Disrupted visceral feedback reduces locomotor activity and influences background contextual fear conditioning in C57BL/6JOlaHsd mice.

The present experiments were designed to study fear conditioning as an emotional learning task with disrupted visceral feedback. For that purpose we used the peripherally acting beta1-adrenoceptor blocker atenolol and studied its effects on the behavior of male C57BL/6JOlaHsd mice in an exploration-related test and during fear-conditioning. In the first experiment, we treated mice with saline or different doses of the beta1-adrenergic blocker atenolol (5mg/kg and 20mg/kg body weight i.p.) 30 min before behavioral testing in a motility box. Only the high but not the low dose of atenolol led to a reduction of locomotor activity (p<0.02). Factors known to be related to emotionality (rearing, area preference) were unaffected. In a second experiment, saline- and atenolol-treated mice (same dosages and mode of application) were trained for auditory fear conditioning, and 24h later they were retested in the same environment. We found differences between the effects of atenolol upon contextual- and cue-fear conditioning. Animals treated with 20mg/kg BW doses of atenolol showed significantly decreased background contextual fear compared to saline-treated control animals. In contrast, no differences were found during CS presentation in the conditioning context between atenolol-treated animals and saline-treated controls, independent from a paired or an unpaired conditioning paradigm. Thus, the blockade of peripheral beta1-adrenoceptors by atenolol may have disrupted the positive feedback to the central nervous system via visceral afferents resulting in a decreased locomotor activity and background contextual fear.

Disruption of latent inhibition in rats with postnatal hippocampal lesions.

Disruption of latent inhibition has been proposed as a possible model of cognitive abnormalities that underlie positive symptoms of a schizophrenia. We tested neonatal hippocampal lesioned rats in a latent inhibition paradigm. Lesions of the ventral hippocampus were induced by bilateral injections of ibotenic acid in 7 days old rats. The behavior of lesioned rats was tested postpubertally. We found a hyperresponsiveness to dopaminergic stimulation by apomorphine in locomotion tests. Latent inhibition was tested using the acquisition of a conditioned reaction in a two-way shuttle box. Sham operated control animals showed after preexposure of the to-be-conditioned stimulus (combined tone and light stimulus) a low acquisition. Ibotenic acid lesioned animals learned the conditioned reaction with and without preexposure in the same way, indicating disturbed latent inhibition. These results demonstrate disturbances in early postnatal hippocampal lesioned rats comparable with those seen in schizophrenic patients, thus further validating this procedure as a useful animal model of some aspects of schizophrenia.

Threshold to elicit seizures by picrotoxin is lowered in pentylenetetrazol-kindled mice.

The convulsant effect of different doses of picrotoxin was investigated in pentylenetetrazol-kindled and saline-treated mice. The percentage of convulsions was greater in the kindled mice, compared to controls.

Effects of nicardipine, an antagonist of L-type voltage-dependent calcium channels, on kindling development, kindling-induced learning deficits and hippocampal potentiation phenomena.

Kindling is considered to be a useful experimental model for investigating drug effects on the convulsive component of epilepsy and related alterations at the behavioural level. It was demonstrated that pentylenetetrazol (PTZ)-kindled rats show diminished learning performance in shuttle-box training. We used this model to study the influence of nicardipine, an antagonist of L-type voltage-dependent calcium channels, on kindling seizure development as well as related learning impairments. Additionally, we tested the influence of nicardipine on kindling-induced potentiation, a special form of long-term enhancement of evoked potentials in the dentate gyrus after kindling. Therefore, monosynaptic evoked field potentials in the dentate area upon test stimuli to the perforant pathway were recorded in freely moving kindled and control rats at different times after injection of PTZ. The results indicate that the blockade of L-type voltage-dependent Ca2+-channels during the kindling procedure attenuates PTZ-kindling, antagonizes a kindling-induced learning deficit in an active avoidance test and decreases a novel form of kindling-related potentiation, the long-lasting amplitude enhancement of the monosynaptic evoked field potential in the dentate gyrus after injection of a small test dose of PTZ. This potentiation can also be prevented in kindled animals by nicardipine injection in an acute experiment.

