Brain GABA and glutamate levels in workers of two ant species (Hymenoptera: Formicidae): interspecific differences and effects of queen presence/absence.

Presence of amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate (Glu) in ant brains was reported in very few studies. To learn more about factors influencing GABA and Glu levels in ant brains, we applied high-performance liquid chromatography to measure levels of these compounds in single brains of workers of 2 ant species, Myrmica ruginodis (subfamily Myrmicinae) and Formica polyctena (subfamily Formicinae) taken from queenright/queenless colony fragments and tested in dyadic aggression tests consisting of an encounter with a nestmate, an alien conspecific or a small cricket. Brain glutamate levels were higher than those of GABA in both tested species. Brain GABA levels (in µmol/brain) and GABA : Glu ratio were higher in M. ruginodis (a submissive species) than in F. polyctena (a dominant, aggressive species) in spite of smaller brain weight of M. ruginodis. Brain glutamate levels (in µmol/brain) did not differ between the tested species, which implies that glutamate concentration (in µmol/mg of brain tissue) was higher in M. ruginodis. Queen absence was associated with increased worker brain GABA levels in F. polyctena, but not in M. ruginodis. No significant effects of opponent type were discovered. As GABA agonists enhance friendly social behavior in rodents, we hypothesize that elevated brain GABA levels of orphaned workers of F. polyctena facilitate the adoption of a new queen. This is the first report providing information on GABA and glutamate levels in single ant brains and documenting the effects of queen presence/absence on brain levels of amino acid neurotransmitters in workers of social Hymenoptera.

Pharmacological characteristics of zolpidem-induced catalepsy in the rat.

Zolpidem is a non-benzodiazepine hypnotic drug acting preferentially at α1-containing GABAA receptors expressed in various parts of the brain, including the basal ganglia. The aim of the present study was to provide preliminary characteristics of zolpidem-induced catalepsy in Wistar rats. Zolpidem (2.5-10.0mg/kg), but not diazepam and midazolam, produced dose-dependent cataleptic responses in the bar test, which were similar to those produced by a reference antipsychotic drug, haloperidol. Zolpidem-induced catalepsy was abolished by a benzodiazepine site antagonist, flumazenil (5.0mg/kg), D2/3 receptor agonist, quinpirole (1.0mg/kg), and a non-competitive NMDA receptor antagonist, MK-801 (0.1mg/kg), but not by a non-selective opioid receptor antagonist, naltrexone (3.0mg/kg). The present results indicate that systemic injections of zolpidem may produce short-lasting, neuroleptic-like catalepsy in the rat.

The effects of serotonin, dopamine, octopamine and tyramine on behavior of workers of the ant Formica polyctena during dyadic aggression tests.

We investigated the effect of injections of four biogenic amines (serotonin, dopamine, octopamine and tyramine) on behavior patterns displayed by workers of the red wood ant Formica polyctena during dyadic confrontations with four types of opponents: a nestmate, an alien conspecific, an allospecific ant (Formica fusca), and a potential prey, a nymph of the house cricket (Acheta domesticus). Significant effects of biogenic amine administration were observed almost exclusively in the case of confrontations with allospecific opponents. Serotonin treatment exerted stimulatory effects on behavior patterns involving physical aggression (biting accompanied by gaster flexing, dragging and formic acid spraying), but these effects were relatively weak and/or documented by indirect evidence. Dopamine administration exerted a stimulatory effect on open-mandible threats directed by F. polyctena to F. fusca and to cricket nymphs, and on biting behavior directed to cricket nymphs. Surprisingly, octopamine treatment did not exert significant effects on aggressive behavior of the tested ants. Tyramine administration exerted a suppressing effect on threatening behavior directed to F. fusca, but led to shortening of the latencies to the first open-mandible threat during the tests with cricket nymphs. Biogenic amine administration also influenced non-aggressive behavior of the tested ants. Our findings confirmed the role of serotonin and dopamine in the mediation of ant aggressive behavior and documented for the first time significant effects of tyramine treatment on ant aggressive behavior. We also demonstrated that not only specific patterns of ant aggressive behavior, but also behavioral effects of biogenic amine treatments are as a rule strongly context-dependent.

