The effect of valproate on the amino acids, monoamines, and kynurenic acid concentrations in brain structures involved in epileptogenesis in the pentylenetetrazol-kindled rats.

BACKGROUND: The study aimed to assess the influence of a single valproate (VPA) administration on inhibitory and excitatory neurotransmitter concentrations in the brain structures involved in epileptogenesis in pentylenetetrazol (PTZ)-kindled rats. METHODS: Adult, male Wistar rats were kindled by repeated intraperitoneal (ip) injections of PTZ at a subconvulsive dose (30 mg/kg, three times a week). Due to the different times required to kindle the rats (18-22 injections of PTZ), a booster dose of PTZ was administrated 7 days after the last rats were kindled. Then rats were divided into two groups: acute administration of VPA (400 mg/kg) or saline given ip. The concentration of amino acids, kynurenic acid (KYNA), monoamines, and their metabolites in the prefrontal cortex, hippocampus, amygdala, and striatum was assessed by high-pressure liquid chromatography (HPLC). RESULTS: It was found that a single administration of VPA increased the gamma-aminobutyric acid (GABA), tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and KYNA concentrations and decreased aspartate (ASP) levels in PTZ-kindled rats in the prefrontal cortex, hippocampus, amygdala and striatum. CONCLUSIONS: Our results indicate that a single administration of VPA in the PTZ-kindled rats restored proper balance between excitatory (decreasing the level of ASP) and inhibitory neurotransmission (increased concentration GABA, KYNA) and affecting serotoninergic neurotransmission in the prefrontal cortex, hippocampus, amygdala, and striatum.

microRNA-dependent regulation of gene expression in GABAergic interneurons.

Information processing within neuronal circuits relies on their proper development and a balanced interplay between principal and local inhibitory interneurons within those circuits. Gamma-aminobutyric acid (GABA)ergic inhibitory interneurons are a remarkably heterogeneous population, comprising subclasses based on their morphological, electrophysiological, and molecular features, with differential connectivity and activity patterns. microRNA (miRNA)-dependent post-transcriptional control of gene expression represents an important regulatory mechanism for neuronal development and plasticity. miRNAs are a large group of small non-coding RNAs (21-24 nucleotides) acting as negative regulators of mRNA translation and stability. However, while miRNA-dependent gene regulation in principal neurons has been described heretofore in several studies, an understanding of the role of miRNAs in inhibitory interneurons is only beginning to emerge. Recent research demonstrated that miRNAs are differentially expressed in interneuron subclasses, are vitally important for migration, maturation, and survival of interneurons during embryonic development and are crucial for cognitive function and memory formation. In this review, we discuss recent progress in understanding miRNA-dependent regulation of gene expression in interneuron development and function. We aim to shed light onto mechanisms by which miRNAs in GABAergic interneurons contribute to sculpting neuronal circuits, and how their dysregulation may underlie the emergence of numerous neurodevelopmental and neuropsychiatric disorders.

Molecular pattern of a decrease in the rewarding effect of cocaine after an escalating-dose drug regimen.

BACKGROUND: Long-term cocaine exposure leads to dysregulation of the reward system and initiates processes that ultimately weaken its rewarding effects. Here, we studied the influence of an escalating-dose cocaine regimen on drug-associated appetitive behavior after a withdrawal period, along with corresponding molecular changes in plasma and the prefrontal cortex (PFC). METHODS: We applied a 5 day escalating-dose cocaine regimen in rats. We assessed anxiety-like behavior at the beginning of the withdrawal period in the elevated plus maze (EPM) test. The reinforcement properties of cocaine were evaluated in the Conditioned Place Preference (CPP) test along with ultrasonic vocalization (USV) in the appetitive range in a drug-associated context. We assessed corticosterone, proopiomelanocortin (POMC), ß-endorphin, CART 55-102 levels in plasma (by ELISA), along with mRNA levels for D2 dopaminergic receptor (D2R), κ-receptor (KOR), orexin 1 receptor (OX1R), CART 55-102, and potential markers of cocaine abuse: miRNA-124 and miRNA-137 levels in the PFC (by PCR). RESULTS: Rats subjected to the escalating-dose cocaine binge regimen spent less time in the cocaine-paired compartment, and presented a lower number of appetitive USV episodes. These changes were accompanied by a decrease in corticosterone and CART levels, an increase in POMC and ß-endorphin levels in plasma, and an increase in the mRNA for D2R and miRNA-124 levels, but a decrease in the mRNA levels for KOR, OX1R, and CART 55-102 in the PFC. CONCLUSIONS: The presented data reflect a part of a bigger picture of a multilevel interplay between neurotransmitter systems and neuromodulators underlying processes associated with cocaine abuse.