Repeated application of ketamine to rats induces changes in the hippocampal expression of parvalbumin, neuronal nitric oxide synthase and cFOS similar to those found in human schizophrenia.

Treatment with the phencyclidine derivative ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a well known anesthetic, has recently been introduced to mimic schizophrenia in animals. Using rats repeatedly treated with sub-anesthetic doses we demonstrate in the hippocampal formation the cellular distribution patterns of proteins being relevant to the pathogenesis of schizophrenia. Compared with controls an increase in the density of reduced nicotinamide adenine dinucleotide phosphate diaphorase-, neuronal nitric oxide synthase- and cFOS-positive hippocampal interneurons was found, whereas the density of parvalbumin expressing cells was decreased. Our experiments show that repeated injections of sub-anesthetic doses of ketamine induce significant changes in the nitrergic and GABAergic system which, in part, resemble those described in postmortem brains of human schizophrenics indicating that sub-chronic treatment with sub-anesthetic doses of ketamine might be a useful animal model to study schizophrenia.

Influence of olfactory bulbectomy and subsequent imipramine treatment on 5-hydroxytryptaminergic presynapses in the rat frontal cortex: behavioural correlates.

1. Alterations of 5-hydroxytryptaminergic mechanisms are thought to play a special role in the pathogenesis of depression and antidepressant treatments are assumed to restore these changes. 2. We have used one of the most reliable models of depression, the olfactory bulbectomized rat to study the long term consequences of this manipulation and of subchronic imipramine treatment on two parameters of 5-hydroxytryptaminergic presynapses, 5-hydroxytryptamine (5-HT) transporter density and tryptophan hydroxylase apoenzyme concentration, in the frontal cortex as well as on active avoidance learning several weeks after bulbectomy. 3. The Bmax value of [3H]-paroxetine binding and the concentration of the 5-HT synthesizing enzyme were both significantly elevated in the frontal cortex of bulbectomized rats compared to sham-operated controls. 4. Imipramine treatment, either by daily injections or by subcutaneous implantation of slow release imipramine-containing polymers reduced the elevated tryptophan hydroxylase apoenzyme levels in the frontal cortex of bulbectomized, but not of sham-operated control rats and restored the deficient learning performance of bulbectomized rats. 5. Both effects were more pronounced after continuous drug administration by imipramine-releasing polymers compared to daily i.p. injections. 6. These findings indicate that bulbectomy leads to a compensatory 5-hydroxytryptaminergic hyperinnervation of the frontal cortex. Chronic antidepressant treatment seems to attenuate the increased output of the 5-hydroxytryptaminergic projections in the frontal cortex through the destabilization of the rate limiting enzyme of 5-HT synthesis of the 5-hydroxytryptaminergic nerve endings in this brain region.

Identification of brain regions that are markedly activated by morphine in tolerant but not in naive rats.

The induction of c-fos mRNA in rat brain due to morphine treatment was analyzed by in situ hybridization. A single dose of up to 100 mg/kg given to naive rats elicited only a weak c-fos expression. However, rats that were repeatedly pretreated with morphine displayed a marked c-fos induction in a few brain areas in response to morphine application. These brain areas essentially comprised the dorsal striatum, the shell of the nucleus accumbens, and some cortical areas. The c-fos signal was transient and not due to a residual withdrawal. Naloxone-precipitated withdrawal led to a more intense c-fos expression which also encompassed a greater range of brain areas. A similar but weaker pattern was observed in case of spontaneous withdrawal. A low morphine dose suppressed the c-fos expression nearly completely and was not sufficient to elicit the morphine-like expression pattern of c-fos. The brain areas which responded strongly to withdrawal included the piriform cortex, septal and hypothalamic nuclei and parts of the thalamus. Taken together, our data indicate that in certain circumscribed brain areas including the dorsal striatum and the shell of the nucleus accumbens, a sensitization towards morphine takes place at the molecular level. These areas responded to morphine with an elevated c-fos expression only when morphine was repeatedly given previously. Sensitization processes are thought to be important for opiate dependence, in particular for the increased craving for the drug. Furthermore, our data indicate that in case of repeated application signs of withdrawal appear after each morphine dose at the molecular level. Repeated events of withdrawal were also implicated in the establishment of a drug dependence state.

