The localization of brain sites of anxiogenic-like effects of urocortin-2.

The influence of intracerebroventricullary-administered urocortin-2, a selective corticotropin-releasing factor receptor 2 (CRF(2)) agonist, on rat anxiety-like behaviour, the expression of c-Fos and CRF, and plasma corticosterone levels was examined in the present study. When applied to animals exposed to the conditioned fear-induced context, urocortin-2 enhanced a conditioned freezing fear response. Urocortin-2 also significantly decreased rat exploratory activity in the open field test. Exogenous urocortin-2 increased the conditioned fear-induced expression of c-Fos in the central amygdala (CeA), and parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN), and revealed the effect of conditioned fear in the medial amygdala (MeA). In the fear-conditioned animals, immunocytochemistry showed an increase in the density of CRF-related immunoreactive complexes in the lateral septum (LS), 35min after urocortin-2 administration and 10min after the conditioned fear test, compared with saline-pretreated fear-conditioned animals. These data suggest a role of urocortin-2 in the behavioural and immunocytochemical responses to stress, in which it strengthens the measures 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.

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.

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.

Effects of buspirone, diazepam, and zolpidem on open field behavior, and brain [3H]muscimol binding after buspirone pretreatment.

The effects of 5-HT(1A) receptor agonist buspirone, a nonselective (diazepam), and a selective (zolpidem) GABA(A) receptor agonist were compared in the open field test of neophobia. Unhabituated rats were pretreated with the drugs once, prior to a first exposure to the open field, and their behavior was recorded both during this test and during a second trial 24 h later. It has been hypothesized that the decrease in exploratory activity observed during the second test session may be considered an adaptive reaction to the first day aversive experience (neophobia). If so, a selective modulation of 5-HT and GABA systems activity during the test could bring about significant changes in animal behavior on the retest. Buspirone at the lowest dose of 0.3 mg/kg revealed anxiolytic-like properties on the first day, whereas the action of diazepam and zolpidem was modulated by the dose-related sedative effect. At the dose of 2.4 mg/kg buspirone elicited delayed in time anxiolytic-like action, i.e., produced the antithigmotactic effect during the retrial 24 h later. Diazepam and zolpidem failed to exhibit similar profile of action. Autoradiography of [3H]muscimol binding after pretreatment of rats with buspirone showed a significant increase in the selective radioligand binding within the frontal cortex and a similar, near-significant tendency in the dentate gyrus of the hippocampus. The behavioral data validate buspirone as important drug for the treatment of anxiety disorders, devoid of disruptive influence on motor and cognitive processes. The open field test, as modified by us, appeared sensitive in distinguishing the behavioral profiles of action of different anxiolytic compounds, including 5-HT(1A) receptor agonist. The present results support the assumption that reduced turnover of 5-HT due to stimulation of 5-HT(1A) autoreceptors, may bring about changes in GABA(A) receptor system activity, in some brain structures, leading to the anxiolytic effect.

Locomotor activity and a conditioned fear response: correlation with cortical and subcortical binding of the dopamine D1 receptor antagonist.

Rat behavior in the open field and conditioned fear response test was correlated with specific binding of dopamine D1 receptor antagonist [3H]SCH 23390 within different brain structures assayed with autoradiography. A significant positive correlation was found between the ligand binding in the substantia nigra pars reticulata and both animal motor activity (r = 0.67, p < 0.05) and the number of entries into the central sector of the open field (r = 0.59, p < 0.05). On the other hand, rat motility and the central entries were negatively correlated with [3H]SCH 23390 binding within the caudate putamen (r = -0.64, p < 0.05 and r = -0.61 p <0.05, respectively). No correlation was revealed between the ligand binding in the examined brain areas and freezing reaction in the contextual fear conditioning test. The present data indicate for the first time a significant, structure-dependent correlation between rat motor behavior and the dopamine D1 receptor ligand binding within the nigrostriatal system.

The effects of neurosteroids on rat behavior and 3H-muscimol binding in the brain.