The effects of the recurrent social isolation stress on fear extinction and dopamine D2 receptors in the amygdala and the hippocampus.

BACKGROUND: The present study assessed the influence of recurrent social isolation stress on the aversive memory extinction and dopamine D2 receptors (D2R) expression in the amygdala and the hippocampus subnuclei. We also analyzed the expression of epigenetic factors potentially associated with fear extinction: miRNA-128 and miRNA-142 in the amygdala. METHODS: Male adult fear-conditioned rats had three episodes of 48 h social isolation stress before each fear extinction session in weeks intervals. Ninety minutes after the last extinction session, the D2R expression in the nuclei of the amygdala and the hippocampus (immunocytochemical technique), and mRNA levels for D2R in the amygdala were assessed (PCR). Moreover, we evaluated the levels of miRNA-128 and miRNA-142 in the amygdala. RESULTS: It was found that recurrent social isolation stress decreased the fear extinction rate. The extinguished isolated rats were characterized by higher expression of D2R in the CA1 area of the hippocampus compared to the extinguished and the control rats. In turn, the isolated group presented higher D2R immunoreactivity in the CA1 area compared to the extinguished, the control, and the extinguished isolated animals. Moreover, the extinguished animals had higher expression of D2R in the central amygdala than the control and the extinguished isolated rats. These changes were accompanied by the increase in miRNA-128 level in the amygdala in the extinguished isolated rats compared to the control, the extinguished, and the isolated rats. Moreover, the extinguished rats had lower expression of miRNA-128 compared to the control and the isolated animals. CONCLUSIONS: Our results suggest that social isolation stress impairs aversive memory extinction and coexists with changes in the D2R expression in the amygdala and hippocampus and increased expression of miRNA-128 in the amygdala.

The role of the dorsal hippocampus in resistance to the development of posttraumatic stress disorder-like behaviours.

This study aimed to determine the activity of the dorsal hippocampus (dHIP) in resistance to the development of posttraumatic stress disorder (PTSD)-like behaviours. Rats were divided into resistant, PTSD(-), and susceptible, PTSD(+) groups based on the time spent in the central area in an open field test and freezing duration during exposure to an aversive context one week after stress experience (electric foot shock). The PTSD(-) rats, compared to the PTSD(+) group, had an increased concentration of corticosterone in plasma and changes in the activity of the dHIP, specifically, increased c-Fos expression in the dentate gyrus (DG) and increased Neuroligin-2 (marker of GABAergic neurotransmission) expression in the DG and CA3 area of the dHIP. Moreover, in the hippocampus, the PTSD(-) group showed decreased mRNA expression for corticotropin-releasing factor receptors type 1 and 2, increased mRNA expression for orexin receptor type 1, and decreased miR-9 and miR-34c levels compared with the PTSD(+) group. This study may suggest that the increase in GABA signalling in the hippocampus attenuates the activity of the CRF system and enhances the function of the orexin system. Moreover, decreased expression of miR-34c and miR-9 could facilitate fear extinction and diminishes the anxiety response. These effects may lead to an anxiolytic-like effect and improve resistance to developing PTSD-like behaviours.

Increased Hippocampal Afterdischarge Threshold in Ketogenic Diet is Accompanied by Enhanced Kynurenine Pathway Activity.

The efficacy of a ketogenic diet (KD) in controlling seizure has been shown in many experimental and clinical studies, however, its mechanism of action still needs further clarification. The major goal of the present study was to investigate the influence of the commercially available KD and caloric restriction (CR) on the hippocampal afterdischarge (AD) threshold in rats, and concomitant biochemical changes, specifically concerning the kynurenine pathway, in plasma and the hippocampus. As expected, the rats on the KD showed higher AD threshold accompanied by increased plasma ß-hydroxybutyrate level compared to the control group and the CR rats. This group presented also lowered tryptophan and elevated kynurenic acid levels in plasma with similar changes in the hippocampus. Moreover, the KD rats showed decreased levels of branched chain amino acids (BCAA) and aromatic amino acids (AAA) in plasma and the hippocampus. No regular biochemical changes were observed in the CR group. Our results are analogous to those detected after single administrations of fatty acids and valproic acid in our previous studies, specifically to an increase in the kynurenine pathway activity and changes in peripheral and central BCAA and AAA levels. This suggests that the anticonvulsant effect of the KD may be at least partially associated with those observed biochemical alternations.

