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.

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 physostigmine and cholinergic receptor ligands on novelty-induced neophobia.

The aim of the study was to analyse in a well-established model of neophobia the effects of peripheral and central (ICV) administration of a prototypical and easily penetrating to the brain acetylcholinesterase inhibitor (AChE-I)--physostigmine, hemicholinium, a selective blocker of the high affinity choline uptake sites, as well as muscarinic and nicotinic receptor ligands. Thus, an attempt was made to address the question whether anxiolytic-like effects of AChE-I, reported in the clinic, are directly related to the anti-emotional action. The effects of peripherally and centrally administrated cholinergic ligands on novelty-induced decrease in exploratory behaviour were examined in rats. It was found that in a limited dose-range physostigmine and nicotine given peripherally or ICV selectively disinhibited rat exploration in the open field, whereas scopolamine stimulated animal motor activity and increased thigmotaxis. Locomotor effects of physostigmine and nicotine appeared at the higher doses and could be easily separated from their anti-neophobic action. The rat's exploratory behaviour tended to be attenuated by central administration of hemicholinium (a choline uptake blocker), and it was significantly inhibited by mecamylamine (a nicotinic receptor antagonist), and pirenzepine (a selective M1 receptor antagonist). Gallamine, a selective M2 receptor antagonist, did not influence on animal novelty-induced anxiety-related behaviour. It is concluded that AChE-I can selectively affect brain emotional processes evoked by neophobia-related stimuli. Probably both nicotinic and M1 cholinergic receptors mediate such an action of AChE-I.

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.

The role of the hippocampus and 5-HT/GABA interaction in the central effects of benzodiazepine receptor ligands.

The effects of an intrahippocampal administering of a nonselective full (midazolam), a partial benzodiazepine (BDZ) receptor agonist (bretazenil), and a BDZ1 selective (zolpidem) receptor ligand were examined in the open field test (OFT) of neophobia and Vogel's test (VT) of conflict behavior in rats. Moreover, the influence of local injections of a noncompetitive GABA(A) receptor antagonist, picrotoxin, on the anxiolytic-like effect of serotonin (5-HT) depletion (p-chlorophenylalanine, p-CPA) in the Vogel test was studied. It was found that in the OFT only midazolam (0.1 microg/site) given to the hippocampus (HP) disinhibited rat exploratory behavior, whereas all the examined compounds inhibited animal motor activity when injected locally at 10.0 microg/site, the highest dose used in the tests. In the VT, again, only midazolam disinhibited rat conflict behavior on a dose-dependent basis. Picrotoxin administered to the HP produced a tendency to increase locomotor activity in rats, and significantly attenuated the anti-conflict action of serotonin depletion without changing the pain threshold and spontaneous drinking of the animals. p-CPA induced potent, dose-dependent and selective 5-HT and 5-hydroxyindoleacetic acid decrease in the HP after administering the dose used in the behavioral experiment. Thus, the present data provide evidence for the lack of selective anxiolytic activity of a partial non-selective agonist and a full selective agonist at the BDZ1 receptor after their administration to the HP. The model of intra-HP drug injections appeared effective in discriminating the anxiolytic spectrum of activity of new psychotropic compounds. Moreover, the obtained results indicate that the dorsal HP is one of the central sites important for GABA/5-HT interaction that modulates rat emotional behavior.

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.

Some behavioral effects of AMPA/kainate receptor agonist and antagonists.

The effects of an agonist (S-AMPA, i.c.v.), as well as competitive (CNQX, NBQX, DNQX, i.p.), and noncompetitive antagonists (GYKI 52466, i.p.) at the AMPA/kainate receptors were examined in the open field and the Vogel tests of anxiety. It was found that both kinds of antagonists inhibited rat exploratory behavior in a dose-dependent manner, at the dose range exhibiting a clear-cut tendency to decrease rat locomotor activity. They appeared inactive in the Vogel test over an examined dose-range. S-AMPA, whereas not changing in a significant way rat behavior in the open field, significantly enhanced the suppressive influence of a shock on drinking in the Vogel test. The drug administered at the dose of 2 micrograms/5 microliters, i.c.v., revealed also a tendency to decrease the motor activity followed by prodromal symptoms of epileptic-like activity in some subjects. It is concluded that AMPA/kainate receptors probably are not directly involved in the control of rat emotional behavior. Thus, their primary role as putative neuroprotective and anticonvulsant agents is indirectly confirmed.

Combined perfusion and percolation of embalmed animal bodies for removing formaldehyde.

Excess formaldehyde was extracted from embalmed animal cadavers by vascular perfusion or combined perfusion and percolation with 20% ethanol. The perfusion was undertaken through a carotid artery and the percolation through the serous body cavities for 1-2 h. Nineteen goats were perfused once and one goat and three cows were perfused and percolated twice a day for 3 days. The extracted cadavers were stored in 20% ethanol or in a freezer at -20 degrees C. The mean value of the atmospheric concentration of formaldehyde after extraction was 0.27 +/- 0.05 mg/m3 for goats and 0.39 +/- 0.12 mg/m3 for cows.