The effects of ICV administration of metabolites of progesterone and deoxycorticosterone [i.e., neurosteroids: AP (3alpha-hydroxy-5alpha-pregnan-20-one, allopregnanolone), 5alpha(-THDOC (3alphat-21-dihydroxy-5alpha-pregnan-20-one, 5alpha-tetrahydrodeoxycorticosterone), 5beta-THDOC (3alpha-21-dihydroxy-5beta-pregnan-20-one, 5beta-tetrahydrodeoxycorticosterone), and PS (3beta-hydroxy-5-pregnen-20-one sulfate, pregnenolone sulfate] were studied in the open-field test of neophobia and Vogel's test of conflict behavior in rats. The influence of in vivo administered 5beta-THDOC, a positive allosteric modulator of the GABA(A) receptor complex, on 3H-muscimol binding in different brain structures, was also studied with the help of quantitative autoradiography. The presented data did not reveal any anxioselective effects for a range of centrally active neurosteroids, in the ethologically orientated and conflict models of anxiety, after intracerebral drug administration. Their central effects appeared secondary to changes in rat gross behavior. It is possible that high local concentration of neurosteroids after ICV injection and production of a narrower range of behavioral effects than that of benzodiazepines, precluded manifestation of the antianxiety effects of AP, 5alpha-THDOC and 5beta-THDOC. Autoradiography did not reveal any significant changes in the specific binding of 3H-muscimol in brain structures after in vivo ICV administration of 5beta-THDOC at the behaviorally active dose. Thus, the possibility that neuroactive neurosteroids may provide a novel potential site for therapeutic interventions in anxiety disorders is not supported. The part of the experiment with 5beta-THDOC is interpreted as contributing to other results, suggesting the existence of a new category of neurosteroids acting as partial agonists of the GABA(A) receptor.

The influence of serotonin depletion on rat behavior in the Vogel test and brain 3H-zolpidem binding.

The influence of p-chlorophenylalanine (p-CPA) and 5,7-dihydroxytryptamine (5,7-DHT)-induced serotonin depletion on rat behavior as well as on zolpidem's the behavioral effects and binding to some brain areas of zolpidem, was examined with the help of Vogel's punished drinking test and autoradiography, respectively. Moreover, changes in the serotonin levels and turnover rate were studied in the forebrain and brainstem of rats pretreated with various ligands at the benzodiazepine (BDZ) receptors (midazolam, bretazenil, abecarnil, zolpidem). These drugs were given at doses shown previously to significantly disinhibit animal behavior suppressed by punishment in the Vogel test (Nazar et al., 1997). It was found that serotonin decrease in the frontal cortex and hippocampus after p-CPA significantly and inversely correlated with rat behavior controlled by fear in the VT. p-CPA produced an anticonflict activity in the absence of effect on spontaneous drinking, pain threshold and motility of animals. All applied benzodiazepine receptor ligands decreased the 5-HT turnover rate in the frontal cortex and hippocampus, whereas in the brainstem only abecarnil and zolpidem diminished 5-hydroxyindoleacetic acid levels. This part of the study replicated earlier data with neurotoxins and indicated that the anxiolytic-like effect of 5-HT depletion in some models of anxiety did not depend on changes in animal appetitive behavior or stimulus control. Moreover, the fact that all nonselective and selective (zolpidem) agonists of the type 1 benzodiazepine receptors seemed to produce the same anticonflict effect and decreasing 5-HT turnover indicates that this subtype of benzodiazepine receptor may be important for the interaction between brain 5-HT and GABA/BDZ systems. Accordingly, it was found that serotonin decrease enhanced the anticonflict effect of zolpidem in the Vogel test and increased 3H-zolpidem binding to the occipital cortex and substantia nigra. Altogether, the present study provides more arguments for the role of changes in the activity of brain 5-HT innervation in the control of emotional processes. Moreover, it points to the BDZ1 receptor subtype as a possible target of interaction between brain 5-HT and GABA(A)/BDZ systems.

Alcohol intake and brain [3H]muscimol binding sites in alcohol-preferring and non-preferring rats.

The present study has employed in vitro autoradiography to determine the distribution and density of [3H]muscimol binding sites in the brains of alcohol high-preferring line of rats (WHP) and alcohol low-preferring line of rats (WLP). While the density of [3H]muscimol binding was found to be similar in the frontal cortex, caudate-putamen, nucleus accumbens, lateral and medial septum, the density of [3H]muscimol binding was lower in cingulate cortex of alcohol low-preferring rats as compared to alcohol high preferring rats. Moreover, the density of muscimol binding sites within this area was positively correlated with the intensity of ethanol consumption.

The effect of serotonin depletion on motor activity habituation, and [3H]muscimol binding in the rat hippocampus.

The effect of serotonin depletion (p-chlorophenylalanine pretreatment) on habituation of exploratory motor activity, and on cortical and hippocampal [3H]muscimol binding in vitro, was examined in rats. It appeared that the very strong decrease in serotonin concentration abolished motor habituation in the open field and decreased [3H]muscimol binding to cortical and hippocampal brain slices. The GABA(A) receptor down-regulation was due to a decrease in the apparent affinity of the radioligand for the receptors. p-Chlorophenylalanine-induced biochemical changes were selective and most probably secondary to serotonin depletion, as the serotonin synthesis inhibitor did not displace [3H]muscimol from its binding sites in neural membranes taken from the occipital cortex. It is concluded that there is a functional interaction between brain serotonin and GABA (gamma-aminobutyric acid) systems, both at behavioral and biochemical levels, that is involved in the motor activity habituation process.

