic plasticity, including drugs of abuse. In drug-addicted individuals, associative memories can drive relapse to drug use. The present study investigated the potential of the mGlu5 receptor positive allosteric modulator (PAM), VU-29 (30 mg/kg, i.p.), to inhibit the maintenance of a learned association between ethanol and environmental context by using conditioned place preference (CPP) in rats. The ethanol-CPP was established by the administration of ethanol (1.0 g/kg, i.p. &times, 10 days) using an unbiased procedure. Following ethanol conditioning, VU-29 was administered at various post-conditioning times (ethanol free state at the home cage) to ascertain if there was a temporal window during which VU-29 would be effective. Our experiments indicated that VU-29 did not affect the expression of ethanol-induced CPP when it was given over two post-conditioning days. However, the expression of ethanol-CPP was inhibited by 10-day home cage administration of VU-29, but not by first 2-day or last 2-day injection of VU-29 during the 10-day period. These findings reveal that VU-29 can inhibit the maintenance of ethanol-induced CPP, and that treatment duration contributes to this effect of VU-29. Furthermore, VU-29 effect was reversed by pretreatment with either MTEP (the mGlu5 receptor antagonist), or MK-801 (the N-methyl-D-aspartate-NMDA receptor antagonist). Thus, the inhibitory effect of VU-29 is dependent on the functional interaction between mGlu5 and NMDA receptors. Because a reduction in ethanol-associated cues can reduce relapse, mGlu5 receptor PAM would be useful for therapy of alcoholism. Future research is required to confirm the current findings.

The global increase in resorting to artificial nutritional formulas replacing breastfeeding has been identified among the complex causes of the obesity epidemic in infants and children. One of the factors recently recognized to influence metabolism and weight gain is kynurenic acid (KYNA), an agonist of G protein-coupled receptor (GPR35). Therefore the aim of the study was to determine the concentration of KYNA in artificial nutritional formulas in comparison with its level in human breast milk and to evaluate developmental changes in rats exposed to KYNA enriched diet during the time of breastfeeding. KYNA levels were measured in milk samples from 25 heathy breast-feeding women during the first six months after labor and were compared with 21 time-adjusted nutritional formulas. Animal experiments were performed on male Wistar rats. KYNA was administered in drinking water. The content of KYNA in human milk increases more than 13 times during the time of breastfeeding while its level is significantly lower in artificial formulas. KYNA was detected in breast milk of rats and it was found that the supplementation of rat maternal diet with KYNA in drinking water results in its increase in maternal milk. By means of the immunoblotting technique, GPR35 was evidenced in the mucosa of the jejunum of 1-day-old rats and distinct morphological changes in the jejunum of 21-day-old rats fed by mothers exposed to water supplemented with KYNA were found. A significant reduction of body weight gain of rats postnatally exposed to KYNA supplementation without changes in total body surface and bone mineral density was observed. The rat offspring fed with breast milk with artificially enhanced KYNA content demonstrated a lower mass gain during the first 21 days of life, which indicates that KYNA may act as an anti-obesogen. Further studies are, therefore, warranted to investigate the mechanisms regulating KYNA secretion via breast milk, as well as the influence of breast milk KYNA on mass gain. In the context of lifelong obesity observed worldwide in children fed artificially, our results imply that insufficient amount of KYNA in baby formulas could be considered as one of the factors associated with increased mass gain.

Among the methods valuable for assessing spatial learning and memory impairments in rodents, the Barnes maze (BM) task deserves special attention. It is based on the assumption that the animal placed into the aversive environment should learn and remember the location of an escape box located below the surface of the platform. Different phases of the task allow to measure spatial learning, memory retrieval, and cognitive flexibility. Herein, we summarize current knowledge about the BM procedure, its variations and critical parameters measured in the task. We highlight confounding factors which should be taken into account when conducting BM task, discussing briefly its advantages and disadvantages. We then propose an extended version of the BM protocol which allows to measure different aspects of spatial learning and memory in rodents. We believe that this review will help to standardize the BM methodology across the laboratories and eventually make the results comparable.

