Characterization of the Roman lines/strains of rats as a genetic model of psychiatric disorders: a behavioral and brain dialysis study

State of the art Depressive disorders are fairly prevalent in the general population, with a higher rate in women compared to men, are disabling, since they can significantly impair psychosocial functioning, and are typically associated with high mortality due mainly to the high rate of suicides but also to the negative impact that depression has on the course of co-occurring illnesses. In addition, pharmacological and psychological antidepressant therapies in use have a limited efficacy and/or are associated with side effects that reduce the compliance in many patients. Although the etiology and pathogenesis of depression are poorly understood, it is most likely that the combination of genetics, early life adverse events, and ongoing stress may ultimately determine the individual vulnerability to stress-related disorders, such as depression. Therefore, the development and characterization of animal models of vulnerability and resistance to the effects of stress, including early life stress, is a major challenge for depression research. One such model is represented by the Roman high- (RHA) and low-avoidance (RLA) lines/strains of rats, which are psychogenetically selected for, respectively, rapid versus extremely poor acquisition of active avoidance in a shuttle box. A large body of evidence indicates that a major reason for their divergent performance in this test is their different reactivity to stressful stimuli, that is, their coping style. Thus, when exposed to aversive stimuli, RLA rats display a reactive coping strategy, associated with a strong activation of the HPA axis; moreover, they display robust depressive-like behaviors in the forced swim test (FST) that are normalized by the subacute administration of antidepressants. In contrast, compared with their RLA counterparts, RHA rats display a proactive coping strategy in the face of aversive conditions, associated with higher baseline levels of impulsivity, a more robust sensation/novelty seeking profile, a marked preference for, and intake of, natural and drug rewards along with a greater responsiveness of the mesolimbic dopaminergic system.Aims The behavioral and neurochemical traits that distinguish the two lines/strains suggest that RLA rats may represent a model of vulnerability to stress-induced depression, whereas RHA rats may model resistance to stress-induced depression-like behaviour. To test this hypothesis, in the first study we evaluated the performance of RLA and RHA rats in the FST in response to chronic antidepressant treatments, since clinical evidence indicates that several weeks of treatment with antidepressant drugs are required to achieve an adequate therapeutic response. Furthermore, one of the cardinal symptoms of depression observed in many patients is anhedonia, which is defined as the loss of interest in once enjoyable activities, including sexual activity. Accordingly, depressive episodes are frequently associated with sexual dysfunctions. In consideration of this clinical evidence, and given the well-established role of dopamine in sexual behavior, the second study was aimed at characterizing the sexual behavior of RHA and RLA rats and its correlation with the functional state of their mesolimbic dopaminergic system. In keeping with the long-term consequences on mental health elicited by early-life adverse events, it has been observed that post weaning social isolation in rodents may lead to a later increment in the prevalence of anxiety/fear related behaviors. Thus, in the third study we evaluated the impact of post weaning isolation on the anxiety-related behaviors of inbred RHA and RLA rats in the Elevated Zero Maze, and in motility cages used to asssess locomotor activity in a new environment. Results In study I we demonstrated that chronic treatments with low doses of antidepressants, that were ineffective when given subacutely, were able to decrease immobility and also to increase climbing (desipramine) or swimming (fluoxetine) in RLA rats. Conversely, neither subacute nor chronic antidepressant treatments affected the behavior of RHA rats in the FST. iii In addition, the results of study II showed that, compared with their RLA counterparts, RHA rats displayed higher levels of sexual motivation and a better copulatory performance, associated with a greater release of DA in the AcbSh. These line-related differences were attenuated but not abolished by sexual experience. Moreover, RLA rats were more responsive than their RHA counterparts to both, the facilitatory effect of apomorphine and the inhibitory effect of haloperidol on sexual behavior. Finally, in study III we found that the isolation-rearing procedure significantly increased the level of anxiety of RHA-I rats in the EZM, as reflected by a smaller number of entries and a shorter time spent in the open space, associated with decreased head dipping, increased latency to enter in the open space, and reduced novelty-induced locomotor activation, whereas it failed to produce significant changes in the behavior of RLA-I rats. Conclusions The results of these studies show that the Roman lines/strains of rats may represent a valid experimental approach to investigate the neural substrates and molecular mechanisms involved in the individual vulnerability and resistance to stress-induced depression, with the aim of identifying both, potential biomarkers for an early diagnosis of depression and potential molecular targets for novel antidepressant treatments. Moreover, the Roman lines/strains may be used to study the neurophysiology of the appetitive and consummatory aspects of sexual behaviour, in order to better understand the mechanisms underlying the psychological and pathological causes of sexual dysfunctions. Finally, the Roman rats may provide a useful model to identify the mechanisms whereby early-life adverse events interact with the genetic make up to induce psychiatric disorders in adulthood

