Active avoidance learning differentially activates ERK phosphorylation in the primary auditory and visual cortices of Roman high- and low-avoidance rats.

Understanding the mechanisms underlying conditioned avoidance is a critical step toward the development of novel treatments of anxiety. In this context, the two-way active avoidance (2WAA) task is a validated paradigm to investigate uncontrolled avoidance, a hallmark of anxiety disorders. The outbred Roman high- (RHA) and low-avoidance (RLA) rat lines are selected for respectively rapid vs. poor acquisition of active avoidant behavior, and emotional reactivity appears to be the most prominent behavioral difference between the two lines, with RLA rats being more fearful/anxious than their RHA counterparts. This study was aimed at assessing the relationship between the different performance of RHA and RLA rats in the 2WAA task and the number of phosphorylated ERK positive (pERK+) neurons in the primary auditory and visual cortices, in three sub-nuclei of the amygdala, as well as in the nucleus accumbens, and the prefrontal cortex. The results indicate that: (1) RHA rats, but not their RLA counterparts, learn very rapidly to avoid mild electric foot-shocks by crossing to the opposite compartment of the shuttle-box during the presentation of the conditioned stimulus and (2) the different behavior of the Roman lines during active avoidance training is associated with differential changes in the number of pERK+ neurons in the primary auditory and visual cortices (where the proactive coping of RHA rats is associated with increased ERK phosphorylation), but not in the other brain areas examined. These results are consistent with the hypothesis that the activation of the ERK signaling cascade in the auditory and visual cortices may be involved in the acquisition of aversive learning in RHA rats.

Effect of Acute Stress on the Expression of BDNF, trkB, and PSA-NCAM in the Hippocampus of the Roman Rats: A Genetic Model of Vulnerability/Resistance to Stress-Induced Depression.

The Roman High-Avoidance (RHA) and the Roman Low-Avoidance (RLA) rats, represent two psychogenetically-selected lines that are, respectively, resistant and prone to displaying depression-like behavior, induced by stressors. In the view of the key role played by the neurotrophic factors and neuronal plasticity, in the pathophysiology of depression, we aimed at assessing the effects of acute stress, i.e., forced swimming (FS), on the expression of brain-derived neurotrophic factor (BDNF), its trkB receptor, and the Polysialilated-Neural Cell Adhesion Molecule (PSA-NCAM), in the dorsal (dHC) and ventral (vHC) hippocampus of the RHA and the RLA rats, by means of western blot and immunohistochemical assays. A 15 min session of FS elicited different changes in the expression of BDNF in the dHC and the vHC. In RLA rats, an increment in the CA2 and CA3 subfields of the dHC, and a decrease in the CA1 and CA3 subfields and the dentate gyrus (DG) of the vHC, was observed. On the other hand, in the RHA rats, no significant changes in the BDNF levels was seen in the dHC and there was a decrease in the CA1, CA3, and DG of the vHC. Line-related changes were also observed in the expression of trkB and PSA-NCAM. The results are consistent with the hypothesis that the differences in the BDNF/trkB signaling and neuroplastic mechanisms are involved in the susceptibility of RLA rats and resistance of RHA rats to stress-induced depression.

Effects of morphine on place conditioning and ERK1/2 phosphorylation in the nucleus accumbens of psychogenetically selected Roman low- and high-avoidance rats.