Neurobiological Basis of Increased Risk for Suicidal Behaviour.

According to the World Health Organization (WHO), more than 700,000 people die per year due to suicide. Suicide risk factors include a previous suicide attempt and psychiatric disorders. The highest mortality rate in suicide worldwide is due to depression. Current evidence suggests that suicide etiopathogenesis is associated with neuroinflammation that activates the kynurenine pathway and causes subsequent serotonin depletion and stimulation of glutamate neurotransmission. These changes are accompanied by decreased BDNF (brain-derived neurotrophic factor) levels in the brain, which is often linked to impaired neuroplasticity and cognitive deficits. Most suicidal patients have a hyperactive hypothalamus-pituitary-adrenal (HPA) axis. Epigenetic mechanisms control the above-mentioned neurobiological changes associated with suicidal behaviour. Suicide risk could be attenuated by appropriate psychological treatment, electroconvulsive treatment, and drugs: lithium, ketamine, esketamine, clozapine. In this review, we present the etiopathogenesis of suicide behaviour and explore the mechanisms of action of anti-suicidal treatments, pinpointing similarities among them.

Individual susceptibility or resistance to posttraumatic stress disorder-like behaviours.

The aim of this study was to explore the neurobiological background of individual susceptibility and resistance to the development of posttraumatic stress disorder (PTSD)-like behaviours. Rats were divided into susceptible, PTSD(+), and resistant, PTSD(-), groups based on freezing duration during exposure to aversive context and the time spent in the central area in open field test one week after threefold stress experience (modified single prolonged stress). PTSD(-) rats showed increased concentrations of corticosterone in plasma and changes in GAD67 expression: decreased in the infralimbic cortex (IL) and increased in the lateral amygdala (LA), dentate gyrus (DG), and CA1 area of the hippocampus. Moreover, in this group, we found an increase in the number of CRF-positive nuclei in the parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN). The PTSD(+) group, compared to PTSD(-) rats, had decreased concentrations of corticosterone in plasma and reduced CRF expression in the pPVN, higher CRF expression in the CA1, increased expression of CRF-positive nuclei and GR receptors in the CA3 area of the hippocampus, and increased expression of GR receptors in the DG and the central amygdala (CeA). Biochemical analysis showed higher concentrations of noradrenaline, glutamic acid in the dorsal hippocampus and amygdala and lower levels of dopamine and its metabolites in the amygdala of the PTSD(+) group than in the PTSD(-) group. The study revealed different behavioural and biochemical profiles of PTSD(+) and PTSD(-) rats and suggested that individual differences in hypothalamic-pituitary-adrenal (HPA) axis activity may determine hippocampal- and amygdala-dependent memory and fear processing.

The role of UCH-L1, MMP-9, and GFAP as peripheral markers of different susceptibility to seizure development in a preclinical model of epilepsy.

In our study, we assessed the potency of the brain-derived proteins ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), matrix metalloproteinase 9 (MMP-9), glial fibrillary acidic protein (GFAP) and the immune activation indicators interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) as peripheral biomarkers of different susceptibilities to kindling in a preclinical model. We observed increased plasma UCH-L1 levels in kindled vs. control animals. Furthermore, MMP-9 and IL-1ß concentrations were the lowest in rats resistant to kindling. In summary, UCH-L1 is an indicator of neuronal loss and BBB disruption after seizure. MMP-9 and IL-1ß may indicate resistance to kindling. UCH-L1, MMP-9 and IL-1ß may have utility as peripheral biomarkers with translational potency in the clinic.

Stress changes amphetamine response, D2 receptor expression and epigenetic regulation in low-anxiety rats.