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

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

Cocaine self-administration in Warsaw alcohol high-preferring (WHP) and Warsaw alcohol low-preferring (WLP) rats.

Individuals prone to drug self-administration may be vulnerable not only to a single drug reinforcer but to a variety of drug reinforcers. It has been shown that two thirds of alcoholics regularly use drugs other than ethanol (alcohol). Up to 30% of alcohol-dependent patients report concurrent misuse of cocaine. The aim of the present study was to investigate intravenous cocaine self-administration in selectively bred, alcohol-preferring WHP (Warsaw high-preferring) and non-preferring WLP (Warsaw low-preferring) rats. It was hypothesized that WHPs could be more prone to cocaine self-administration in comparison to WLPs. Rats from both lines were allowed to nose-poke for cocaine infusions (0.33 mg/kg/infusion) under the FR-1, FR-2, and FR-3 schedule of reinforcement. Dose-response curves were assessed with increasing doses of cocaine (0.03, 0.1, 0.33, 1.0mg/kg/infusion). The WHP and WLP rats did not differ in cocaine self-administration. Both groups quickly acquired nose-poke responding for cocaine, presented a similar response profile when the schedule of reinforcement was increased from FR-1 to FR-3, and similar sensitivity to cocaine in the dose-response test. The present results may indicate that the selective breeding of alcohol-preferring WHP and alcohol non-preferring WLP rats did not lead to differences in cocaine's rewarding effects as assessed in the self-administration procedure.

The influence of neonatal serotonin depletion on emotional and exploratory behaviours in rats.

Recent studies have shown that neurodevelopmental disturbances in the structure and function of the brain are significant factors in the onset of psychiatric disorders. Such deficits may also affect neurotransmission. Among the different neurotransmitter systems, serotonin (5-HT) plays an important role in the organisation and maturation of brain structures during development. The aim of the present study was to examine the influence of neonatal 5-HT depletion on emotional and exploratory behaviours in adult rats. Three-day-old Wistar male rats received intraventricular injections of the selective serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Littermates given saline injections acted as controls. After three months, rat behaviour was analysed in an open field test, a social interaction test and a novel object test. Moreover, contextual-conditioned freezing and ultravocalisation fear responses were studied. The pain reactivity was measured in a flinch-jump test. Biochemical analysis of 5,7-DHT-treated rats revealed a significant decrease in the concentration of 5-HT and its metabolite in the frontal cortex, hippocampus and striatum, with a decreased dopamine level in striatum. Early serotonin depletion reduced locomotor activity in the open field test and attenuated social interaction in non-aversive conditions and exploration of a novel object in adult rats. Ultravocalisation, but not freezing, was increased in the contextual fear-conditioning paradigm in 5-HT-depleted rats. There was no difference in the pain threshold between groups. These data demonstrate that neonatal 5-HT depletion resulted in subtle alterations in the locomotor, exploratory and conditioned fear response of adult animals.

Neonatal serotonin (5-HT) depletion does not disrupt prepulse inhibition of the startle response in rats.

