Oxytocin and carbetocin ameliorating effects on restraint stress-induced short- and long-term behavioral changes in rats.

OBJECTIVES: Carbetocin (CBT), an oxytocin (OXY) analog, was designed to exert prolonged peripheral actions. It has also been proposed as potential therapeutic mean in certain psychiatric disorders where OXY role has been implicated. This study examined the effects of both peptides on behavior of naive and restraint stress exposed rats in the open field (OF) and elevated plus maze (EPM) tests. METHODS: Spontaneous behavior in the OF and EPM was measured in Wistar rats after intraperitoneal (i.p.) application of OXY or CBT and/or repeated restraint stress. Behavioral parameters were recorded and subsequently elaborated by an automated activity monitoring system (AnyMaze, Stoelting, U.S.A.). Changes in the total movement distance (TMD) and movement in the center area (CMD) were postulated as indicators of the anxiety level. RESULTS: OXY (0.05 mg/kg) and CBT (0.3 mg/kg) increased TMD but not CMD 60 min after the i.p. treatment; the increased locomotion/exploration indicate participation of arousal/vigilance. Daily stress exposures for three consecutive days, followed by behavioral tests, reduced locomotion of rats in OF and EPM tests; OXY and CBT partly prevented these effects. Five days after the last stress, rats exhibited an increase of both TMD and CMD in the OF. CBT but not OXY prevented these long-term post-stress changes. In the EPM the stressed rats exhibited an increase in time spent in open arms; CBT accelerated this time development. Similar prevention of stress behavioral sequel in OF were obtained in study when stress and peptides were applied for three consecutive days but behavioral testing was postponed for several days to determine the long-lasting effects. CBT reduced the developed locomotor enhancement (6-11 days post-stress) irrespectively whether injected before or after stress. CONCLUSIONS: Repeated restraint stress exposure produced acute and persisting effects on Wistar rat behavior in the OF and EPM tests. CBT either injected before or after stress practically abolished the developed changes in the mobility parameters. The CBT effectiveness to ameliorate the late post-stress behavioral alteration supports the notion of its therapeutic potential in psychiatric disorders in which the role of OXY has been implicated.

Novel and simple behavioral paradigm for assessing anxiety in rats: effect of diazepam.

OBJECTIVES: Anxiety is an emotional state experienced by people, and is not readily modeled in animals. In order to extend till now ethologically derived paradigms used in the evaluation of anxiety and fear in rodents, a modified open-field was designed. METHODS: Spontaneous behavior of male rats was investigated in the elevated arena; the bottom was divided by inter-space of different width (2, 4, 6, 8 and 10 cm) into two identical parts. Anxiolytic effects of diazepam (DZP) at doses 0.5, 1.0, 2.5 and 5.0 mg/kg were investigated in the newly designed device and compared with the effects of similar doses in a large circular open-field arena. RESULTS: In Experiment 1 the progressive extension of the inter-space prolonged the first passing, decreased the total number of passing, and increased the inter-space sniffing in intact animals. In Experiment 2 DZP at doses 1.0, 2.5 and 5.0 mg/kg significantly enhanced the readiness to cross, the frequency of passing the inter-space and decreased inter-space sniffing as compared to controls. In Experiment 3 we found that DZP at doses 0.1 and 0.3 mg/kg increased behavioral activity both along the perimeter and in the center of the arena, thus indicating lower level of anxiety. CONCLUSION: The presented modified open-field test is a useful paradigm to investigate risk assessment behavior in rats, and may provide a sensitive novel model of anxiety and fear level.

Oxytocin and carbetocin effects on spontaneous behavior of male rats: modulation by oxytocin receptor antagonists.