Differential effects of voluntary ethanol consumption on dopamine output in the nucleus accumbens shell of Roman high- and low-Avoidance rats: A behavioral and brain microdialysis study

The Roman high- (RHA) and low-Avoidance (RLA) rats were selectively bred for rapid vs poor acquisition of two-way active avoidance behavior. These lines differ in numerous behavioral traits, with RLA rats being more fearful/anxious than RHA rats, and the latter being novelty-seekers and showing larger intake of, and preference for, addictive substances including ethanol (ETH). Moreover, several differences in central dopaminergic, serotonergic, and GABAergic functions have been reported in these two lines. Since those neural systems are involved in the regulation of ETH consumption, it was considered of interest to investigate: 1) the differences in ETH intake and preference between RHA and RLA rats, 2) the effects of ETH on DA release in the shell of the nucleus accumbens (AcbSh) using brain microdialysis. ETH solutions of increasing concentrations (2%- 10%) were presented on alternate days in a free choice with water. To examine ETH intake and preference stability, animals were subsequently switched to daily presentations of 10% ETH for 10 consecutive days. RHA rats consumed significantly larger amounts of ETH and displayed higher ETH preference than did RLA rats throughout the acquisition and maintenance phases. Following chronic exposure to ETH the animals were habituated to a restricted access to ETH schedule (2% ETH, 2 h per day × 4 days) before surgical implantation of a dialysis probe in the AcbSh. Under these experimental conditions, voluntary ETH intake (2%, 1 h, p.o.) produced a significant increase in accumbal DA output in RHA rats but not in their RLA counterparts. Finally, the i.p. administration of ETH (0.25 g/kg) to naïve Roman rats produced a significant increment in accumbal DA output only in RHA rats. These results indicate that the mesolimbic dopaminergic system of RHA rats is more responsive to the effects of ETH than that of RLA rats

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

Immunochemical detection of BDNF in the brain of a rat model of depression

Several lines of evidence show a relationship between alterations in the mechanisms that control the expression of neurotrophic factors and mood disorders (1). In particular, support for the role of brain-derived neurotrophic factor (BDNF) in the pathogenesis of depression and related deficits in neuronal plasticity comes from evidence that a reduction of BDNF expression has been found in postmortem brains and serum of depressed subjects and that the BDNF gene is required for the response to antidepressant drugs. With the aim to contribute to the characterization of the molecular and neuronal systems involved in the pathogenesis of depression and in the mechanism of action of the antidepressant treatments, here we use the outbread Roman High- (RHA) and Roman Low-Avoidance (RLA) rat lines, psychogenetically selected for rapid versus poor acquisition of active avoidance, respectively, and bearing several behavioral characteristics closely resembling the cardinal symptoms of depression (2), to investigate on the immunochemical occurrence of BDNF in selected areas of the RHA and RLA rat brain by means of western blot (WB) and immunohistochemistry. WB analysis indicates that the relative levels of BDNF patently and markedly differed in the hippocampus, where they were significantly lower by 58% in RLA vs RHA rats (p = 0.0014). In the remaining examined areas, namely the prefrontal cortex, the caudate-putamen complex proper, the core and shell regions of the nucleus accumbens and the ventral tegmental area, the relative BDNF levels did not show statistically significant differences. In tissue sections, BDNF-like immunoreactive (LI) material labelled neuronal cell bodies, proximal processes and varicose nerve fibers, with an uneven distribution in telencephalic cerebral cortex, hippocampus, amygdala, nucleus accumbens, caudate-putamen complex proper, thalamus and ventral tegmentum of the midbrain. Densitometric analysis of immunostained brain sections were used to quantify differences among the two rat lines. The results obtained provide a morphological evidence for a differential expression of BDNF in specific areas of RLA vs RHA rat brains and may form the morphological basis to understand the regulation of the trophic machinery in depression