The neurodevelopmental hypothesis of many brain disorders is based on the notion that environmental factors have significant effects on brain maturation. Because serotonin (5-HT) dysfunction in development may be involved in disease etiology, the present investigation assessed the effects of neonatal 5-HT depletion on prepulse inhibition of the startle response (PPI) in rats. Three-day-old Sprague-Dawley rats were pretreated with desipramine (20 mg/kg), followed by an intraventricular injection of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, 70 µg dissolved in 2 µl of 0.1% saline solution in ascorbic acid) on each side. Three months later, the rats' PPI was tested. Despite a severe and permanent decrease (80-100%) in hippocampal, prefrontal and striatal 5-HT levels, treatment with 5,7-DHT caused no disruption of PPI. In contrast to this lack of effect, the 5,7-DHT treatment increased basal startle activity, as measured in response to a 120 dB stimulus. Thus, our results clearly indicate that neonatal 5-HT depletion does not interrupt prepulse inhibition in rats. Studies involving lesions of brain structures or chemical systems run the risk of inducing compensatory changes in brain function, resulting in an amelioration of any deficit. The development of such compensatory mechanisms seems likely in the current study, due to the severe and long-lasting effect of neonatal 5,7-DHT-induced reduction on 5-HT levels.

Diverse behavioral, monoaminergic and Fos protein responses to opioids in Warsaw high-alcohol preferring and Warsaw low-alcohol preferring rats.

Predisposition to addictions is presumably related to a dysfunction of the brain reward system, which can be 'compensated' by the intake of different psychoactive drugs. Hence, animals showing propensity for developing dependence to a specific drug class may also be useful for modeling other addictions. We compared the effects of repeated (14 daily doses) morphine (10 mg/kg) or methadone (2 mg/kg) treatment followed by a 2-week withdrawal and a morphine challenge (5 mg/kg) on locomotor activity, brain Fos expression and selected brain regional levels of dopamine, serotonin and their metabolites in the 38th generations of selectively bred Warsaw low-alcohol-preferring (WLP) and Warsaw high-alcohol-preferring (WHP) rat lines. The rats were given the opioids during the active (i.e. dark) phase of their daily cycle. Drug-naïve WHP rats compared to their WLP counterparts showed higher locomotor activity in an open field test and higher propensity for lasting behavioral sensitization to morphine. Morphine did not significantly enhance, but suppressed Fos expression in certain brain regions of drug-naïve WLP and WHP rats. Fos expression revealed considerable differences in the responses of WLP and WHP rats to morphine challenge, particularly after methadone pretreatment. These differences were associated with differences in monoamine metabolite levels that were suggestive of elevated basal ganglia and lowered frontal cortical dopamine function, and of lowered somatosensory cortex serotonin function, in the morphine-challenged WHP rats (irrespective of the pretreatment type). Hence, the WLP/WHP line pair may be useful for the search of factors that underlie the propensity for developing opiate dependence.

Stress-opioid interactions: a comparison of morphine and methadone.

The utility of methadone and morphine for analgesia and of methadone for substitution therapy for heroin addiction is a consequence of these drugs acting as opioid receptor agonists.We compared the cataleptogenic and antinociceptive effects of single subcutaneous doses of methadone hydrochloride (1-4 mg/kg) and morphine sulfate (2.5-10 mg/kg) using catalepsy and hot-plate tests, and examined the effects of the highest doses of the drugs on Fos protein expression in selected brain regions in male Sprague-Dawley rats. Methadone had greater cataleptogenic and analgesic potency than morphine. Fos immunohistochemistry revealed substantial effects on the Fos response of both the stress induced by the experimental procedures and of the drug exposure itself. There were three response patterns identified: 1) drug exposure, but not stress, significantly elevated Fos-positive cell counts in the caudate-putamen; 2) stress alone and stress combined with drug exposure similarly elevated Fos-positive cell counts in the nucleus accumbens and cingulate cortex; and 3) methadone and morphine (to a lesser extent) counteracted the stimulatory effect of nonpharmacological stressors on Fos protein expression in the somatosensory cortex barrel field, and Fos-positive cell counts in this region correlated negatively with both the duration of catalepsy and the latency time in the hot-plate test. The overlap between brain regions reacting to nonpharmacological stressors and those responding to exogenous opioids suggests that stress contributes to opioid-induced neuronal activation.

Post-session injections of a protein synthesis inhibitor, cycloheximide do not alter saccharin self-administration.

