Paweł Krząścik

The effects of neurosteroids on picrotoxin-, bicuculline- and NMDA-induced seizures, and a hypnotic effect of ethanol

The effects of intraperitoneally (IP) or intracerebroventricularly (ICV) administered neurosteroids [allopregnanolone (AP); 5beta-tetrahydrodeoxycorticosterone (5beta-THDOC); dehydroepiandrosterone sulfate (DHEAS); pregnenolone sulfate (PS)] and their precursors [progesterone (PROG), pregnanedione (PREG)] on N-methyl-D-aspartic acid (NMDA)-, picrotoxin (PTX)- and bicuculline (BIC)-induced seizures and ethanol-induced sleep were studied in mice. It was found that IP injections of (+)MK-801 most potently antagonized NMDA-, PTX- and BIC-induced seizures, as compared to diazepam (DZP), PROG and PREG. Both precursors of neurosteroids appeared only marginally active in the applied models of convulsions. ICV injections of AP selectively blocked PTX- and BIC-induced seizures, whereas 5beta-THDOC and (+)MK-801 also antagonized NMDA-induced convulsions. ICV administered DHEAS induced seizures in a dose-dependent way. ICV injections of AP and midazolam shortened the latency and prolonged the duration of sleep induced by IP injections of ethanol (5.0 g/kg). On the contrary, DHEAS and PS significantly reduced the hypnotic-like effect of ethanol. The obtained results suggest that neurosteroids may modulate in an agonistic (AP, 5beta-THDOC), or antagonistic way (PS, DHEAS), the GABA(A) receptor complex functions. Some of them (5beta-THDOC) also interact with NMDA receptors. AP appeared to be the most selectively acting compound, with its profile of action fully comparable to that of midazolam. AP also enhanced the hypnotic effect of ethanol, pointing out to the propensity to interact with centrally depressant agents. These findings, together with the possibility of conversion of some neurosteroids in the brain to other steroid hormones (testosterone, estradiol and aldosterone), indicate the limitations of their use for the treatment of neurological and psychiatric disorders.

Changes in the brain expression of alpha-2 subunits of the GABA-A receptor after chronic restraint stress in low- and high-anxiety rats

This study assessed the mechanisms underlying the behavioral differences between high- (HR) and low-anxiety (LR) rats selected for their behavior in the contextual fear test (i.e., the duration of the freezing response was used as a discriminating variable). Rats were subjected to chronic restraint stress (21 days, 3h daily). We found that in the HR group, chronic restraint stress decreased rat activity in the Porsolt test and reduced the concentration of corticosterone in the prefrontal cortex. The behavioral changes were accompanied by a lower expression of alpha-2 GABA-A receptor subunits in the secondary motor cortex (M2 area) and in the dentate gyrus of the hippocampus (DG) compared to LR restraint animals. Moreover, restraint stress increased the density of alpha-2 GABA-A subunits in the basolateral amygdala (BLA) in HR rats and decreased the expression of these subunits in the DG and M2 areas compared to the HR control group. The present results suggest that, in HR rats exposed to chronic restraint stress, the function of hippocampal and cortical GABAergic neurotransmission is attenuated and that this effect could have important influences on the functioning of the hypothalamic-pituitary-adrenal axis and on depressive symptoms.

Mapping of c-Fos expression in the rat brain during the evolution of pentylenetetrazol-kindled seizures

c-Fos protein immunocytochemistry was used to map the brain structures recruited during the evolution of seizures that follows repeated administration of a subconvulsive dose (35mg/kg, ip) of pentylenetetrazol in rats. c-Fos appeared earliest in nucleus accumbens shell, piriform cortex, prefrontal cortex, and striatum (stages 1 and 2 of kindling in comparison to control, saline-treated animals). At the third stage of kindling, central amygdala nuclei, entorhinal cortex, and lateral septal nuclei had enhanced concentrations of c-Fos. At the fourth stage of kindling, c-Fos expression was increased in basolateral amygdala and CA1 area of the hippocampus. Finally, c-Fos labeling was enhanced in the dentate gyrus of the hippocampus only when tonic-clonic convulsions were fully developed. The most potent changes in c-Fos were observed in dentate gyrus, piriform cortex, CA1, lateral septal nuclei, basolateral amygdala, central amygdala nuclei, and prefrontal cortex. Piriform cortex, entorhinal cortex, prefrontal cortex, lateral septal nuclei, and CA3 area of the hippocampus appeared to be the brain structures selectively involved in the process of chemically induced kindling of seizures.

Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment

Behavioral sensitization and tolerance to repetitive exposure to addictive drugs are commonly used for the assessment of the early stages of the drug dependence progress in animals. The orchestra of tools for studying the progress of drug dependence in laboratory rodents has been considerably enriched in the 1980s by the introduction of ultrasonic vocalization (USV) detection and characterization. However, the relationship between the results of this technology and those of traditional behavioral tests is not clear. We attempted to elucidate some of the respective ambiguities by comparing the effects of an intermittent amphetamine treatment, which was aimed both at the induction of sensitization and tolerance to this drug and at testing the persistence of these effects, on the locomotor activity and 50-kHz USV responses to both the drug and the context of drug exposure in adult male rats showing diverging susceptibility for sensitization to amphetamine. Categorization of the rats into low and high responders/callers based on sensitization of their frequency-modulated 50-kHz USV responsiveness showed some correspondence with conditioned place preference effects, but not with responses to amphetamine. The study showed distinct changes in the rate and latency of the frequency-modulated 50-kHz USV responses to repetitive amphetamine treatment, which were reminiscent of classical behavioral signs of sensitization and tolerance. These results show the utility of the appetitive USV for monitoring of early phases of complex processes leading to drug dependence. However, USV, locomotor activity and conditioned place preference seem to reflect different aspects of these phenomena.

The effect of chronic administration of corticosterone on anxiety- and depression-like behavior and the expression of GABA-A receptor alpha-2 subunits in brain structures of low- and high-anxiety rats

The aim of this study was to examine changes in rat emotional behavior and determine differences in the expression of GABA-A receptor alpha-2 subunits in brain structures of low- (LR) and high-anxiety (HR) rats after the repeated corticosterone administration. The animals were divided into LR and HR groups based on the duration of their conditioned freezing in a contextual fear test. Repeated daily administration of corticosterone (20 mg/kg) for 21 days decreased activity in a forced swim test, reduced body weight and decreased prefrontal cortex corticosterone concentration in both the LR and HR groups. These effects of corticosterone administration were stronger in the HR group in comparison with the appropriate control group, and compared to LR treated and LR control animals. Moreover, in the HR group, chronic corticosterone administration increased anxiety-like behavior in the open field and elevated plus maze tests. The behavioral effects in HR rats were accompanied by a decrease in alpha-2 subunit density in the medial prefrontal cortex (prelimbic cortex and frontal association cortex) and by an increase in the expression of alpha-2 subunits in the basolateral amygdala. These studies have shown that HR rats are more susceptible to anxiogenic and depressive effects of chronic corticosterone administration, which are associated with modification of GABA-A receptor function in the medial prefrontal cortex and basolateral amygdala. The current data may help to better understand the neurobiological mechanisms responsible for individual differences in changes in mood and emotions induced by repeated administration of high doses of glucocorticoids or by elevated levels of these hormones associated with chronic stress or affective pathology.

The effects of morphine and morphine conditioned context on 50 kHz ultrasonic vocalisation in rats

The 50 kHz ultrasonic vocalisations (USVs) that are emitted by rats are dependent on activity of dopaminergic neurons projecting from the ventral tegmental area to the limbic and cortical structures. According to many experimental data, emission of the 50 kHz USV reflects a positive emotional state. The appetitive calls are also emitted in response to the administration of drugs of abuse, e.g., cocaine or amphetamine (AMPH), or in a reply to a positively conditioned context. However, there is no strong evidence in the literature that morphine can also modulate 50 kHz USVs. The aim of this paper is to study the effects of morphine and morphine-conditioned context on 50 kHz USVs, using spontaneously or drug-modulated 50 kHz USVs. Our results showed that acute administration of morphine to rats after withdrawal period inhibited the emission of 50 kHz USVs. The stimulating effect of morphine-conditioned context on 50 kHz USVs appeared on the post-withdrawal challenge day immediately before drug injection, 14 days after the last episode of morphine-induced context conditioning. The context-induced 50 kHz USVs can be used as a sensitive test for drug dependency. The current study also shows that 50 kHz USVs can be useful tool for studying the mechanisms of long lasting central effects of morphine.

The effects of central administration of physostigmine in two models of anxiety.