Glutamate is essential for learning and memory processes, and acute and chronic exposures to ethanol (or protracted abstinence) alter glutamatergic transmission. In the current study, we investigated the effects of VU-29, positive allosteric modulator of metabotropic glutamate 5 (mGlu5) receptor, on the acute ethanol- and ethanol withdrawal-induced impairment of novel object recognition (NOR) task in rats. The influence of VU-29 (30 mg/kg) on memory retrieval was measured (a) at 4-h delay after acute ethanol administration, as well as (b) after acute withdrawal (24 and 48 h) of repeated (2.0 g/kg, once daily for 7 days) ethanol administration. Additionally, the effects of VU-29 on expression of mGlu5 and mGlu2 receptor proteins in the hippocampus, prefrontal cortex, and striatum were determined 48 h after ethanol withdrawal. Our results indicated that VU-29, given before acute ethanol administration, prevented the ethanol-induced impairments in spatial memory retrieval. Furthermore, VU-29 given before the testing session on the first day of abstinence facilitated NOR performance in ethanol-withdrawn rats at 4- and 24-h delay after administration. Our ELISA results show that VU-29 normalized ethanol withdrawal induced increase in expression of mGlu5 receptor protein in the hippocampus, prefrontal cortex, and striatum, as well as expression of mGlu2 receptor protein in the hippocampus. Thus, results from our study indicate that positive modulation of mGlu5 receptor prevented and reversed ethanol-induced memory impairment. Moreover, mGlu5 (hippocampus, prefrontal cortex, and striatum) and mGlu2 (hippocampus) receptors play an important role in the ethanol-induced recognition memory impairment induced by ethanol withdrawal.

Kissorphin (KSO) is a new peptide derived from kisspeptin-10. This peptide possesses neuropeptide FF (NPFF)-like biological activity in vitro ; NPFF, in many cases, inhibits opioid and ethanol effects in rodents. Therefore, the current study explored the influence of KSO on acute ethanol- and morphine-induced hyperactivity, and on the development and expression of locomotor sensitization induced by these drugs. In the present study, sensitization to locomotor effects was induced by repeated exposure to ethanol (2.4 g/kg, intraperitoneally [i.p.], 1 × 4 days) or morphine (10 mg/kg, subcutaneously [s.c.], 1 × 7 days). We found that KSO (1–10 nmol/300 μL, intravenously [i.v.]) did not have an impact on locomotor activity of naive mice. However, it reduced both acute ethanol- (10 nmol/300 μL) and morphine-induced hyperactivity (3 and 10 nmol/300 μL). Pretreatment of animals with KSO (10 nmol/300 μL), before every ethanol or morphine injection during development of sensitization or before the ethanol or morphine challenge, attenuated the development, as well as the expression of locomotor sensitization to both substances. Moreover, prior administration of the NPFF receptor antagonist RF9 (10 nmol/300 μL, i.v.) inhibited the ability of KSO (10 nmol/300 μL) to reduce the expression of ethanol and morphine sensitization. KSO given alone, at all used doses, did not influence the motor coordination measured via the rotarod test. The results from this study show that KSO effectively attenuated acute and repeated effects of ethanol and morphine. Thus, KSO possesses NPFF-like anti-opioid activity in these behavioral studies.

The present study was conducted to evaluate the influence of AMN082, the metabotropic glutamate receptor subtype 7 (mGlu7) allosteric agonist on different stages of memory processes connected with fear conditioning in the passive avoidance (PA) learning task in mice and negative emotional state (anxiety-like) induced by ethanol- and morphine-withdrawal in the elevated plus maze (EPM) test in rats. To perform the PA test, AMN082 (1.25, 2.5 and 5 mg/kg, i. p.) was injected to interfere with (or inhibit) acquisition, consolidation, and retrieval processes. The retention latency in each group was recorded using a step-through passive avoidance task 24 h after training. In turn, in ethanol- and morphine-withdrawal rats, the influence of AMN082 on anxiety-like behavior was estimated in the EPM test 24 h- (ethanol) and 72- h (morphine) after the last dose of repeated drug administrations. In all experimental groups, AMN082 at the dose of 5 mg/kg significantly decreased the step-through latency of long-term memory in the PA task. These AMN082 effects were reversed by MMPIP (10 mg/kg), the antagonist of mGlu7 receptor. AMN082 (2.5 and 5 mg/kg) also decreased ethanol- and morphine withdrawal-induced anxiety-like behavior in the EPM test, and this AMN082 (5 mg/kg) effect was counteracted by MMPIP pretreatment. Taken together, the results show that mGlu7 is involved in fear learning to the context and anxiety-like state connected with unpleasant experiences after ethanol- and morphine withdrawal in rodents. However, it appears that functional dissociation exists between these two AMN082 effects.

