Chronic stress changes prepulse inhibition after amphetamine challenge: the role of the dopaminergic system.

The goal of this research was to examine the influence of chronic mild stress (CMS) on prepulse inhibition (PPI). We used an amphetamine challenge to study the role of the dopaminergic system in limbic structures. Chronic stress caused a reduction in both sucrose preference and body weight. It was found that the initially strong response to amphetamine in the control rats was weakened after stress on both the behavioural and biochemical levels: improved PPI, decreased dopamine D2 receptor expression in the central nucleus of amygdala (CeA) and nucleus accumbens (NAC), and decreased dopamine and 3-MT (3-methoxytyramine) levels in NAC. We observed that the stress-evoked attenuation of amphetamine-induced stimulation was also paralleled by changes in corticosterone level. These effects were accompanied by a decrease in both glutamate and the glutamate/gamma-aminobutric acid (GABA) ratio in the NAC. The interpretation of these results is that prolonged stress induces compensatory mechanisms in the mesolimbic system which are responsible for psychostimulant (amphetamine) effects.

Corticotropin releasing factor receptor 1 antagonist differentially inhibits freezing behavior and changes gamma-aminobutyric acidergic activity in the amygdala in low- and high-anxiety rats.

The aim of this study was to examine the effects of non-peptide corticotropin-releasing factor receptor 1 (CRF1) antagonist (antalarmin) administration on rat conditioned fear responses and gamma-aminobutyric acid (GABA)-ergic brain activity (GAD67 expression and GABA concentration) in low-anxiety (LR) and high-anxiety (HR) rats. The animals were divided into the LR and HR groups based on the duration of their conditioned freezing response in the first contextual fear test. After 28 days, the animals were re-subjected to the contextual fear training and test. The rats received an antalarmin injection (10 mg/kg or 20 mg/kg) 80 min before the second exposure to the aversive context. Antalarmin significantly attenuated the conditioned fear response only in the HR rats. The behavioral effect of a lower dose (10 mg/kg) of antalarmin was accompanied by increased GAD67 expression in the prelimbic cortex (PL) and central nucleus of the amygdala (CeA) and an increased GABA concentration in the amygdala. These studies showed that HR rats were more susceptible to the anxiolytic effects of CRF1 antagonist administration, which were associated with increased GABAergic activity in the medial prefrontal cortex and amygdala. The current data may provide insights into the neurobiological mechanism operating within the mesolimbic CRF-GABA neurotransmitter systems, which may be responsible for individual differences in stress-related diseases. This knowledge can be applied to further elucidate the pathophysiology of anxiety and trauma/stress-related disorders.

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.

The kynurenine pathway: a missing piece in the puzzle of valproate action?

The present study was designed to determine the role of the kynurenine pathway (KP) in the mechanism of action of valproate (VPA). Therefore, we investigated changes in the concentrations of tryptophan (TRP), kynurenic acid (KYNA), and kynurenine (KYN) in the brain and plasma following VPA administration (50, 250 and 500mg/kg i.p.). The most important findings of our study were that VPA administration produced a progressive and strong increase in the central concentrations of KYNA, KYN and TRP. Simultaneously, the TRP level in plasma declined, while the peripheral increase of KYNA in plasma was weaker and occurred earlier than in the hippocampus. Bearing in mind that the observed effect may be a result of a strong VPA-induced displacement of TRP from its binding sites to plasma albumin, we checked the effect of ibuprofen (IBU) administration (a prototypic drug used to study drug binding to serum albumin) on the KP. We found that IBU evoked a similar pattern of change in the KP activity as VPA. These new findings indicate the existence of a mechanism that could stimulate the production of KYNA in the brain after VPA administration, and may partially contribute to the mechanisms of VPA action. The results of our experiment indicate that an increase in the brain's KYNA level may be achieved by TRP displacement from its binding site on plasma albumin with the administration of different drugs, including VPA, IBU, or short-chain fatty acids, with important clinical consequences.

Corticosterone modulates fear responses and the expression of glucocorticoid receptors in the brain of high-anxiety rats.