The aim of this study was to assess the influence of chronic restraint stress on amphetamine (AMPH)-related appetitive 50-kHz ultrasonic vocalisations (USVs) in rats differing in freezing duration in a contextual fear test (CFT), i.e. HR (high-anxiety responsive) and LR (low-anxiety responsive) rats. The LR and the HR rats, previously exposed to an AMPH binge experience, differed in sensitivity to AMPH's rewarding effects, measured as appetitive vocalisations. Moreover, chronic restraint stress attenuated AMPH-related appetitive vocalisations in the LR rats but had no influence on the HR rats' behaviour. To specify, the restraint LR rats vocalised appetitively less in the AMPH-associated context and after an AMPH challenge than the control LR rats. This phenomenon was associated with a decrease in the mRNA level for D2 dopamine receptor in the amygdala and its protein expression in the basal amygdala (BA) and opposite changes in the nucleus accumbens (NAc) - an increase in the mRNA level for D2 dopamine receptor and its protein expression in the NAc shell, compared to control conditions. Moreover, we observed that chronic restraint stress influenced epigenetic regulation in the LR and the HR rats differently. The contrasting changes were observed in the dentate gyrus (DG) of the hippocampus - the LR rats presented a decrease, but the HR rats showed an increase in H3K9 trimethylation. The restraint LR rats also showed higher miR-494 and miR-34c levels in the NAc than the control LR group. Our study provides behavioural and biochemical data concerning the role of differences in fear-conditioned response in stress vulnerability and AMPH-associated appetitive behaviour. The LR rats were less sensitive to the rewarding effects of AMPH when previously exposed to chronic stress that was accompanied by changes in D2 dopamine receptor expression and epigenetic regulation in mesolimbic areas.

Differences in the dopaminergic reward system in rats that passively and actively behave in the Porsolt test.

The aim of the study was to assess appetitive responses and central dopaminergic neurotransmission in passive and active rats divided according to their immobility time in the Porsolt swim test and exposed to restraint stress. Passive rats had more episodes of appetitive 50-kHz ultrasonic vocalization (USV) during rat encounter after social isolation and spent significantly more time in the amphetamine-associated context in conditioned place preference test, compared to active rats. Restraint stress decreased sucrose preference, but increased appetitive vocalization and reinforced the conditioned place preference only in passive animals that was associated with increased dopamine concentration in the amygdala. Restraint stress increased also the level of Cocaine- and Amphetamine Regulated Transcript (CART) peptide, a neuromodulator linked to dopamine neurotransmission, in the central nucleus of amygdala, while decreasing it the nucleus accumbens shell in passive rats. In the parvocellular region of paraventricular nucleus of the hypothalamus passive animals had a higher expression of CART compared to passive restraint rats and active control rats. The obtained results show that active and passive rats in the Porsolt test differ significantly in response to appetitive stimuli, which can be additionally changed under stress conditions. The underlying mechanisms are probably associated with differences in dopaminergic activity and CART signaling in reward system.

Altered expression of GABA-A receptor subunits in the hippocampus of PTZ-kindled rats.

BACKGROUND: Changes in the expression of the GABA-A receptor subunits involved in phasic and tonic inhibition have been studied in a wide spectrum of animal models of epilepsy. However, there is no exhaustive data regarding the pentylenetetrazole (PTZ) kindling model of epilepsy. METHODS: The aim of our study was to analyse the hippocampal changes in the expression of GABA-A receptor subunits involved in phasic (α1, γ2) or tonic (α4 and δ) inhibition in rats subjected to the PTZ kindling using immunohistochemistry method as well as in animals subjected to a single injection of a subconvulsive (30mg/kg) or convulsive (55mg/kg) dose of PTZ. Moreover, the expression of GABA transporters (GAT-1 and GAT-3) was also assessed. RESULTS: In kindled animals, we observed an increase in the expression of α1 (in CA1, DG (dentate gyrus) and CA3 regions) and γ2 (CA1 and CA3) subunits as well as in the expression of GAT-1 (CA1). On the other hand, the expression of the δ subunit in the DG was reduced. The single injection of PTZ at a dose of 30mg/kg increased the expression of the α4 subunit in the DG, while at a dose of 55mg/kg, PTZ increased the expression of the α1 and α4 subunits in the DG and reduced expression of the γ2 subunit in the CA1 and CA3 regions. CONCLUSIONS: The pattern of changes observed in our study indicates that changes in tonic inhibition are involved in abnormal neuronal activity observed in PTZ model of epilepsy.

The role of IL-1ß and glutamate in the effects of lipopolysaccharide on the hippocampal electrical kindling of seizures.

In our study, we used rapid electrical hippocampal kindling and in vivo microdialysis methods to assess the involvement of inflammatory mediators: lipopolysaccharide (LPS) and proinflammatory interleukin-1ß (IL-1ß) in mechanisms of epileptogenesis. We observed, that both, LPS and IL-1ß, administered into stimulated hippocampus, accelerated kindling process. LPS also increased the expression of IL-1ß in stimulated hippocampus in kindled rats. In vivo acute LPS perfusion, via a microdialysis cannula implanted into the naïve rat's hippocampus, produced an increase in extracellular glutamate release. We suppose, that particularly IL-1ß action and increased glutamate concentration may significantly contribute to LPS effects on kindling development.