OBJECTIVES: Oxytocin (OXY) in addition to peripheral actions has many central regulatory functions which can be studied on animal models. In the present study we examined in rats, which behavioral actions of OXY and long-acting carba-analog of OXY carbetocin (CBT) in the open-field can be inhibited by OXY-receptor antagonists. Our interest focused on the behavioral patterns considered indicative of anxiety-related behavior. To determine what is the participation of OXY receptor on OXY and CBT induced behavioral changes, we used two peptide and one nonpeptide OXY antagonists differing in selectivity for OXY receptor. METHODS: OXY, CBT as well as OXY antagonists were injected intraperitoneally, and spontaneous behavior (horizontal and vertical activity, grooming) of Wistar rats was observed in the circular open-field arena 60 min after application of drugs; in some experiments testing was performed without treatment few days after drug administration. RESULTS: OXY at the dose 0.05 mg/kg increased locomotion indicating anxiety attenuation, but 1.0 mg/kg reduced both locomotion and rearing. CBT in the dose range 0.1-3.0 mg/kg either did not change or increased horizontal activity. The increase in exploration after both peptides persisted for several days. A marked difference in the behavioral effects of the two peptides was grooming enhancement induced by OXY compared with the absence of this effect after CBT. The increase of the activity induced by OXY and CBT indicating anxiolytic-like action was blocked by OXY antagonists. However, the reduction of exploration induced by 1.0 mg dose of OXY was only partially reversed. The OXY induced enhanced grooming was completely antagonized by all used antagonists. CONCLUSIONS: Behavioral effects of OXY and its antagonists after their i.p. application indicate that they penetrate blood brain barrier. The diversity in potency of OXY antagonists to inhibit grooming and other behaviors induced by OXY suggests that receptors participating in these behaviors may differ in brain localization, receptor conformation and/or in the utilized signaling pathways.

Effects of melanotan II, a melanocortin agonist, on grooming and exploration in rats after repeated restraint/immobilization.

2mg/kg melanotan II (MTII, administered i.p.), a cyclic peptide analog of alpha-melanocyte stimulating hormone, at a single dose increased grooming in naive rats placed in an unfamiliar open-field device without changing locomotion or rearing. Male rats exposed to restraint/immobilization stress (IS) for 1h on three consecutive days displayed increased grooming after the second stressor exposure, compared to pre-stress levels. MTII, administered to the rats after IS, enhanced the grooming response compared both to the pre- and post-stress values. The increase was greatest after the first dose and declined over the following two applications. As to the locomotion of rats in the entire experimental space, IS reduced the distance moved only after the first two stressor exposures; MTII did not influence these alterations. Locomotion in the central part of arena was not reduced by the stressor or by MTII, on the contrary, there was an increase in both groups after the third intervention. The only observed change in rearing was an increase in the MTII group after the third restraint exposure. Thus, MTII selectively increased grooming without markedly affecting the spatio-temporal structure of locomotor behavior in the open-field. The decline of MTII enhanced grooming over the three test days may be interpreted in terms of adaptation to the stressor and of the developing tolerance to the peptide.

Dipeptide "alaptide" prevented impairments in spontaneous behavior produced with trimethyltin in male rats.

OBJECTIVES: On the animal model of trimethyltin (TMT) induced behavioral deficits the effect of chronic treatment with spirocyclic dipeptide cyclo/alanyl-l-amino-l-cyclopentane-carbonyl (alaptide, AL) was evaluated in adult male rats. METHODS: Changes in the spontaneous behavioral repertoire were investigated in the open-field test on Day 21 (Session 1) and Day 28 (Session 2) after a single oral TMT administration. RESULTS: In Experiment 1, rats given the highest TMT dose (7.5 mg/kg) exhibited significantly increased total number of behavioral patterns, the floor sniffing being the most frequent pattern. While the medium TMT dose (5 mg/kg) had a similar effect only in Session 1, the lowest TMT dose (2.5 mg/kg) was entirely ineffective. In Experiment 2, an explicit beneficial influence of both AL doses (5 and 10 mg/kg) given for 10 days before and 10 days after TMT (7.5 mg/kg) on the spontaneous behavior repertoire was observed in both Session 1 and Session 2. The total number of patterns and the time spent in individual patterns of AL+TMT treated animals did not differ from the controls and those given AL alone. CONCLUSION: We conclude that sufficiently long AL treatment interfered with deleterious effects of TMT and forestalled changes in the structure and timing of spontaneous behavioral patterns. Thus, AL can be designated as a substance having "neuroprotective" effects.

Spontaneous alternation behaviour in rats: kynurenic acid attenuated deficits induced by MK-801.