The aim of our experiments was to assess the effect of acutely administered corticosterone on the expression of glucocorticoid receptors (GRs) in the brain of rats with high (HR) and low (LR) levels of anxiety. The rats were divided into groups according to their conditioned fear-induced freezing responses and then were subjected to a second conditioned fear session one week after the initial fear conditioning. Immunocytochemical analysis revealed that the second exposure to contextual aversive stimuli resulted in higher levels of GRs expression in cingulate cortex area 1 (Cg1), the secondary motor cortex (M2) of the prefrontal cortex and the dentate gyrus of the hippocampus (DG) in LR rats compared with HR rats. The pretreatment of HR rats with corticosterone (20mg/kg, sc) increased the expression levels of GRs in Cg1, the M2 area and the DG to the levels observed in the LR vehicle group. The increase in the GRs levels was accompanied by a significant decrease in the conditioned fear response in the HR group. The control animals that were not exposed to aversive stimuli had similar levels of receptor-related immunoreactivity in all brain regions, and corticosterone did not change these expression levels. Our results suggest that HR animals may have deficits in the expression of stress-induced GRs in the prefrontal cortex and the DG. In addition, pretreatment with corticosterone increases the expression of GRs and normalizes the fear response in HR rats.

Corticosterone attenuates conditioned fear responses and potentiates the expression of GABA-A receptor alpha-2 subunits in the brain structures of rats selected for high anxiety.

The aim of the experiment was to assess the effects of an acutely administered corticosterone on the expression of GABA-A receptor alpha-2 subunits in the brain structures of high (HR) and low (LR) anxiety rats (divided according to their conditioned fear-induced freezing response) subjected to a second conditioned fear session (1 week after fear conditioning). We found that corticosterone (20 mg/kg, sc) given to rats prior to the second conditioned fear session significantly enhanced a decrease in fear expression in the HR group. The behavioural effect of fear was accompanied by the increased expression of alpha-2 subunits in the basolateral amygdala (BLA) and the dentate gyrus of the hippocampus (DG) of the HR group. Corticosterone potentiated the effect of fear on alpha-2 subunit expression in the BLA, DG, the cingulate cortex area 1 and the secondary motor cortex (areas Cg1 and M2). The current study provides insight into the mechanisms that may be responsible for the beneficial effects of glucocorticoids in the therapy of some anxiety disorders.

The effects of electrical hippocampal kindling of seizures on amino acids and kynurenic acid concentrations in brain structures.

Our study demonstrated that the development of seizures during the electrically induced kindling of seizures is associated with significant changes in the concentration of kynurenic acid (KYNA) and its precursor, tryptophan (TRP). The primary finding of our study was an increase in KYNA levels and the KYNA/TRP ratio (a theoretical index of activity of the kynurenine pathway) in the amygdala and hippocampus of kindled animals. We also found decreases in the concentration of tryptophan in the hippocampus and prefrontal cortex. Changes in the concentration of KYNA and TRP in the amygdala were accompanied by a significant decrease in γ-Aminobutryic Acid (GABA) levels and an increase in the glutamate/GABA ratio. Moreover, we found a significant negative correlation between the local concentrations of KYNA and glutamate in the amygdala of kindled rats. However, there were no changes in the local concentrations of the following amino acids: glutamate, aspartate, glutamine, glycine, taurine and alanine. In conclusion, these new results suggest a modulatory influence of KYNA on the process of epileptogenesis, characterized by a negative relationship between the KYNA and glutamate systems in the amygdala.

Expression of N-methyl-D-aspartate (R)(GluN2B) - subunits in the brain structures of rats selected for low and high anxiety.

In this paper, we studied differences in the density of N-methyl-D-aspartate (NMDA) receptor GluN2B subunits in the brains of low (LR) and high (HR) anxiety rats subjected to extinction trials and re-learning of a conditioned fear response, modeling a natural course of anxiety disorders. Classifications of animals as LR or HR was determined by fear-induced freezing responses in the contextual fear test. Increased basal concentrations of GluN2B subunits were observed in the amygdala of HR rats as compared to the unconditioned control group by Western blot analysis. Re-exposure of HR animals to the fear-conditioned context resulted in elevated concentrations of GluN2B subunits in the amygdala, hippocampus and the prefrontal cortex compared to LR rats as well as in the hippocampus and prefrontal cortex vs. the control group. In addition, it was shown that re-test of a conditioned fear increased the number of cells expressing GluN2B subunits in the basolateral amygdala, dentate gyrus of the hippocampus and secondary motor cortex (M2) in the HR group relative to the LR group. Together, these data suggest that animals that are more anxious have altered patterns of GluN2B subunit expression in the frontal cortex and limbic structures, which control emotional behaviour.