RATIONALE: Extracellular signal-regulated kinase (ERK1/2) phosphorylation is critical for neuronal and behavioural functions; in particular, phosphorylated ERK1/2 (pERK1/2) expression in the nucleus accumbens (Acb) of the rat is stimulated by addictive drugs with the exception of morphine, which decreases accumbal ERK1/2 phosphorylation in the Sprague-Dawley and Wistar rats. The psychogenetically selected Roman low- (RLA) and high-avoidance (RHA) rats differ behaviourally and neurochemically in many responses to addictive drugs. In particular, morphine elicits a greater increment in locomotor activity and in dopamine transmission in the Acb of RHA vs RLA rats. However, the effects of morphine on place conditioning (conditioned place preference (CPP)) and ERK1/2 phosphorylation in the Roman lines remain unknown. OBJECTIVES AND METHODS: To characterize in the Roman lines the reinforcing properties of morphine (i.e. morphine-elicited CPP acquisition) and the relationship between these properties and its effects on ERK1/2 phosphorylation in the Acb, the behavioural effects of morphine were evaluated in a place-conditioning apparatus and ERK1/2 phosphorylation was assessed by immunohistochemistry in the shell and core subregions of the Acb of rats both acutely administered with morphine or undergoing conditioning. RESULTS: Morphine elicited CPP in both Roman lines and decreased pERK1/2 expression in the Acb of RLA but not RHA rats. Such decrease was prevented by conditioning. CONCLUSIONS: These findings indicate that the selective breeding of the Roman lines has generated a divergence, in terms of morphine-elicited pERK1/2 expression but not of morphine-elicited CPP, between RLA and RHA rats and sustain the observation that changes in pERK1/2 expression in the Acb are not a requisite for the reinforcing effects of morphine.

Differential effects of antipsychotic and propsychotic drugs on prepulse inhibition and locomotor activity in Roman high- (RHA) and low-avoidance (RLA) rats.

RATIONALE: Animal models with predictive and construct validity are necessary for developing novel and efficient therapeutics for psychiatric disorders. OBJECTIVES: We have carried out a pharmacological characterization of the Roman high- (RHA-I) and low-avoidance (RLA-I) rat strains with different acutely administered propsychotic (DOI, MK-801) and antipsychotic drugs (haloperidol, clozapine), as well as apomorphine, on prepulse inhibition (PPI) of startle and locomotor activity (activity cages). RESULTS: RHA-I rats display a consistent deficit of PPI compared with RLA-I rats. The typical antipsychotic haloperidol (dopamine D2 receptor antagonist) reversed the PPI deficit characteristic of RHA-I rats (in particular at 65 and 70 dB prepulse intensities) and reduced locomotion in both strains. The atypical antipsychotic clozapine (serotonin/dopamine receptor antagonist) did not affect PPI in either strain, but decreased locomotion in a dose-dependent manner in both rat strains. The mixed dopamine D1/D2 agonist, apomorphine, at the dose of 0.05 mg/kg, decreased PPI in RHA-I, but not RLA-I rats. The hallucinogen drug DOI (5-HT2A agonist; 0.1-1.0 mg/kg) disrupted PPI in RLA-I rats in a dose-dependent manner at the 70 dB prepulse intensity, while in RHA-I rats, only the 0.5 mg/kg dose impaired PPI at the 80 dB prepulse intensity. DOI slightly decreased locomotion in both strains. Finally, clozapine attenuated the PPI impairment induced by the NMDA receptor antagonist MK-801 only in RLA-I rats. CONCLUSIONS: These results add experimental evidence to the view that RHA-I rats represent a model with predictive and construct validity of some dopamine and 5-HT2A receptor-related features of schizophrenia.

Effects of Forced Swimming Stress on ERK and Histone H3 Phosphorylation in Limbic Areas of Roman High- and Low-Avoidance Rats.

Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent.

Neonatal handling enduringly decreases anxiety and stress responses and reduces hippocampus and amygdala volume in a genetic model of differential anxiety: Behavioral-volumetric associations in the Roman rat strains.

The hippocampus and amygdala have been proposed as key neural structures related to anxiety. A more active hippocampus/amygdala system has been related to greater anxious responses in situations involving conflict/novelty. The Roman Low- (RLA) and High-avoidance (RHA) rat lines/strains constitute a genetic model of differential anxiety. Relative to RHA rats, RLA rats exhibit enhanced anxiety/fearfulness, augmented hippocampal/amygdala c-Fos expression following exposure to novelty/conflict, increased hippocampal neuronal density and higher endocrine responses to stress. Neonatal handling (NH) is an environmental treatment with long-lasting anxiety/stress-reducing effects in rodents. Since hippocampus and amygdala volume are supposed to be related to anxiety/fear, we hypothesized a greater volume of both areas in RLA than in RHA rats, as well as that NH treatment would reduce anxiety and the volume of both structures, in particular in the RLA strain. Adult untreated and NH-treated RHA and RLA rats were tested for anxiety, sensorimotor gating (PPI), stress-induced corticosterone and prolactin responses, two-way active avoidance acquisition and in vivo 7 T 1H-Magnetic resonance image. As expected, untreated RLA rats showed higher anxiety and post-stress hormone responses, as well as greater hippocampus and amygdala volumes than untreated RHA rats. NH decreased anxiety/stress responses, especially in RLA rats, and significantly reduced hippocampus and amygdala volumes in this strain. Dorsal striatum volume was not different between the strains nor it was affected by NH. Finally, there were positive associations (as shown by correlations, factor analysis and multiple regression) between anxiety and PPI and hippocampus/amygdala volumes.