The present study was undertaken to investigate the effects of pharmacological modulation of the NMDA receptors on spontaneous alternation behaviour. The performance of rats treated with MK-801 and kynurenic acid (KYNA) was assessed in the cross-arm-maze. We evaluated: (a) the total number of arm entries representing locomotor activity, (b) spontaneous variation of different arms thought to reflect alternation performance. In the first experiment, MK-801 (0.01, 0.025, 0.05, 0.1 and 0.2 mg/kg, i.p.) was given 30 min prior to the testing. Beginning the dose of 0.05 mg/kg the drug increased locomotion and impaired alternation performance. An ability of animals to enter subsequently three or four different arms was reduced significantly. In the second experiment, the dose of 0.05 mg/kg was chosen as the lowest possible dose of MK-801 producing marked behavioural impairment. KYNA (0.3, 3 and 30 mg/kg, s.c.) was administered 60 min prior to the MK-801. While all KYNA doses prevented hyperlocomotion, only the highest dose (30 mg/kg) maintained alternation score at the control levels, i.e. the KYNA plus MK-801 treated animals alternated regularly three or four different arms. The results suggest different sensitivity of the two behavioural systems, i.e. locomotion and space orientation, towards pharmacological insult. In conclusion, the study confirmed protective behavioural effects of KYNA given in sufficient amounts and sufficiently long prior MK-801.

Timing of stress and testing influence the long-lasting behavioral performance in rats.

Three exposures (Days 1, 2 and 3) of rats to immobilization or immobilization combined with cold induced an alteration of exploratory behavior in an open space arena. When tested 1h after both stressors exposure, rats displayed a decrease in locomotor and rearing score. The deficit disappeared when rats were tested five days later and the performance remained unchanged in trials performed on days 9, 10, 15, 22 and 29 of the study. When testing was postponed five days after the third stressor exposure, a gradual reduction of the performance developed and the deficit persisted until the last trial on Day 29. Amphetamine, in a dose of 0.3 mg/kg revealed a sensitized response to the drug in the stressed animals. The results showed short- and long-lasting behavioral consequences of the used stressors, the long-term manifestation of the sequelae being dependent on the sequence and timing of stressor exposure and open space testing.

Oxiracetam pre- but not post-treatment prevented social recognition deficits produced with trimethyltin in rats.

The social recognition paradigm was used to investigate the effect of trimethyltin (TMT) in adult male rats. Consequently, the effect of chronic oxiracetam (OXI) treatment in TMT impaired animals was evaluated. In all experiments, a behavioural testing was performed 3 weeks after TMT administration. Experiment 1: A single TMT oral dose, 5 and 7.5 but not 2.5mg/kg, impaired the natural ability of the adults to recognize a juvenile conspecific that they encountered 30 min before. The dose of 5mg/kg TMT was chosen to be used in subsequent experiments. Experiment 2: Chronic OXI pre-+post-treatment, daily 3 or 30 mg/kg sc for 7 days before and 7 days after the insult, protected the adults against recognition deficit produced by TMT. Experiment 3: OXI pre- but not post-treatment (always 3 and 30 mg/kg) had beneficial effects on the social recognition. The findings suggest that social recognition ability of adult male rats pre-treated sufficiently long with OXI is resistant to the neurotoxicity effect of TMT.

Behavioral deficits in adult rats treated neonatally with glutamate.

The present study evaluated long-term behavioral consequences of neonatal monosodium-l-glutamate (MSG) treatment in rats. The pups received MSG (3 mg/g sc) daily from postnatal day (PD) 5-12. Data from an automatic activity monitor showed that locomotion of MSG-treated females and males aged 56 and 84 days was significantly reduced. Beginning PD 120, three behavioral tests were performed. As compared to the controls, in the elevated plus maze test, modified to evaluate the adaptive form of spatial memory, MSG-treated animals of both sex had significantly prolonged start and transfer latencies. In the social recognition test, assessing olfactory working memory, MSG-treated males displayed a reduced interest in the juvenile conspecific as the stimulus partner during both the initial exposure and re-exposure performed 30 min later. In the open field test, a significant decrease in the habituation rate was found in MSG-treated animals. Sex-dependent differences in behavioral performance were suggested in the open field and elevated plus maze tests. Behavioral changes are discussed in light of the deficits in perception and processing of visual and olfactory stimuli.