The effect of CRF2 receptor antagonists on rat conditioned fear responses and c-Fos and CRF expression in the brain limbic structures.

The influence of intracerebroventricular-administered selective corticotropin-releasing factor receptor 2 (CRF(2)) antagonists (antisauvagine-30, astressin-2B), on rat anxiety-like behavior, expression levels of c-Fos and CRF, and plasma corticosterone levels were examined in the present study. In fear-conditioned animals, both CRF receptor antagonists enhanced a conditioned freezing fear response and increased the conditioned fear-elevated concentration of serum corticosterone. Exogenously administered antisauvagine-30 increased the aversive context-induced expression of c-Fos in the 1 and 2 areas of the cingulate cortex (Cg1, Cg2), the central amygdala (CeA) and parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), and it enhanced the effect of conditioned fear in the secondary motor cortex (M2) and medial amygdala (MeA). Immunocytochemistry demonstrated an increase in CRF expression in the Cg1, M2 areas of the cortex, and pPVN, and it revealed the effect of conditioned fear in the CeA 35 min after antisauvagine-30 administration and 10 min after the conditioned fear test. Furthermore, astressin-2B, another CRF(2) receptor antagonist, enhanced expression of c-Fos and CRF in the CeA and pPVN, and revealed the effect of conditioned fear in the Cg1. These data support a model in which an excess in CRF(1) receptor activation, combined with reduced CRF(2) receptor signaling, may contribute to stronger expression of anxiety-like responses.

The opposite role of hippocampal mGluR1 in fear conditioning in kindled and non-kindled rats.

In the present paper, we analyzed the effects of hippocampal mGluR1 on the consolidation of a fear-conditioned response and on hippocampal glutamate and GABA concentration in rats subjected to the chemically-induced kindling of seizures. We hypothesized the important role of this glutamate receptor subpopulation in behavioural disturbances accompanying epilepsy. To this end, the behavioural and biochemical effects of selective mGluR1 and 5 receptor ligands were compared in sham and kindled animals (pentylenetetrazol-induced seizures). It was found that despite the fact that the freezing response to the aversively conditioned context was not changed by kindling itself, post-training intrahippocampal (dentate gyrus) injection of AIDA (a mGluR1 antagonist) oppositely influenced rat freezing behaviour in the non-kindled and kindled animals (i.e. the receptor ligand increased and decreased duration of the fear reaction, respectively). Kindling of seizures also enhanced the Glutamate/GABA ratio in the dorsal hippocampus (in vivo microdialysis), indicating an enhancement of excitatory processes in the brain. Altogether, the results showed that kindling of seizures led the potentiation of excitatory processes in the hippocampus, changing the role of the local mGluRs1 population in the conditioned fear learning.

Glutamate concentration in whole saliva and taste responses to monosodium glutamate in humans.

It is universally accepted that saliva plays an important role in taste sensations. However, interactions between constituents of whole saliva and the five basic taste modalities are still poorly understood. The aim of the present study was to evaluate possible relationship between endogenous glutamate (Glu) levels in whole saliva and taste responses to a prototypic umami substance, monosodium glutamate (MSG; 0.03-10.0%). Rated intensity and pleasantness of MSG taste was studied in healthy volunteers divided into a high glutamate (HG) in saliva (HG; n = 19) and low glutamate in saliva (LG; n = 18) group based on the median split level of salivary Glu. The HG and LG group did not differ in terms of electrogustometric thresholds, rated intensity of the MSG samples and pleasantness of distilled water and the lower MSG concentrations (0.03-1.0%). Perceived intensity of water taste was significantly (P < 0.05) higher in the LG subjects. The LG group rated the higher MSG concentrations (3.0-10.0%) as more unpleasant (P < 0.01). The difference remained significant after controlling for a between-group difference in age. The present results suggest that individual differences in salivary Glu levels may alter hedonic responses to suprathreshold MSG concentrations.

Midazolam inhibits neophobia-induced Fos expression in the rat hippocampus.