The effects of intracerebroventricular and intraseptal (the medial septum) administration of a prototypical acetylcholinesterase inhibitor (AChE-I), physostigmine, and a classic benzodiazepine midazolam on rat behavior in the open field test of neophobia and in the conditioned fear test (freezing reaction) were examined in rats. In the open field test of neophobia midazolam and physostigmine increased at a limited dose range, rat exploratory activity, after intracerebroventricular injection. Physostigmine produced in addition the hyperlocomotory effect. Following intraseptal injections, only physostigmine selectively prolonged the time spent by animals in the central sector of the open field. In the model of a conditioned fear, both midazolam and physostigmine inhibited rat freezing reaction to the aversively conditioned context after intracerebroventricular, but not after intraseptal, pretrial drug administration. The presented data support the notion about the selective anxiolytic-like effects of some AChE-Is. It appears, therefore, that the calming and sedative effects of AChE-Is observed in patients with Alzheimers disease may be directly related to their anxiolytic action, independent of an improvement in cognitive functions, which in turn may decrease disorientation-induced distress and anxiety.

Neonatal serotonin (5-HT) depletion does not affect spatial learning and memory in rats

BACKGROUND Extensive previous research has suggested a role for serotonin (5-HT) in learning and memory processes, both in healthy individuals and pathological disorders including depression, autism and schizophrenia, most of which have a developmental onset. Since 5-HT dysfunction in brain development may be involved in disease etiology, the present investigation assessed the effects of neonatal 5-HT depletion on spatial learning and memory in the Morris water maze (MWM). METHODS Three days old Sprague-Dawley rats were pretreated with desipramine (20 mg/kg) followed by an intraventricular injection of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, 70 μg). Three months later rats were tested in the MWM. RESULTS Despite a severe and permanent decrease (80-98%) in hippocampal, prefrontal and striatal 5-HT levels, treatment with 5,7-DHT caused no spatial learning and memory impairment. CONCLUSIONS Limited involvement of chronic 5-HT depletion on learning and memory does not exclude the possibility that this neurotransmitter has an important neuromodulatory role in these functions. Future studies will be needed to identify the nature of the compensatory processes that are able to allow normal proficiency of spatial learning and memory in 5-HT-depleted rats.

Antagonism of picrotoxin-induced changes in dopamine and serotonin metabolism by allopregnanolone and midazolam.

The effects of allopregnanolone and midazolam, given intracerebroventricularly, on the behavioral and biochemical effects of picrotoxin, were examined in a model of neurotoxin-induced seizures, in mice. After acute injections, midazolam (ED(50)=39.8 nmol) and allopregnanolone (ED(50)=11.0 nmol) produced similar and dose-dependent protection against picrotoxin-induced seizures. Picrotoxin given intraperitoneally at the ED(85) dose decreased significantly the concentration of serotonin (5-HT), dopamine (DA), homovanilic acid (HVA) and 3,4-dihydroxyindolacetic acid (DOPAC), in the mouse striatum and the frontal cortex, in the period of time immediately preceding the onset of seizures. A single injection of allopregnanolone more potently, in comparison to midazolam, antagonized the biochemical action of picrotoxin, abolishing its effects on DA, HVA and 5-HT concentration, in the mouse striatum and the frontal cortex. These results for the first time provide a direct argument for an involvement of central dopaminergic and serotonergic systems in the seizure development. The present data add also to the accumulating evidence suggesting a favorable pharmacological profile for some neurosteroids currently considered to have a future role in the management of epilepsy.

Is the interaction between fatty acids and tryptophan responsible for the efficacy of a ketogenic diet in epilepsy? The new hypothesis of action

The effects of a ketogenic diet in controlling seizure activity have been proven in many studies, although its mechanism of action remains elusive in many regards. We hypothesize that the ketogenic diet may exert its antiepileptic effects by influencing tryptophan (TRP) metabolism. The aim of this study was to investigate the influence of octanoic and decanoic fatty acids (FAs), the main components in the MCT diet (medium-chain triglyceride diet, a subtype of the ketogenic diet), on the metabolism of TRP, the activity of the kynurenic pathway and the concentrations of monoamines and amino acids, including branched-chain amino acids (BCAA) and aromatic amino acids (AAA) in rats. The acute effects of FA on the sedation index and hippocampal electrical after-discharge threshold were also assessed. We observed that intragastric administration of FA increased the brain levels of TRP and the central and peripheral concentrations of kynurenic acid (KYNA), as well as caused significant changes in the brain and plasma concentrations of BCAA and AAA. We found that the administration of FA clearly increased the seizure threshold and induced sedation. Furthermore, we have demonstrated that blocking TRP passage into the brain abolished these effects of FA but had no similar effect on the formation of ketone bodies. Given that FAs are major components of a ketogenic diet, it is suggested that the anticonvulsant effects of a ketogenic diet may be at least partly dependent on changes in TRP metabolism. We also propose a more general hypothesis concerning the intracellular mechanism of the ketogenic diet.