Current studies indicate that the cholinergic and opioid systems interact to modulate pain. In the present work, we investigated the influence of the cholinesterase inhibitors, donepezil (0.5; 1 or 3mg/kg, i.p.) and rivastigmine (0.03; 0.5 or 1mg/kg, i.p.), on the acute antinociceptive effects of morphine (5mg/kg, i.p.) in the hot plate test in mice. Herein, both inhibitors were found to enhance and prolong the analgesic effects of morphine without affecting latencies themselves. In an extension of this work, we determined which cholinergic receptors subtype mediates the enhancement of analgesic effects of morphine, following inhibition of cholinesterases. In this part of the study, scopolamine (0.5mg/kg, i.p.), a muscarinic cholinergic receptors antagonist, but not mecamylamine (3mg/kg, i.p.), a nicotinic cholinergic receptors antagonist, reversed the enhancing effects of donepezil (3mg/kg, i.p.) and rivastigmine (1mg/kg, i.p.) on the morphine antinociception. Moreover, both cholinesterase inhibitors attenuated the development of tolerance to the antinociceptive effects of morphine. In contrast, acute administration of donepezil (3mg/kg, i.p.) or rivastigmine (1mg/kg, i.p.) on the day of expression of tolerance, had no effect on the already developed morphine tolerance. What is more, in both set of experiments, rivastigmine was slightly more potent than donepezil due to the broader inhibitory spectrum of this drug on acetylcholine degradation. Thus, our results suggest that the cholinesterase inhibitors, donepezil and rivastigmine, may be administered with morphine in order to enhance the latter's analgesic effects for the treatment of acute and chronic pain.

Amphetamine (AMPH) induces deficits in cognition, and depressive-like behavior following withdrawal. The aim of the present study was to investigate whether pre-treatment with memantine (5mg/kg, i.p.), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, attenuates memory impairment induced by withdrawal from a 1 day binge regimen of AMPH (2mg/kg, four times every 2h, i.p.), in the novel object recognition test in rats. Herein, the influence of scopolamine (0.1mg/kg), an antagonist of the muscarinic cholinergic receptors, and the impact of MK-801 (0.1mg/kg), an antagonist of the NMDA receptors, on the memantine effect, were ascertained. Furthermore, the impact of memantine (5; 10; 20mg/kg, i.p.) was measured on depression-like effects of abstinence, 14 days after the last AMPH treatment (2mg/kg×1×14 days), in the forced swim test. In this test, the efficacy of memantine was compared to that of tricyclic antidepressant imipramine (10; 20; 30mg/kg, i.p.). Our study indicated that withdrawal from a binge regimen of AMPH impaired recognition memory. This effect was attenuated by administration of memantine at both 72h and 7 days of withdrawal. Moreover, prior administration of scopolamine, but not MK-801, decreased the memantine-induced recognition memory improvement. In addition, memantine reversed the AMPH-induced depressive-like behavior in the forced swim test in rats. The antidepressant-like effects of memantine were stronger than those of imipramine. Our study indicates that memantine constitutes a useful approach towards preventing cognitive deficits induced by withdrawal from an AMPH binge regimen and by depressive-like behavior during AMPH abstinence.

Background microRNA (miRNA) belongs to the non-coding RNAs family responsible for the regulation of gene expression. Renalase is a protein composed of 342 amino acids, secreted by the kidneys and possibly plays an important role in the regulation of sympathetic tone and blood pressure. The aim of the present study was to investigate plasma renalase concentration, and explore the relationship between miRNA-146a-5p expression and plasma renalase levels in hemodialyzed patients. Methods The study population comprised 55 subjects who succumbed to various cardiac events, 27 women and 28 men, aged 65–70 years. The total RNA including miRNA fraction was isolated using QiagenmiRNEasy Serum/Plasma kit according to the manufacturer’s protocol. The isolated miRNAs were analyzed using a quantitative polymerase chain reaction (qRT-PCR) technique. The plasma renalase levels were measured using a commercial ELISA kit. Results In the group of patients with high levels of renalase, higher miRNA-146a expression was found, compared with those with low concentration of renalase. Patients with simultaneous low miRNA-146a expression and high level of renalase were confirmed to deliver a significantly longer survival time compared with other patients. Conclusions miRNA-146a and plasma renalase levels were estimated as independent prognostic factors of hemodialyzed patients’ survival time. Patients with low miRNA-146a expression demonstrated a significantly longer survival time in contrast to the patients with a high expression level of miRNA-146a. Moreover, a significantly longer survival time was found in patients with high renalase activity compared with patients with low activity of the enzyme.

Zebrafish are now widely accepted as a valuable animal model for a number of different central nervous system (CNS) diseases. They are suitable both for elucidating the origin of these disorders and the sequence of events culminating in their onset, and for use as a high-throughput in vivo drug screening platform. The availability of powerful and effective techniques for genome manipulation allows the rapid modelling of different genetic epilepsies and of conditions with seizures as a core symptom. With this review, we seek to summarize the current knowledge about existing epilepsy/seizures models in zebrafish (both pharmacological and genetic) and compare them with equivalent rodent and human studies. New findings obtained from the zebrafish models are highlighted. We believe that this comprehensive review will highlight the value of zebrafish as a model for investigating different aspects of epilepsy and will help researchers to use these models to their full extent.