The effect of midazolam on expression of c-Fos protein was examined in the rat hippocampus, following the open field test of neophobia. It was found that pretreatment of rats with midazolam, at the dose of 0.5 mg/kg, enhanced rat exploratory behavior, and inhibited neophobia related stimulation of c-Fos in the CA-1 and CA-3 areas of the hippocampus. The presented results provide new immunocytochemical data on the involvement of hippocampus in emotional processes related to neophobia, and indicate a possible site of action of benzodiazepines.

Pregnenolone sulfate potentiates the effects of NMDA on hippocampal alanine and dopamine.

The aim of the present study was to analyze biochemical effects of a neurosteroid, pregnenolone sulfate (PS), which accompany changes in the threshold of seizures, and to establish the contribution of local, hippocampal monoaminergic and amino acid systems, to the control of convulsive activity. Pretreatment of mice with PS (intracerebroventricularly) selectively enhanced the potency of peripherally (intraperitoneally) administered NMDA at the LD16 (88.0 mg/kg) to induce clonic-tonic convulsions (PS, LD84 = 184.7 nM; 95% CL = 181.4-188.1). The proconvulsive actions of picrotoxin and bicuculline, the GABA-A receptor antagonists, were not modified by pretreatment of mice with PS. Administration of PS alone (up to 240 nM icv) did not show any seizure-like activity. PS given at LD84, together with NMDA (at the LD16), increased the hippocampal concentration of alanine, and enhanced local metabolism of dopamine in a period immediately preceding the onset of seizures significantly stronger than did NMDA alone. These and other data indicate that the enhancement by PS of hippocampal levels of alanine may contribute to the seizures development as this amino acid is a precursor of glutamate, and a co-agonist of the NMDA receptors. On the other hand, simultaneously occurring stimulation of hippocampal dopaminergic system may be considered a compensatory phenomenon, limiting seizures propagation through the limbic forebrain. Summarizing, our results show that PS-induced potentiation of NMDA seizures is accompanied by selective changes in hippocampal dopamine turnover and alanine concentration.

Pentylenetetrazol-kindling of seizures selectively decreases [3H]-citalopram binding in the CA-3 area of rat hippocampus.

The present study was aimed at determining the changes in the 5-HT transporter activity, in different brain structures after pentylenetetrazol induced kindling of seizures. We examined [3H]-citalopram binding in the rat brain structures, and the neurodegenerative effects in the hippocampal formation using autoradiographic and immunohistochemical methods. A statistically significant and selective reduction in the binding of [3H]-citalopram was found in the CA3 field of the hippocampus (P=0.009), and a similar tendency, close to the significance level, in the dentate gyrus (P=0.05). This effect was accompanied by a loss of neurons and activation of microglia in the hippocampal formation. The present data suggest the important role for CA3- serotonergic innervation in pentylenetetrazol induced kindling of seizures.

The anxiolytic-like effect of nicotine undergoes rapid tolerance in a model of contextual fear conditioning in rats.

The effects of repeated administration of nicotine on contextual fear conditioning, locomotor activity, and pain threshold, were examined in rats. It was found that a single injection of nicotine prior to the training session (three 0.7-mA footshocks, each 0.5 s long), decreased the freezing reaction during the retest 24 h later. The locomotor activity was moderately enhanced, and the pain threshold remained unchanged. The baseline freezing measured immediately after administration of a single dose of nicotine was not significantly different from the saline-treated group. The anxiolytic-like effect of nicotine was as potent as that of midazolam, a benzodiazepine derivative. After five day-by-day injections, the anxiolytic-like effect of nicotine (0.6 mg/kg, sc) was no longer present, independently whether the last drug injection was given 24 h or 5 min (i.e., the sixth, additional, nicotine injection), prior to the training session. Thus, it appeared that the expression of tolerance to the nicotine-induced anxiolytic-like action did not require a direct stimulation of nicotinic receptors. Simultaneously, in this group of animals, nicotine caused a potent stimulation of locomotor activity in the open field test. The applied dosage and regimen of nicotine administration did not change rat pain threshold (flinch-jump test). Collectively, the present data showed for the first time, that short-term, intermittent, administration of nicotine was sufficient to induce tolerance to the anxiolytic-like effect of this drug, in the model of fear conditioning to context. Importantly, a clear dissociation between the locomotor and anxiolytic-like effects of nicotine was present. This effect appeared independent also of changes in rat pain threshold. The possible mechanisms of this phenomenon are discussed.