The effects of repeated administration of diazepam, MK-801 and CGP 37849 on rat behavior in two models of anxiety.

The effects of repeated administration of diazepam, MK-801 and CGP 37849 on rat behavior in the Vogel conflict test, and in the open field test of neophobia, were studied in rats. The drugs were given at doses active acutely, for 5 days, the last dose was administered 30 or 60 min prior to testing. It appeared that diazepam and MK-801 treated animals showed clear-cut signs of behavioral tolerance and motor sensitization, respectively. CGP 37849 was characterized by the best pharmacological profile, in that on repeated administration the drug not only retained its anxiolytic-like potency in the Vogel test, but even enhanced rat exploratory behavior in a new environment, independently of changes in animal motor activity. Repeated injections of the examined agents did not cause any significant modifications in monoamine levels and their turnover rates, in the striatum and limbic forebrain. It is concluded that the new class of competitive NMDA receptor antagonists, exemplified by CGP 37849, is the most promising candidate for clinical trials in anxiety disorders.

Central serotonergic system and mechanism of anxiolytic action.

The results clearly indicate that the hippocampus, rather than nucleus accumbens is involved in mediating anxiolytic-like effects of the 5-HT1A receptor agonists. Furthermore, hippocampal postsynaptic 5-HT1A receptors may account for the anti-emotional influence of this groups of drugs. As far as the 5-HT3 receptor antagonists are concerned, it was more difficult to localize their central anti-anxiety like action. More clear and unequirocal effects could be observed after intra-accumbens, rather than after intrahippocampal injections of tropisetron and ondansetron.

Alcohol drinking in rats injected ICV with 6-OHDA: effect of 8-OHDPAT and tropisetron (ICS 205930).

6-Hydroxydopamine (6-OHDA) was administered ICV to Wistar male rats. Lesioned animals displayed lower preference for ethanol (ETOH) than sham-operated rats. Among 6-OHDA lesioned rats only 9% became high-preferring whereas 20% of sham-operated animals became high-preferring ones. Both tropisetron (the antagonist of 5-HT3 receptors) and 8-OHDPAT (the 5-HT1A receptor agonist) reduced ETOH drinking in high-preferring rats. However, in 6-OHDA lesioned rats the effect of tropisetron was reduced although 8-OHDA retained its effect on ETOH consumption. These results suggest that brain DA neurons are involved in tropisetron action but are not responsible for antipreference effect of 8-OHDPAT.

Alcohol drinking in rats treated with 5,7-dihydroxytryptamine: effect of 8-OH-DPAT and tropisetron (ICS 205-930).

5,7-Dihydroxytryptamine (5,7-DHT) was administered ICV to Wistar male rats. Lesioned rats displayed higher preference for ETOH than sham-lesioned animals. Among 5,7-DHT-pretreated rats 38% became high-preferring, while only 22% of sham-lesioned rats displayed this behavioural pattern (p < 0.05). Both 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; the agonist of serotonin 5-HT1A receptors) and tropisetron (ICS 205-930, the antagonist of 5-HT3 receptors) reduced ETOH consumption in high-preferring, sham-lesioned rats. However, in 5,7-DHT rats the effect of 8-OH-DPAT was completely abolished, while tropisetron retained its antipreference activity. Therefore, it seems that 5-HT1A autoreceptors are critically involved in 8-OH-DPAT action, while 5-HT3 receptor sites responsible for tropisetron action are located beyond the 5-HT system.

Antidepressant treatment and limbic serotonergic mechanisms regulating rat locomotor activity.

The effects of chronic administration of desipramine, citalopram, and electroconvulsive shocks (ECS) on changes in rat motility after intraaccumbens (NAS) injections of selective serotonergic drugs were studied in intact and 5.7-DHT lesioned animals. It was shown that local injections of 8-OHDPAT and DOI-HCl depressed rat locomotor activity. Their effects appeared to be mediated postsynaptically, and could be antagonized by NAN-190 and ritanserin, respectively. Chronic but not acute pretreatment of rats with antidepressants (21 days long; the experiment was performed 24 h after the last dose) as well as repeated ECS (shocks were applied five times every second day), antagonized behavioral depression after 8-OHDPAT and DOI-HCl. The influence of antidepressant treatment was prevented by serotonergic lesions. Chronic administration of antidepressants and ECS did not equivocally affect the levels or metabolism of 5-HT, dopamine, and noradrenaline in the rat limbic forebrain. It is concluded that the present data indicate diminished activity of 5-HT systems related to the 5-HT1A and 5-HT2 receptors in the limbic nucleus, after chronic antidepressant treatment. This effect of drugs and ECS concerns nervous processes linked with the function of postsynaptically localized 5-HT receptor subtypes, and it probably depends on intact presynaptic 5-HT innervation.