A large body of evidence indicates that reactivation of aversive memories leads to protein synthesis-dependent memory reconsolidation which can be disrupted by cycloheximide (CHX) and other protein synthesis inhibitors. The aim of the present study was to investigate whether CHX would alter maintenance of well-trained instrumental responding for 0.1% saccharin. Male Wistar rats were trained to lever press for saccharin. When lever pressing stabilized, experimental self-administration sessions with CHX (3 mg/kg, s.c.) started. The animals received four experimental sessions, with each session separated by 5 days. The protein synthesis inhibitor was injected immediately after the experimental sessions 1-3. Repeated post-session injections of CHX did not alter saccharin self-administration. A two-bottle choice test conducted after the last experimental session revealed that CHX had not induced any conditioned taste aversion to 0.1% saccharin. The present results suggest that well-consolidated long-term memory of an appetitive instrumental task does not depend on de novo protein synthesis.

Effects of cycloheximide on extinction in an appetitively motivated operant conditioning task depend on re-exposure duration.

Little is known about the role of new protein synthesis in extinction of operant responding for natural and chemical reinforcers. In the present study, the authors investigated whether the effects of a protein synthesis inhibitor, cycloheximide (CHX) on extinction of operant responding for sweet reward depended on the duration of re-exposure sessions. In addition, the authors investigated whether the effects of CHX on extinction could generalize to relapse of saccharin seeking induced by discrete cues. CHX injected after short re-exposure sessions (5min) accelerated extinction of non-reinforced responding. In contrast, the drug injected after long re-exposure sessions (30min) partially inhibited extinction. Reinstatement of saccharin seeking induced by the saccharin-paired discrete cues was not altered by the previous treatment with CHX. Concluding, the results of the present study indicate that: (i) the protein synthesis inhibitor, CHX can alter extinction of operant responding for sweet reward in rats; (ii) the effects of CHX on extinction critically depend on the duration of re-exposure/extinction sessions and do not generalize to relapse of saccharin seeking induced by discrete cues.

The effect of cannabinoid CB(1) receptor antagonist rimonabant (SR-141716) on ethanol drinking in high-preferring rats.

The present study investigated the effect of the cannabinoid CB(1) receptor antagonist, rimonabant (SR-141716) on ethanol intake in selectively bred alcohol-preferring Warsaw High-Preferring rats. Ethanol (10% vol/vol) and food were available in daily 4-h limited access period while water was available ad libitum. The administration (i.p.) of single 2.5 and 5.0-mg/kg doses of rimonabant preferentially reduced ethanol intake, whereas a 10 mg/kg dose of rimonabant similarly reduced both ethanol and food intake. Our result extends the suppressive effect of cannabinoid CB(1) receptor antagonist to the ethanol drinking behavior in Warsaw High-Preferring line of rats. The result also supports a growing body of literature indicating that the cannabinoid CB(1) receptor is involved in motivational and appetitive properties of ethanol.

Warsaw high-preferring (WHP) and Warsaw low-preferring (WLP) lines of rats selectively bred for high and low voluntary ethanol intake: preliminary phenotypic characterization.

The Warsaw High Preferring (WHP) and Warsaw Low Preferring (WLP) lines were bred from Wistar foundation stock to obtain lines of rats that differ in their preference for ethanol solutions. The WHP line has met several major criteria for an animal model of alcoholism. The WHP rats voluntarily drink excessive amounts of ethanol while the WLP rats consume negligible amounts of ethanol. The WHP rats attain physiologically active blood ethanol concentrations with chronic free-choice drinking. They also develop subtle but visible signs of physical dependence (the withdrawal signs). The patterns of ethanol consumption in WHP and WLP lines are stable in time and independent of the manner of access to ethanol solutions. Notably, when exposed to the increasing ethanol concentrations WHP rats gradually increased total ethanol intake whereas the WLP rats consumed invariably very low amounts of ethanol. Furthermore, the WHP rats show an increased responsiveness to the stimulatory effects of low dose of ethanol.