Tolerance to the anticonvulsant activity of midazolam and allopregnanolone in a model of picrotoxin seizures.

The effects of an intracerebroventricular (i.c.v.) administration of a non-selective full benzodiazepine receptor agonist, midazolam, and a neuroactive steroid, allopregnanolone, on picrotoxin-induced seizures and striatal dopamine metabolism, were studied in mice. It was found that acute i.c.v. injections of midazolam (ED50=38.25 nmol) and allopregnanolone (ED50=26.34 nmol) blocked picrotoxin-induced seizures to a similar extent. After repeated administration at the ED(85) doses (midazolam-56.6 nmol, allopregnanolone-94.2 nmol; once or twice daily for 5 days) tolerance developed to the anticonvulsant activity of midazolam (ED50=94.14 nmol) and allopregnanolone (ED50=186.70 nmol). Acute i.c.v. injections of midazolam and allopregnanolone (at the ED50 doses established in the model of picrotoxin seizures: 38.25 and 26.34 nmol, respectively), significantly decreased the concentration of dopamine metabolites: 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, as well as the dopamine turnover rate (homovanilic acid/dopamine ratio; by about 20%), in the mouse striatum. These findings together with the recently published data on the potentiation by midazolam and allopregnanolone of ethanol-induced sleep [Pharmacol. Biochem. Behav. 67 (2000) 345] indicate a very similar central effect profile of benzodiazepines and neurosteroids. Moreover, similar efficacy of allopregnanolone and midazolam at the GABA(A) receptors has been found. Overall, the results of the present study, along with the possibility of neurosteroid conversion in the brain into other steroid hormones (testosterone, estradiol, aldosterone), add to the accumulating evidence suggesting a less favorable pharmacological profile for this class of drugs than was previously thought.

Opposite effects of olanzapine and haloperidol in rat ultrasonic vocalization test.

The opposite effects of the classical antipsychotic, haloperidol, and atypical neuroleptic, olanzapine, in the rat ultrasonic vocalization test of anxiety were observed. The present data are discussed in relation to growing body of evidence of specific brain biochemical changes after pretreatment with different antipsychotics.

Role of serotonergic and noradrenergic systems in a model of visceral pain.

The effects of selective manipulations of activity of the serotonergic and noradrenergic systems were examined in the rat model of visceral pain. It was found that neither p-chlorophenylalanine(p-CPA)- nor N-chloro-ethyl-2,2--bromo-benzylamine(DSP-4)-induced strong and selective depletion of the brain and spinal cord serotonin and noradrenaline, respectively, changed in a significant way rat visceral pain perception. On the other hand, 8-OH-DPAT, a full selective 5-HT1A receptor agonist, prazosin, an alpha1-adrenoceptor antagonist, clonidine, an alpha2-adrenoceptor agonist, and two beta-adrenoceptor antagonists: propranolol and metoprolol, dose-dependently reduced the number of body writhes induced by intraperitoneally administered 2% solution of acetic acid (the writhing test). The results obtained with selective receptor ligands, DSP-4 and p-CPA, indicate that the noradrenergic and serotonergic innervation of the central nervous system contribute in a complex way to the animal behavior in the writhing test. The 5-HT1A receptors and alpha2-adrenoceptors play an inhibitory role in the expression of rat behavior in this model of visceral pain. On the other hand, adrenergic alpha1 and beta1 receptors facilitate the behavioral effects of the irritant agent.

Rapid down-regulation of GABA-A receptors after pretreatment of mice with progesterone.

The effect of a single administration of a high dose of progesterone on brain [3H]muscimol binding, was examined in mice using quantitative autoradiography. It was found that progesterone given at the dose of 150 mg/kg ip (the ED85 dose established previously in the model of picrotoxin seizures, Czlonkowska et al., Pharmacol. Biochem. Behav., 2000, 67, 345-353), significantly decreased the specific binding of [3H]muscimol to the nucleus caudatus and nucleus accumbens, as early as 1 h after injection. A similar tendency, close to the statistically significant level, was also present in the dentate gyrus of the hippocampus (p = 0.07). It is suggested that a high dose of progesterone and ensuing excessive stimulation of GABAA receptors by its metabolites, neurosteroids, could bring about rapid changes in the GABAA receptor number and/or affinity.

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.