The role of interleukin-1ß in the pentylenetetrazole-induced kindling of seizures, in the rat hippocampus.

Because the contribution of inflammatory mediators to seizure disorders is unclear, we investigated the changes in the expression of interleukin-1ß (IL-ß) and its receptor - IL-1 receptor type 1 (IL-1R1), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the rat hippocampus at different stages of pentylenetetrazole (PTZ)-induced kindling. The occurrence and progressive development of seizures were induced by repeated systemic administration of PTZ, a non-competitive antagonist of the γ-aminobutyric acid type A (GABAA) receptor at a subconvulsive dose of 30 mg/kg. We also examined the effects of continuous intracerebroventricular administration of IL-1ß and lipopolysaccharide (LPS) in this model of epilepsy using subcutaneously implanted osmotic mini-pumps. We observed enhanced IL-1R1 expression in the dentate gyrus (DG) at different stages of kindling, whereas the elevated IL-1ß level was distinctive to fully kindled seizures. We did not detect significant changes in the concentration of IL-6 or TNF-α throughout the kindling process. LPS accelerated transiently the process of kindling, while IL-1ß showed a predisposition to delay kindling acquisition. Our study supports the concept of seizure-related modifications in brain cytokine production during epileptogenesis. Although some evidence indicates a proconvulsant property of IL-1ß activity, it cannot be ruled out that the alterations in IL-1 system reflect the activation of endogenous protective mechanisms with respect to the kindling of seizures.

Valproate disturbs the balance between branched and aromatic amino acids in rats.

We investigated the effect of valproate (ip, 500 mg/kg), which is regarded as a potent plasma protein tryptophan (TRP) displacer, on the central nervous system (hippocampal) and peripheral (plasma) levels of the aromatic amino acids (AAAs; e.g. TRP, tyrosine and phenylalanine) and branched-chain amino acids (BCAAs; e.g. valine, isoleucine and leucine) as well as the other amino acids (glutamate, GABA, alanine, glutamine, glycine, aspartate and taurine) involved in the regulation of neurotransmission. Furthermore, we investigated whether the changes in the BCAA/AAA ratio affected the hippocampal concentration of monoamines [serotonin (5-HT), dopamine (DA) and noradrenaline (NA)]. Valproic acid (VPA) administration potently modified the balance between the BCAA and AAA. In the brain, the significantly increased AAA and decreased BCAA concentrations were followed by a decrease in the BCAA/AAA ratio. In the plasma, VPA significantly decreased the BCAA and AAA levels. The changes in the BCAA and AAA levels were accompanied by an increase in the NA, DA and 5-HT levels as well as hippocampal 5-HT metabolism. This novel finding indicates that VPA, through the displacement of TRP from its protein-binding sites, could disturb the BCAA/AAA ratio, with central nervous system consequences, including the possible contribution to VPAs effects in affective disorders.

Time course of changes in the concentrations of amino acids in the brain structures of pentylenetetrazole-kindled rats.

The results showed that the development of seizures (pentylenetetrazole, PTZ-induced kindling; PTZ was administered to rats at a subconvulsive dose of 35mg/kg three times a week, i.p.) was accompanied by a progressive recruitment of limbic structures and a characteristic pattern of changes in the brain tissue concentration of examined amino acids (ex vivo measurements, 1.5h after the last dose of PTZ). The earliest and homogenous increase in the excitatory (glutamate and alanine) and inhibitory (GABA and taurine) amino acids was observed in the entorhinal cortex (at stages 1 and 2 according to Racine's scale), and this effect was maintained at the fifth stage of kindling (except for glutamate). At the fifth stage of kindling, glutamate was elevated in the amygdala, nucleus accumbens and piriform cortex, whereas alanine content was increased in the hippocampus, amygdala, striatum, nucleus accumbens and piriform cortex. In the case of GABA, a significant increase in the local concentration of this amino acid was found in rats with stage 1 and 2 seizures in the prefrontal and entorhinal cortices and a decrease was present in amygdala. Kindling raised the local level of taurine in the entorhinal cortex (stage 1 and 2 seizures), amygdala, nucleus accumbens and piriform cortex (stage 5 seizures). These data confirm the conclusion that separate seizure circuitries in the forebrain structures mutually interact to facilitate and/or inhibit one another. Overall, these data suggest that there is a shift in the balance between neurotransmitters toward increased production of excitatory amino acids.