MK-801 induced amnesia for the elevated plus-maze in mice.

MK-801, a non-competitive NMDA receptor antagonist, has been shown to have amnesic properties in animal models. The purpose of the present study was to examine potential amnesic effects of MK-801 in mice using the modified elevated plus-maze paradigm. An animal was placed on the distal end of an open arm, and the transfer latency, i.e. the time in which it moves to the enclosed arm, was measured. Four different experimental schedules (i.e. the combination of the treatment and the testing) were used: MK-801 (0.075, 0.15, 0.25 and 0.4 mg/kg or saline) were given (a) 30 min before the acquisition session, (b) immediately after the acquisition session, (c) 60 min after the acquisition session, and (d) 30 min before the retention session. The retention session always followed 24 h after the acquisition session. Analysis of data showed a significant shortening of the transfer latency in saline-treated animals during the retention session. Further, MK-801 (at the dose range of 0.15--0.4 mg/kg) administered before and immediately after the acquisition session as well as before the retention session prolonged the transfer latency during the retention session. In fact, transfer latencies in MK-801 treated mice did not differ from those measured during the acquisition session. Thus, prolongation of the transfer latency in MK-801-treated mice indicates deficits in 'memorization' processes. On the contrary, MK-801 given 60 min after the acquisition session failed to increase the transfer latency, which suggests that the memory trace was sufficiently consolidated at this time. Based on the present results, the glutamatergic NMDA receptor mechanisms play an important role in a spatial orientation of mice placed on the elevated plus-maze.

Oxiracetam prevented the scopolamine but not the diazepam induced memory deficits in mice.

In mice, the elevated plus-maze paradigm was used to investigate the effect of scopolamine hydrobromide and diazepam and their interaction with oxiracetam on the retrieval of spatial memory trace. This paradigm measures (using the transfer latency) an animal's capacity to escape from the open arm to the enclosed one. The retention session followed 24 h after the acquisition one. Experiment 1: Scopolamine (0.25 and 0.5 mg/kg) and diazepam (0.5 and 1.0 mg/kg) given 30 min before the retention session significantly prolonged the transfer latency as compared with the saline treated mice and those given the lowest dose of scopolamine (0.125 mg/kg) and diazepam (0.25 mg/kg). Experiment 2: Oxiracetam administered at doses of 3, 10 and 30 mg/kg immediately after the acquisition session prevented the scopolamine induced prolongation of the transfer latency. Thus, oxiracetam forestalled the impairment of retrieval of memory trace: the animals were able to remember the spatial configuration of the plus-maze. On the contrary, oxiracetam was not effective in the diazepam treated mice. We suggest that beneficial effect of oxiracetam might be confounded or blocked by the anxiolytic effect of diazepam.

Impaired passive avoidance acquisition in Sprague-Dawley and Lewis rats after restraint and cold stress.

The study examined the effects of restraint combined with cold water stress (IMO+C) on learning and memory of Sprague-Dawley (S-D) and Lewis (LE) rats in the passive avoidance task. The procedure started with 6 days of adaptation to the apparatus during which the recorded latencies to enter the dark compartment were used to assess the process of habituation. On the training day rats were exposed to IMO+C for 60 min and the stressor exposure terminated 1 h before the acquisition trial. Retention trials started 24 h later. To evaluate the possible long-term consequences of the acute and repeated stress presentation on the performance of the two strains with diverse activity of hypothalamic-pituitary-adrenal axis, this procedure was performed three times including stress application (Parts 1-3). Finally, an identical procedure was performed without stress (Part 4). An immediate behavioural effect of the stressor exposure was observed in an increase of latencies to enter the dark compartment before the shock delivery in rats of both strains; this enhancement approached significance after the second and third exposure to the stressor (Parts 2 and 3). Control animals of both strains acquired passive avoidance response after training in Parts 2-4. IMO+C produced significant impairment of this response irrespective of the strain. The three-time repeated exposure did not influence the ability to learn the task in the final procedure without stress. Differences in behaviour of S-D and LE rats were observed already during the first adaptation period. LE rats exhibited longer latencies upon the first exposure to the novel environment compared to S-D rats. Also only LE rats displayed habituation. In Part 4 marked strain differences in the latencies both before and after training were recorded. The results show that the repeated exposure to the IMO+C stressor proved to be a strong amnestic stimulus but without persisting consequences for the ability of rats to acquire the learning task.