Divergent effects of isolation rearing on prepulse inhibition, activity, anxiety and hippocampal-dependent memory in Roman high- and low-avoidance rats: A putative model of schizophrenia-relevant features.

Social isolation of rats induces a constellation of behavioral alterations known as "isolation syndrome" that are consistent with some of the positive and cognitive symptoms observed in schizophrenic patients. In the present study we have assessed whether isolation rearing of inbred Roman high-avoidance (RHA-I) and Roman low-avoidance (RLA-I) strains can lead to the appearance of some of the key features of the "isolation syndrome", such as prepulse inhibition (PPI) deficits, increased anxious behavior, hyperactivity and memory/learning impairments. Compared to RLA-I rats, the results show that isolation rearing (IR) in RHA-I rats has a more profound impact, as they exhibit isolation-induced PPI deficits, increased anxiety, hyperactivity and long-term reference memory deficits, while isolated RLA-I rats only exhibit deficits in a spatial working memory task. These results give further support to the validity of RHA-I rats as a genetically-based model of schizophrenia relevant-symptoms.

Involvement of dopamine in the differences in sexual behaviour between Roman high and low avoidance rats: an intracerebral microdialysis study.

Outbred Roman high- (RHA) and low-avoidance (RLA) rats are selected for respectively rapid vs. poor acquisition of the active avoidance response and display different copulatory patterns when exposed to a sexually receptive female, with RHA rats showing more robust sexual motivation and better performance than RLA rats also after repeated sexual activity. Here we show that the distinct patterns of sexual behaviour of the Roman lines are correlated with differences in the activity of the dopaminergic mesolimbic system, which plays a key role in sexual motivation and copulatory performance. Thus, differential increases in the concentrations of dopamine and its main metabolite 3,4-dihydroxyphenylacetic acid, occurred in dialysates obtained from the nucleus accumbens shell of naïve and sexually experienced Roman rats during the anticipatory and consummatory phases of sexual activity. These differences were particularly evident between sexually naïve RHA and RLA rats and tended to diminish but still persisted between sexually experienced rats, as did the differences in sexual behaviour. Analysis of the biochemical and behavioural findings showed that, while in RHA rats sexual experience caused a shift in the changes in both the dopaminergic activity and copulation towards the first period of the sexual test, in RLA rats sexual experience increased dopaminergic activity and copulation throughout the entire test. Therefore, this study adds experimental support to the view that the different sexual patterns of the Roman lines are due, at least in part, to a more robust functional tone of the mesolimbic dopaminergic system of RHA rats.

Dopamine is involved in the different patterns of copulatory behaviour of Roman high and low avoidance rats: studies with apomorphine and haloperidol.