Alterations of the dopaminergic and glutamatergic neurotransmission in adult rats with postnatal ibotenic acid hippocampal lesion.

In 6-week and 8-week-old rats (pre- and post-pubertally) with neonatal excitotoxic lesions of the ventral hippocampus with ibotenic acid (IBO), we have studied apomorphine-induced motor activity and glutamate and dopamine D1 and D2 binding sites in the hippocampus, striatum, nc. accumbens and frontal cortex as well as K+ -stimulated (3H)-D-aspartate release from hippocampal and frontal cortical slices. Specific glutamate binding was enhanced in the frontal cortex of 8-week-old IBO-treated animals, whereas that in other brain regions remained unchanged. Both D1 and D2 binding sites were downregulated in the striatum without changes in other brain structures. In 6-week-old rats, neither the glutamate nor the dopamine binding sites were altered. The amino acid release from hippocampal and frontal cortical slices of adult IBO treated rats was significantly decreased in comparison to controls, whereas in 6-week-old rats, no significant alterations were detectable. The additionally monitored motor activity was enhanced only in adult IBO-lesioned rats after apomorphine pretreatment. The present data are in agreement with the hypothesis of hyperactive dopamine and hypoactive glutamate systems in schizophrenia and are discussed in the light of schizophrenia-like behavioral changes in rats after postnatal hippocampal IBO lesion.

Social behaviour in rats lesioned with ibotenic acid in the hippocampus: quantitative and qualitative analysis.

RATIONALE: Neonatal ibotenic acid lesion of the ventral hippocampus was proposed as a relevant animal model of schizophrenia reflecting positive as well as negative symptoms of this disease. Before and after reaching maturity, specific alterations in the animals' social behaviour were found. OBJECTIVE: In this study, social behaviour of ventral hippocampal lesioned rats was analysed. For comparison, rats lesioned either in the ventral hippocampus or the dorsal hippocampus at the age of 8 weeks were tested. METHODS: Rats on day 7 of age were lesioned with ibotenic acid in the ventral hippocampus and social behaviour was tested at the age of 13 weeks. For comparison, adult 8-week-old rats were lesioned either in the ventral or the dorsal hippocampus. Their social behaviour was tested at the age of 18 weeks. RESULTS: It was found that neonatal lesion resulted in significantly decreased time spent in social interaction and an enhanced level of aggressive behaviour. This shift is not due to anxiety because we could not find differences between control rats and lesioned rats in the elevated plus-maze. Lesion in the ventral and dorsal hippocampus, respectively, in 8-week-old rats did not affect social behaviour. CONCLUSIONS: The results of our study indicate that ibotenic acid-induced hippocampal damage per se is not related to the shift in social behaviour. We favour the hypothesis that these changes are due to lesion-induced impairments in neurodevelopmental processes at an early stage of ontogenesis.

Enhanced development of morphine tolerance in rats treated with 2-deoxy-D-galactose.

Rats treated subcutaneously for 6 days with morphine developed a weak tolerance which was characterized by a decrease in the analgesic action of the opioid. Under those experimental conditions a simultaneous intracerebroventricular application of 2-deoxy-D-galactose enhanced development of morphine tolerance, while other deoxy-sugars like 2-deoxy-D-glucose and 6-deoxy-D-galactose were ineffective. In contrast, no influence of 2-deoxy-D-galactose on a more enhanced morphine tolerance after a 11-day pretreatment with morphine was found. The results are discussed in the light of a rather specific interference of 2-deoxy-D-galactose with neuronal glycoprotein processing and related cellular mechanism underlying adaptive processes involved in the development of morphine tolerance.

Cellular changes in rat brain areas associated with neonatal hippocampal damage.