Combined restraint and cold stress in rats: effects on memory processing in passive avoidance task and on plasma levels of ACTH and corticosterone.

The effect of restraint stress combined with water immersion (IMO+C), applied at various intervals before and after the acquisition of a passive avoidance task, was studied in rats. The procedure started with two pre-training trials. On the single training trial the rats received a footshock (0.3 mA, 3s) after they entered the preferred dark compartment. The exposure to IMO+C lasting 1 h terminated 4 or 1 h before application of the footshock or started immediately or 3 h after this aversive stimulus. Retention tests were performed 1 and 2 days after the acquisition trial. In an attempt to relate the behavioural responses to the stressor with plasma levels of two stress hormones we measured ACTH and corticosterone under similar conditions as were used in the behavioural experiments. IMO+C exposure terminating 1 h before the training resulted in very short avoidance latencies during retention testing. A similar impairment of retention test performance was found in animals exposed to the stressor immediately after training. When IMO+C exposure terminated 4 h before training the stressed rats exhibited comparably long avoidance latencies as shown by the controls. IMO+C presented 3 h after acquisition trial also did not influence retention of avoidance learning. The hormones were estimated 1 and 4 h after IMO+C, both in the absence and presence of footshock. Both ACTH and corticosterone were significantly increased 1 h after IMO+C termination, and their plasma levels returned to control values within 4 h. Footshock alone increased plasma corticosterone, however, the hormone levels were significantly lower than those estimated after IMO+C terminating 1 h before blood collection. Footshock substantially increased ACTH levels in rats exposed to IMO+C 1 h before footshock, but not in stressed rats with already high levels of corticosterone. In conclusion, IMO+C represents a strong stress stimulus exerting amnesic effect when applied shortly before or after the acquisition trial. Further, the findings indicate the restraint and cold stressor to interfere with consolidation of passive avoidance response. We suggest that the moderate circulating levels of corticosterone found after footshock may be positively related to the memory consolidation, while the exceedingly high levels have an opposite effect.

N-methyl-D-aspartate improved social recognition potency in rats.

Activation of N-methyl-D-aspartate (NMDA) receptors is believed to play an important role in cognitive processes. In the present study we addressed the question of whether systematically administered NMDA can improve social recognition in adult male rats. We utilized the paradigm in which an adult animal is exposed to the same or a novel juvenile during two successive interactions. The adults given NMDA (10, 25 and 50 mg/kg, s.c.) immediately after the initial exposure to a juvenile exhibited significantly reduced social investigation during the second encounter with the same juvenile performed both 120 and 240 min later. Reduction in social investigation was not observed in saline treated animals as well as in those treated with both NMDA and saline but re-exposed to novel juveniles. These results indicate that NMDA improved social recognition potency of adult animals and prolonged retention of previously stored olfactory information.

Effects of two types of restraint stress on the spontaneous behaviour in rats.

Our previous findings suggested the existence of stressor-specific behavioural and cognitive responses in rats. In the present study, restraint stressor (immobilization, IMO) and restraint stressor combined with partial immersion of rats into water (IMO+C) were applied for 1 hour to Wistar male rats and their spontaneous behaviour was examined in the open field test. The classic behavioural parameters were recorded: crossing, rearing, and resting. When tested 1 and 4 hours after IMO+C, animals exhibited strong suppression of locomotor and exploratory activity (crossing and rearing); partial inhibition of both behavioural variables was found after IMO. Thus, substantial differences were observed in dependence on the length of period between the end of stressor application and the start of testing. In testing performed one week later, the locomotor and exploratory activity levels of both IMO and IMO+C animals corresponded to the control ones. These data suggest a differential behavioural response to both used stressors that may result from their different proportion of psychical and physical components. In conclusion, our results provide other data for the support of differential effects of two types of restraint stressors on spontaneous behaviour of animals exposed to a novel environment.