I ratti delle linee Roman (High e Low Avoidance) presentano differenze nel comportamento sessuale: ruolo del sistema dopaminergico mesolimbico

I RATTI DELLE LINEE ROMAN (HIGH E LOW AVOIDANCE) PRESENTANO DIFFERENZE NEL COMPORTAMENTO SESSUALE: RUOLO DEL SISTEMA DOPAMINERGICO MESOLIMBICO. Introduzione. I ratti delle linee Roman (RHA, High avoidance e RLA, Low avoidance) mostrano tratti comportamentali divergenti: i primi sono impulsivi e proni all’abuso di sostanze mentre i secondi sono iper-emotivi e proni a sviluppare sintomi depressivi. I ratti Roman differiscono anche nel comportamento sessuale: gli RHA, infatti, mostrano una maggiore motivazione e migliori prestazioni rispetto agli RLA. L’obiettivo di questo studio era indagare se queste differenze sono relate ad alterazioni nella funzione del sistema dopaminergico mesolimbico, che gioca un ruolo chiave nel comportamento motivato. Metodi. Sono stati misurati con la metodica della microdialisi intracerebrale i livelli di dopamina liberata nel nucleo accumbens di ratti RHA e RLA mai esposti prima a stimoli sessuali (naïve) oppure esperienti, quando esposti ad una femmina recettiva inaccessibile e durante la copula. Contemporaneamente, sono stati misurati diversi indici comportamentali motivazionali e di performance sessuale. L’analisi statistica è stata eseguita con ANOVA per disegni misti, seguita da test post hoc (P < 0.05). Risultati. Nei ratti RHA si riscontra una maggiore liberazione di dopamina nel nucleo accumbens rispetto ai ratti RLA sia in presenza di una femmina recettiva inaccessibile che durante l’interazione sessuale. Similmente a quanto osservato con i parametri comportamentali, le differenze sono maggiori tra i gruppi naïve, tendono a diminuire tra i gruppi esperienti, ma persistono anche dopo stabilizzazione del comportamento dovuta all’esperienza. Conclusioni. Le differenze di comportamento sessuale delle linee Roman possono essere dovute al differente tono funzionale del sistema dopaminergico mesolimbico, che nei ratti RLA appare più debole rispetto ai ratti RHA. Questa differente funzione del sistema dopaminergico mesolimbico può essere implicata anche nelle alterazioni presenti in altri aspetti del comportamento motivato di questi animali (ad es., assunzione di sostanze d’abuso, vulnerabilità alla depressione)

Immunochemical detection of trkB receptor in the brain of a rat model of depression