Alcohol relapse induced by discrete cues activates components of AP-1 transcription factor and ERK pathway in the rat basolateral and central amygdala.

Alcohol-related cues may induce relapse to heavy alcohol drinking and promote molecular adaptations in discrete brain regions. An exact nature of these molecular alterations is still unknown. In the present study, rats trained to self-administer ethanol were tested for cue-induced reinstatement of ethanol seeking after 30 days of abstinence. Next, a detailed immunocytochemical analysis of c-Fos activation was performed within seven nuclei of the amygdala. In the second experiment, c-Fos activation after reinstatement of ethanol seeking induced by discrete cues was compared with the activation pattern of its putative partner (c-Jun) and regulators (extracellular signal-regulated kinases and c-Jun N-terminal kinases). Reexposure to ethanol-associated context cues (an extinction session) potentiated c-Fos expression within the basolateral and central amygdala. Repeated presentation of ethanol-associated discrete cues in an extinction/reinstatement session led to even stronger c-Fos activation in the latter nuclei. In the second experiment, reexposure to the ethanol-associated context and discrete cues activated both c-Jun and extracellular signal-regulated kinases (ERK1/2) in the basolateral amygdala. Our observations suggest that the basolateral and central amygdala may be specifically involved in alcohol-seeking behavior induced by discrete cues.

Active versus passive cocaine administration: differences in the neuroadaptive changes in the brain dopaminergic system.

There is considerable evidence that chronic exposure to cocaine is associated with low striatal dopamine D2 receptor availability. In the present study we wished to determine whether neuroadaptive changes in densities of D2 receptors were due to direct pharmacological actions of cocaine or they reflected motivational states that were present when cocaine injection depended on active drug-seeking behavior and whether these changes were related to the actual expression of D2 mRNA. To achieve this goal we utilized a "yoked" procedure in which rats were tested simultaneously in groups of three, with only one rat actively self-administering cocaine while the other two received yoked injections of either cocaine or saline. Only passively administered cocaine produced a decrease in dopamine D2 receptor levels in the anterior and central regions of caudate/putamen, and both the shell and core of the nucleus accumbens, as measured by in vitro quantitative autoradiography. In contrast, examination of D2 receptor gene expression using in situ hybridization analysis revealed that there was an increase in D2 receptor mRNA levels in the ventral tegmental area of rats actively self-administered cocaine. We conclude that the reductions in striatal D2 receptor densities may be related to the chronic administration of cocaine per se and not to the motivated process of reinforced responding. Our results also suggest that increases in D2 receptor mRNA levels in limbic regions do not necessarily result in increased receptor densities and these changes likely reflect motivational states that were present when cocaine injection dependent on active drug self-administration.

History of cocaine self-administration alters morphine reinforcement in the rat.

It has been shown repeatedly that cocaine pre-exposure may sensitise neurochemical and behavioural responses to opioid drugs. The aim of the present study was to investigate effects of a prior history of cocaine self-administration on morphine reinforcement in the rat. Male Sprague-Dawley rats were allowed to acquire intravenous cocaine self-administration (0.3 mg/kg/infusion) for 20 days. When operant responding for cocaine had stabilised, morphine was introduced instead of cocaine in the next self-administration session. One group of cocaine-exposed rats was allowed to respond for 0.56 mg/kg/infusion of morphine (i.e. the dose which was willingly self-administered by drug-naive controls). The second group was allowed to respond for 0.056 mg/kg/infusion of morphine (i.e. the dose which did not maintain self-administration behaviour in the drug-naive rats). The subjects with the history of cocaine self-administration, in contrast to the drug-naive group, did not maintain operant responding for 0.56 mg/kg/infusion of morphine. These rats easily self-administered the ten times lower dose of the opioid (0.056 mg/kg/infusion). An opioid receptor antagonist, naltrexone (1 mg/kg i.p.) restored the positive reinforcing properties of the higher dose of morphine in the cocaine-exposed rats. Concluding, the present results suggest that prior history of cocaine self-administration sensitises rats to the positive reinforcing properties of morphine.