Outbred Roman high- (RHA) and low-avoidance (RLA) rats, originally selected for rapid vs. poor acquisition of active avoidance in a shuttle box, show differential copulatory patterns when exposed to a receptive female. Indeed, in the first copulation test male RHA rats show more mounts, intromissions and ejaculations than RLA rats. Such differences do not disappear in subsequent copulation tests, with sexually experienced RHA rats always showing higher levels of sexual motivation and performance than their RLA counterparts. This study shows that the different copulatory patterns of sexually experienced RHA and RLA rats are differentially facilitated by apomorphine, a mixed D1/D2-like dopamine receptor agonist, and impaired by haloperidol, a D2-like dopamine receptor antagonist, given at doses which facilitate and impair, respectively, copulatory behaviour in Sprague Dawley rats used as an external reference strain. Accordingly, apomorphine-induced facilitation and haloperidol-induced impairment of copulatory behaviour were more robust in RLA than RHA rats, as indicated by their effects on several copulatory parameters including mount, intromission and ejaculation latencies, mount, intromission and ejaculation frequencies, post ejaculatory interval, inter-intromission interval and copulatory efficacy. Pretreatment with haloperidol also reduced the facilitatory effect of apomorphine more effectively in RLA than RHA rats. These results suggest that the different copulatory patterns of RHA and RLA rats are mainly due to a lower dopaminergic tone at level of the mesolimbic and incerto-hypothalamic dopaminergic systems of RLA vs. RHA rats, which play a key role in sexual behaviour.

Male Roman high and low avoidance rats show different patterns of copulatory behaviour: comparison with Sprague Dawley rats.

Roman high- (RHA) and low-avoidance (RLA) rats, selectively bred for, respectively, rapid vs. extremely poor acquisition of avoidant behaviour in the shuttle-box, display different coping strategies when exposed to aversive environmental conditions: RLA rats are reactive copers and show hyperemotional behaviour characterized by hypomotility and freezing, while RHA rats show a proactive coping behaviour aimed at gaining control over the stressor. RHA rats also display a robust sensation/novelty seeking profile, high baseline levels of impulsivity, and marked preference for, and intake of, natural and drug rewards. This study shows that the Roman lines also differ in sexual behaviour, a main source of natural reward. Thus, male RHA rats engaged in copulatory activity with a receptive female showing more mounts, intromissions and ejaculations in the first copulation test as compared with their RLA counterparts and Sprague Dawley rats used as an external reference strain. Such differences decreased only partially in subsequent copulation tests, with RHA rats always showing higher levels of sexual motivation and performance than RLA rats. Accordingly, analysis of copulatory parameters of five copulation tests performed at 3-day intervals confirmed that the Roman lines display different patterns of copulatory activity that persist after stabilization of copulatory behaviour by sexual experience. Finally, the weight of the testes, epididymides and seminal vesicles increased to a similar extent in both Roman lines after sexual activity. These results are discussed in terms of the relative contribution of differences in brain neurotransmission (mainly dopamine) and neuroendocrine function to the different patterns of copulatory behaviour of the Roman lines.

Dopamine agonist-induced penile erection and yawning: a comparative study in outbred Roman high- and low-avoidance rats.

The effects on penile erection and yawning of subcutaneous (SC) injections of the mixed dopamine D1/D2-like agonist apomorphine (0.02-0.2 mg/kg) were studied in outbred Roman high- (RHA) and low-avoidance (RLA) male rats, two lines selectively bred for their respectively rapid versus poor acquisition of the active avoidance response in the shuttle-box, and compared with the effects observed in male Sprague-Dawley (SD) rats. Apomorphine dose-response curves were bell-shaped in all rat lines/strains. Notably, more penile erections and yawns were recorded mainly in the ascending part of these curves (e.g., apomorphine 0.02-0.08 mg/kg) in both RLA and RHA rats compared to SD rats, with RLA rats showing the higher response (especially for yawning) with respect to RHA rats. Similar results were found with PD-168,077 (0.02-0.2 mg/kg SC), a D4 receptor agonist, which induced penile erection but not yawning. In all rat lines/strains, apomorphine responses were markedly reduced by the D2 antagonist L-741,626, but not by the D3 antagonist, SB277011A, whereas the D4 antagonists L-745,870 and FAUC213 elicited a partial, yet statistically significant, inhibitory effect. In contrast, the pro-erectile effect of PD-168,077 was completely abolished by L-745,870 and FAUC213, as expected. The present study confirms and extends previously reported differences in dopamine transmission between RLA and RHA rats and between the SD strain and the Roman lines. Moreover, it confirms previous studies supporting the view that dopamine receptors of the D2 subtype play a predominant role in the pro-yawning and pro-erectile effect of apomorphine, and that the selective stimulation of D4 receptors induces penile erection.