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Two-way active avoidance (TWAA) acquisition constitutes a particular case of approach -avoidance conflict for laboratory rodents. The present article reviews behavioural, psychopharmacological and neuroanatomical evidence accumulated along more than fifty years that provides strong support to the contention that anxiety is critical in the transition from CS (conditioned stimulus)-induced freezing to escape/avoidance responses during the initial stages of TWAA acquisition. Thus, anxiolytic drugs of different types accelerate avoidance acquisition, anxiogenic drugs impair it, and avoidance during these initial acquisition stages is negatively associated with other typical measures of anxiety. In addition behavioural and developmental treatments that reduce or increase anxiety/stress respectively facilitate or impair TWAA acquisition. Finally, evidence for the regulation of TWAA acquisition by septo-hippocampal and amygdala-related mechanisms is discussed. Collectively, the reviewed evidence gives support to the initial acquisition of TWAA as a paradigm with considerable predictive and (in particular) construct validity as an approach-avoidance conflict-based rodent anxiety model., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Schizophrenia involves positive, negative and cognitive symptoms, as well as comorbidity with anxiety and obsessive-compulsive disorder. Prepulse inhibition (PPI) of the startle response is a measure of sensorimotor gating that is impaired in schizophrenia and animal models of the disease. Remarkably, impaired PPI has been related to other schizophrenia-like features in rodent models, such as cognitive deficits and hyperactivity. However, it remains to be investigated whether deficient PPI and increased exploratory activity are associated in genetically heterogeneous (outbred) naïve animals. This study was undertaken to evaluate the relationships among PPI and other schizophrenia-related symptoms, such as augmented exploratory activity, anxiety and compulsivity in the genetically heterogeneous (outbred) NIH-HS rat stock (HS) and in the genetically-selected inbred Roman High-Avoidance (RHA) and Low-avoidance (RLA) rats. Animals underwent the following tests: open-field (exploratory activity), elevated zero-maze (anxiety-like behavior), marble burying (compulsive-like behavior), and PPI. Three groups of HS rats were formed according to their PPI scores, i.e. Low-PPI, Medium-PPI and High-PPI. The HS Low-PPI group displayed higher exploratory activity in the open-field than the HS Medium-PPI and HS High-PPI groups. Likewise, compared with their RLA counterparts, RHA rats exhibited lower PPI and more intense exploratory activity in the open-field test. Correlational and factorial analyses of the whole HS sample and the RHA/RLA data globally corroborated the results of the PPI-stratified HS subgroups. These data suggest that such a consistent association between impaired PPI and increased exploratory activity in outbred HS and inbred RHA/RLA rats is a relevant parameter that must be taken into account when modeling clusters of schizophrenia-relevant symptoms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To characterize learning/memory profiles for the first time in the genetically heterogeneous NIH-HS rat stock, and to examine whether these are associated with anxiety, we evaluated NIH-HS rats for spatial learning/memory in the Morris water maze (MWM) and in the following anxiety/fear tests: the elevated zero-maze (ZM; unconditioned anxiety), a context-conditioned fear test and the acquisition of two-way active avoidance (conditioned anxiety). NIH-HS rats were compared with the Roman High- (RHA-I) and Low-Avoidance (RLA-I) rat strains, given the well-known differences between the Roman strains/lines in anxiety-related behavior and in spatial learning/memory. The results show that: (i) As expected, RLA-I rats were more anxious in the ZM test, displayed more frequent context-conditioned freezing episodes and fewer avoidances than RHA-I rats. (ii) Scores of NIH-HS rats in these tests/tasks mostly fell in between those of the Roman rat strains, and were usually closer to the values of the RLA-I strain. (iii) Pigmented NIH-HS (only a small part of NIH-HS rats were albino) rats were the best spatial learners and displayed better spatial memory than the other three (RHA-I, RLA-I and NIH-HS albino) groups. (iv) Albino NIH-HS and RLA-I rats also showed better learning/memory than the RHA-I strain. (v) Within the NIH-HS stock, the most anxious rats in the ZM test presented the best learning and/or memory efficiency (regardless of pigmentation). In summary, NIH-HS rats display a high performance in spatial learning/memory tasks and a passive coping strategy when facing conditioned conflict situations. In addition, unconditioned anxiety in NIH-HS rats predicts better spatial learning/memory., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Two recent microarray and qRT-PCR studies showed that inbred Roman high- (RHA-I, low anxiety and frustration vulnerability) and low-avoidance (RLA-I, high anxiety and frustration vulnerability) rats, psychogenetically selected on the basis