Cycloheximide impairs acquisition but not extinction of cocaine self-administration.

The aim of the present study was to assess the role of de novo protein synthesis in the acquisition and extinction of cocaine self-administration. In a first experiment, rats were trained to respond for intravenous cocaine infusions (0.3 mg/kg) and a protein synthesis inhibitor, cycloheximide (CHX; 3 mg/kg, s.c.) was injected immediately after each self-administration session. In a second experiment, rats were allowed to acquire cocaine self-administration and CHX was injected immediately after subsequent extinction sessions. CHX impaired the acquisition, but not extinction, of cocaine self-administration. In control experiments, CHX (3 mg/kg) blocked c-Fos protein expression after foot-shock stress and impaired the acquisition of conditioned freezing but did not inhibit spontaneous locomotor activity and sucrose drinking. Our results suggest that: i) the acquisition and extinction of cocaine-reinforced behaviour have a different molecular basis; and ii) only the former process requires de novo protein synthesis.

Effects of ethanol on nicotine-induced conditioned place preference in C57BL/6J mice.

It has been shown that small doses of ethanol (

Nicotine-induced conditioned taste aversion in the rat: effects of ethanol.

It has been shown that small doses of ethanol antagonise the discriminative stimulus properties of nicotine in the rat. The aim of the present study was to evaluate whether ethanol could antagonise the aversive stimulus effects of nicotine. Wistar rats were trained to associate nicotine injections with a novel tasting fluid (0.1% saccharin) in the conditioned taste aversion procedure. Nicotine (0.3 mg/kg, s.c.) was injected 5 min after the end of a 20-min exposure to the saccharin solution. Ethanol (0.25-0.5 g/kg, i.p.) was administered 5 or 50 min before nicotine. In general, ethanol did not inhibit nicotine-induced conditioned taste aversion. Contrary to the findings in drug discrimination studies, a slight but significant enhancement of nicotine-induced taste aversion conditioning was observed after ethanol pre-treatment. Blood ethanol levels were measured in a separate group of rats. Maximal blood ethanol levels after i.p. administration of 0.25 or 0.5 g/kg ethanol exceeded 20 and 80 mg%, respectively. Concluding, the present results may indicate that ethanol does not attenuate nicotine-induced conditioned taste aversion in the rat.

Contingency does not contribute to the effects of cocaine self-administration on prodynorphin and proenkephalin gene expression in the rat forebrain.

Neuroadaptations in the brain opioid systems produced by chronic exposure to drugs of abuse may contribute to the drug dependence and addiction. Although regulation of the gene expression of the opioid propeptides proenkephalin (PENK) and prodynorphin (PDYN) by psychostimulants has previously been described, little attention has been paid to dissociating effects of pharmacological actions of the drugs from those produced by motivational processes driving active drug intake in self-administration paradigms. In the present study, effects of response-dependent (contingent) and response-independent (noncontingent) cocaine administration on the PENK and PDYN gene expression in the rat forebrain have been directly compared using the "yoked" self-administration procedure. The i.v. cocaine treatment lasted for 5 weeks, and rats were sacrificed 24 h after the last self-administration session. In situ hybridization analysis revealed that levels of the PDYN mRNA were significantly increased in the caudate/putamen, to the same extent in rats self-administering cocaine as in animals receiving noncontingent injections of the drug at the same frequency and dosage. No changes in the expression of the PDYN gene were detected in the nucleus accumbens or in the central nucleus of amygdala. Levels of the PENK mRNA remained unaltered in all the above-mentioned forebrain regions of rats receiving contingent or noncontingent cocaine injections. The obtained data indicate that up-regulation of the PDYN gene expression in the caudate/putamen results from direct pharmacological actions of cocaine rather than from the motivational and cognitive processes underlying active self-administration of the drug.