The neonatal destruction of the ventral hippocampus was introduced as a model to recreate features of schizophrenia in the rat. While behavioral consequences of this intervention have been studied in detail, less is known about the cellular processes underlying the deviant behavior. We studied in rats (neonatally or adult lesioned, controls) brain areas receiving or not receiving hippocampal projections. The number of neurons and the expression of the cell markers L-ornithine decarboxylase, nitric oxide synthase/NADPH diaphorase, calretinin and GFAP were estimated. Reduced numbers of neurons and increased immunostaining for ornithine decarboxylase and nitric oxide synthase in the prefrontal, perirhinal and entorhinal cortex of neonatally but not adult lesioned rats or controls demonstrate persistent cellular changes after ventral hippocampus damage.

dTyr-D-Phe3 (Pro-D-Phe-Pro-Gly) interacts specifically with amygdaloid-kindled seizures and is capable of preventing the learning deficit occurring after kindling.

In amygdala-kindled rats a deficit in learning brightness discrimination was found. We concluded that this result provides a reliable basis for creating an animal model of cognitive dysfunctions in epileptics. Recently, a des-tyrosine D-amino acid substituted derivative of bovine beta-casein(1-5) was reported to exhibit anticonvulsant as well as antidepressant activity. Therefore, we tested the effect of this peptide on kindled seizures and the learning deficit after kindling. It was found that the peptide suppressed the duration of seizures whereas seizure severity was not influenced. Furthermore, the learning performance of peptide-treated rats was significantly higher than that of kindled controls.

Influence of beta-casomorphin derivatives on chemically and electrically induced seizures.

Derivatives of beta-casomorphin(1-5) at a dose level of 1 mumol/kg body weight were tested for their influence on pentylenetetrazol, picrotoxin, or electrically induced seizures after subcutaneous injection in mice. Tyr-Pro-Phe-D-Pro-Gly was found to facilitate pentylenetetrazol-evoked seizures, whereas desTyr derivatives Pro-Phe-D-Pro-Gly and Pro-Phe-Pyr exhibited anticonvulsant properties against those convulsions. The tripeptide was effective only 10 min after application. The beta-casomorphin derivative Pro-D-Phe-Pro-Gly was effective against electrically induced seizures. The protective action of this tetrapeptide lasted for about 5 h. Additionally, we tested the influence of orally administered Pro-Phe-D-Pro-Gly on pentylenetetrazol-induced seizures and Pro-D-Phe-Pro-Gly on electrically induced seizures. Both peptides were effective at a dose of 5 mumol/kg body weight.

The effect of acutely administered beta-casomorphin derivatives on pentylenetetrazol-kindled mice.

Some of the Tyr-containing and desTyr beta-casomorphin derivatives were tested for their anticonvulsant action in pentylenetatrazol-kindled mice. It was demonstrated that some of these substances exert powerful action against kindled seizures, suggesting therapeutical usefulness.

Influence of modified casomorphins on yawning behavior of rats.

Apomorphine-induced yawning was completely suppressed in animals treated with 5 nmol [D-Pro4]casomorphin (CM) (ICV), 10 nmol [D-Phe3]CM (ICV) or 10 nmol [D-Pip4]CM (ICV). The apomorphine-induced yawning was also decreased, by des-Tyr analogs, but only by about 50%. Physostigmine (0.15 mg/kg, IP) induced yawning. The physostigmine-induced yawning was suppressed by 5 nmol [D-Pro4]CM and 10 nmol [D-Phe3]CM. Both [des-Tyr-D-Phe3]CM and [des-Tyr-D-Pip4]CM were without effect, whereas [des-Tyr-D-Pro4]CM increased significantly the physostigmine-induced yawning. The results suggest that dopaminergic transmission can be modulated by beta-casomorphin derivatives, thus resulting in a decrease in yawning. In the case of the des-tyrosine derivatives, we can assume a dopaminergic modulation, too. An increase in serotonergic activity might be supposed for [des-Tyr-D-Pro4]CM.