The outbread Roman High- (RHA) and Roman Low-Avoidance (RLA) rat lines were psychogenetically selected for rapid versus poor acquisition of active avoidance, respectively, and differ in many behavioural traits that closely resemble the cardinal symptoms of depression (1). Beyond the monoamine hypothesis of depression, compelling evidence suggests that mood disorders are characterized by reduced neuronal plasticity. Consistently, it has been shown that exposure to stress and antidepressant treatment modulate the expression of neurotrophic molecules and their relevant receptors, and that these changes show an anatomical specificity (2). With the aim to characterize the molecular and neuronal systems involved in the pathogenesis of depression and in the mechanism of action of the antidepressant treatments, here we investigate on the immunochemical occurrence of trkB, the high affinity tyrosinekinase receptor for brain-derived neurotrophic factor (BDNF), in selected areas of the RHA and RLA rat brain by means of western blot (WB) and immunohistochemistry. WB analysis indicates that the relative levels of trkB patently and markedly differed in the prefrontal cortex and the hippocampus, where they were lower in RLA vs RHA rats, and in the caudate-putamen complex proper where, by contrast, they were higher in RLA vs RHA rats. No statistically significant differences were seen in nucleus accumbens and ventral tegmental area. In tissue sections, trkB-like immunoreactive (LI) labelling was mainly localized to neuronal cell bodies and proximal processes, unevenly distributed in the telencephalic cerebral cortex, the hippocampus, and the ventral tegmentum of the midbrain. Densitometric analysis of immunostained brain sections revealed that differences among the two groups are consistent to a good extent with WB data. As a whole, the finding of a different expression of trkB receptor in the RLA vs RHA rat brains implies the occurrence of an altered neuronal responsiveness to BDNF in specific brain regions and may contribute to outline the molecular and morphological basis for the distinct vulnerability to depression in the two rat lines

Effects of chronic antidepressant treatments in a putative genetic model of vulnerability (Roman low-avoidance rats) and resistance (Roman high-avoidance rats) to stress-induced depression

Introduction: The Roman low- (RLA) and high-avoidance (RHA) rats were selectively bred for, respectively, poor versus rapid acquisition of active avoidance in a shuttle box and, under aversive conditions, display reactive (RLA) versus proactive (RHA) coping behaviors. In the forced swim test (FST), RLA rats exhibit a depression-like behavior characterized by greater immobility and fewer climbing counts when compared with their RHA counterparts. Furthermore, subacute treatments with clinically effective antidepressant drugs decrease immobility and increase climbing or swimming in RLA rats but do not modify the performance of RHA rats. Objective and methods: Because chronic treatment with antidepressants is usually required to produce clinical effects, the present study was designed to compare the behaviors of RLA and RHA rats in the FST after subacute (1 day) and chronic (15 days) administration of desipramine, fluoxetine, and chlorimipramine. Results: In RLA rats, subacute treatments with low doses of desipramine, fluoxetine, and chlorimipramine (2.5-5 mg/kg) were ineffective whereas chronic treatments with the same doses of all three antidepressants decreased immobility and also increased climbing (desipramine) or swimming (fluoxetine). By contrast, neither subacute nor chronic treatments with these antidepressants induced significant changes in the behavior of RHA rats in the FST. Conclusions: RLA and RHA rats represent two divergent phenotypes, respectively susceptible and resistant to develop depression-like behavior under aversive environmental conditions that may be used to identify genetically determined neural substrates and mechanisms underlying vulnerability and resistance to stress-induced depression

Possible role of dopamine in the differences in sexual behaviour between Roman High and Low Avoidance rats: behavioral, pharmacological and neurochemical findings

POSSIBLE ROLE OF DOPAMINE IN THE DIFFERENCES IN SEXUAL BEHAVIOUR BETWEEN ROMAN HIGH AND LOW AVOIDANCE RATS: BEHAVIORAL, PHARMACOLOGICAL AND NEUROCHEMICAL FINDINGS. Roman High (RHA) and Low Avoidance (RLA) rats display opposite behavioral traits: RHA rats are active copers, impulsive and prone to abuse drugs while RLA rats are reactive copers, hyperemotional and prone to develop depressive-like symptoms. These differences are linked to differences in brain monoamine (mainly dopamine) function and neuroendocrine responses to stress. RHA and RLA rats differ also in sexual behavior, with RHA rats displaying higher motivation and better copulatory performance than RLA rats. Moreover, in the two lines sexual behaviour is differentially influenced by dopamine agonists and antagonists, suggesting a different sensitivity/density of dopamine receptors in brain areas involved in sexual behaviour. Taken together and in keeping with the well known role of dopamine in the modulation of different aspects of sexual behaviour, these findings raise the possibility that the differences observed in sexual behaviour between RHA and RLA rats can be due to differences in brain dopamine transmission. In order to test this hypothesis, naive (never exposed to a receptive female) and sexually experienced (which underwent five copulation tests) RHA and RLA rats implanted with a microdialysis probe aimed at the shell of the nucleus accumbens (NAs), were used in a classical appetitive/consummatory test of sexual behavior, during which copulatory parameters were recorded and dialysate aliquots collected from the NAs for the determination of dopamine by HPLC-ECD. The results show that the higher sexual motivation and better performance of RHA vs. RLA rats occurred concomitantly with a higher dopamine release, as shown by the higher dopamine concentrations found in the NAs dialysate of RHA vs. RLA rats. These differences between the two lines were greater in naive animals and persisted, although attenuated, in experienced animals. These findings confirm that a different mesolimbic dopaminergic tone exists in RHA and RLA rats, which may be responsible, at least in part, for their different copulatory patterns. Moreover, as sexual behaviour is one of the most important sources of natural reward due its importance in the preservation of species, these results, taken together with those that show differences between the two lines in the mesolimbic dopaminergic function after the intake of different natural or addictive rewards (as drugs of abuse), strongly support the validity to use these two rats lines as a model for the study of the genetic, neurochemical and behavioural factors at the basis of individual differences in motivated behaviour and its alterations and their interactions with different classes of stimuli as well

Dopamine is involved in the different copulatory patterns 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

Involvement of dopamine in the differences in sexual behaviour between Roman High and Low Avoidance rats: behavioral, pharmacological and neurochemical findings

INVOLVEMENT OF DOPAMINE IN THE DIFFERENCES IN SEXUAL BEHAVIOUR BETWEEN ROMAN HIGH AND LOW AVOIDANCE RATS: BEHAVIORAL, PHARMACOLOGICAL AND NEUROCHEMICAL FINDINGS. Roman High (RHA) and Low Avoidance (RLA) rats display opposite behavioral traits: RHA rats are active copers, impulsive and prone to abuse drugs while RLA rats are reactive copers, hyperemotional and prone to develop depressive-like symptoms. These differences are linked to differences in brain monoamine (mainly dopamine) function and neuroendocrine responses to stress. RHA and RLA rats differ also in sexual behavior, with RHA rats displaying higher motivation and better copulatory performance than RLA rats, and their sexual behavior differentially influenced by dopamine agonists and antagonists [1; 2]. Together with the well known role of dopamine in sexual behavior, these findings suggest that the sexual differences between RHA and RLA rats may be due to differences in dopamine neurotransmission. In order to test this hypothesis, naive (never exposed to a receptive female) and sexually experienced (which underwent five copulation tests) RHA and RLA rats implanted with a microdialysis probe aimed at the shell of the nucleus accumbens (NAs), were used in a classical appetitive/consummatory test of sexual behavior, during which copulatory parameters were recorded and dialysate aliquots collected from the NAs for the determination of dopamine by HPLC-ECD. The results show that the higher sexual motivation and better performance of RHA vs. RLA rats occurred concomitantly with a higher dopamine release, as shown by the higher dopamine concentration found in the NAs dialysate of RHA vs. RLA rats. These differences between the two lines were greater in naive animals and persisted, although attenuated, in experienced animals. These findings confirm that a different mesolimbic dopaminergic tone exists in RHA and RLA rats, which may be responsible, at least in part, for their different copulatory patterns, and provide insights into the differences among individuals in the neural basis of motivated behaviours and their relationship with vulnerability to abuse natural and/or drug rewards or to develop depressive disorders