The anticonvulsive effect of BCH 325 is age dependent.

The two different experimental approaches which were applied to study the anticonvulsive effectiveness of BCH 325, a des-tyrosine derivative of bovine beta-casomorphin-(5), in immature (22-day-old) and mature (7-week-old) female rats revealed that the peptide was able to protect mature females from electrically induced seizures and that it had no effect on pentylenetetrazol-induced convulsions. As opposed to this, immature animals were protected against chemically induced seizures but no effect was found using electrically induced seizures.

Effects of the peptide BCH-325 upon the efficacy of common antiepileptic drugs.

It was shown that BCH-325 (Pro-D-Phe-Pro-Gly), a des-tyrosine derivative of beta-casomorphin, exhibits anticonvulsive effects. In the present study, we combined the peptide with clinically used antiepileptic drugs (phenytoine, carbamazepine, diazepam, phenobarbital, and dipropyl acetate) to investigate possible interactions and, moreover, to draw conclusions concerning the mode of action of BCH-325. There were no interactions with phenytoine and carbamazepine, which might be interpreted to mean that BCH-325 exerted no action on sodium channels. The efficacy of the combinations with the other drugs (diazepam, phenobarbital, dipropyl acetate) was increased. The data suggest that the anticonvulsant effect of BCH-325 might occur by interference with elements of GABA/benzodiazepinergic neurotransmission.

Phe1-substituted beta-casomorphin-5 analogues with analgesic activity.

The antinociceptive potency of linear and cyclic beta-casomorphin-5 (CM-5) analogues, modified in position 1 by substitution of the tyrosine (Tyr) by the phenylalanine (Phe) residue, was studied using the vocalization test. With the exception of the linear [Phe1,D-Orn2]CM-5, the Phe1-substituted linear and cyclic casomorphin analogues exhibit remarkable analgesic potency compared to morphine, although the opioid receptor affinity and the opioid activity in vitro is diminished compared to the corresponding Tyr-containing analogues. The analgesic effect of the compounds is mediated by activation of opioid receptors, because it can be antagonized with naloxone. Furthermore, it was demonstrated that the [Phe1,D-Orn2,D-Pro4]CM-5, which was about sixfold more potent than morphine, developed cross-tolerance to morphine.

Linear and cyclic beta-casomorphin analogues with high analgesic activity.

We investigated the antinociceptive efficacy of casomorphin (CM) derivatives using the vocalization test. Male Wistar rats received chronic microcannulae into the right lateral ventricle. One week later we examined the analgesic effect of CM derivatives 10, 30, 60, and 90 min after intraventricular injection (5 microliters). The analgesic effect was calculated as the individual percent increase in the pain threshold and was compared to controls (saline treatment). The substitution of D-lysine and D-ornithine in position 2 in connection with a cyclization through ring closure of the 2 position side chain amino group to the C-terminal glycine-COOH group resulted in high analgesic potency. The substitution of D-Pro4 was without any effect in the ineffective linear derivatives and decreased the effectiveness in the highly effective cyclic derivatives. The cyclic [D-Orn2]CM-5 and the cyclic [D-Lys2]CM-5 are the CM derivatives with the highest antinociceptive activity. The cyclic [D-Orn2]CM-5 is greater than 1000 times more effective than morphine. We conclude, on the basis of studies of receptor binding and in vitro investigations, that mu receptor activity alone is not responsible for the analgesic activity. The delta receptor and possibly also the kappa receptor could modulate the nociceptive effectiveness.

Social memory is impaired in neonatally ibotenic acid lesioned rats.

Postnatal lesion with ibotenic acid in the ventral hippocampus produces several behavioural effects that resemble the symptoms of schizophrenia. In the present study, we tested neonatally lesioned 1-year-old Sprague-Dawley rats for their social memory. It was found that social memory is worsened in lesioned rats. Subchronic treatment with haloperidol (0.025 mg/kg body weight) partly ameliorated this impairment. It was suggested that social memory might be a useful paradigm to test clinically used and potential antipsychotic drugs for their effects on learning and memory processes.