of their divergence in two-way avoidance performance, differed in basal whole-brain and hippocampal expression of genes related to neurotransmission, emotion, stress, aversive learning, and drug seeking behavior. We have extended these studies by analyzing strain differences in hippocampal gene expression following a frustrative experience involving reward downshift, i.e. instrumental successive negative contrast (iSNC), a phenomenon in which the sudden reduction of an expected reward induces frustration/anxiety. Food-deprived male Roman rats were exposed to a reduction in the amount of solid food presented in the goal of a straight alley (from 12 pellets in "training" trials - i.e. preshift trials- to 2 pellets in "frustration testing" trials - i.e. postshift trials-). The iSNC effect, as measured by response latencies in the "postshift" trials, appeared only in RLA-I rats (i.e. higher response latencies in the 12-2 RLA-I group as compared to the 2-2 RLA-I control group in postshift trials). Two and a half hours after the "postshift" behavioral test, hippocampi were removed and stored (-80°C) until analysis. Microarray analysis of these hippocampi showed that four differentially-expressed, and qRT-PCR-validated genes (TAAR2, THAP1, PKD2L1, NANOS), have relevance for brain function and behavior, including schizophrenia, depression, anxiety, and drug addiction, thus showing the usefulness of Roman strains as a genetic model for research on the neurogenetic basis of frustration., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To identify genes involved in the development/expression of anxiety/fear, we analyzed the gene expression profile in the hippocampus of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock is a unique genetic resource for the fine mapping of quantitative trait loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety, fearfulness or other complex traits. We selected high- and low-anxious NIH-HS rats according to the number of avoidance responses they performed in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety/fearfulness tests, i.e. the elevated zero-maze and a "novel-cage activity" test. Three weeks after behavioral testing, the hippocampus was dissected and prepared for the microarray study. There appeared 29 down-regulated and 37 up-regulated SNC-related genes (fold-change>|2.19|, FDR<0.05) in the "Low-anxious" vs. the "High-anxious" group. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, nine relevant genes (Avpr1b, Accn3, Cd74, Ltb, Nrg2, Oprdl1, Slc10a4, Slc5a7 and RT1-EC12), tested for validation through qRT-PCR, have either neuroendocrinological or neuroinmunological/inflammation-related functions, or have been related with the hippocampal cholinergic system, while some of them have also been involved in the modulation of anxiety or stress-related (neurobiological and behavioral) responses (i.e. Avpr1b, Oprdl1). The present work confirms the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis or mechanisms involved in anxiety and/or fear, and suggest that some MHC-(neuroinmunological/inflammation)-related pathways, as well as the cholinergic system within the hippocampus, may play a role in shaping individual differences in trait anxiety., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To identify genes involved in anxiety/fear traits, we analyzed the gene expression profile in the amygdala of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock has revealed to be a unique genetic resource for the fine mapping of Quantitative Trait Loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety-(or other)-related traits. We selected high- and low-anxious NIH-HS rats differing in their number of avoidances in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety tests (e.g., elevated zero-maze). Three weeks after behavioural testing, the amygdala was dissected and prepared for the microarray study. There appeared 6 significantly down-regulated and 28 up-regulated genes (fold-change >|2|, FDR<0.05) between the low- and high-anxious groups, with central nervous system-related functions. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, six relevant genes were examined with qRT-PCR, four of which (Ucn3, Tacr3, H2-M9 and Arr3) were validated. Remarkably, some of them are characterized by sharing known functions related with hormonal HPA-axis responses to (and/or modulation of) stress, anxiety or fear, and putative involvement in related neurobehavioural functions. The results confirm the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis of anxiety and fear, while suggesting the involvement of some neuropeptide/neuroendocrine pathways on the development of differential anxiety profiles., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The inbred Roman low- (RLA-I) and high-avoidance (RHA-I) rats used in this study were initially selected and bred for extremely poor vs. rapid acquisition of active two-way avoidance behavior in the shuttle box. As a result of the selection for divergent avoidance acquisition, clear behavioral differences have been found between RHA and RLA rats in a variety of tasks related to anxiety and conflict. In rats of these two strains/lines previous brain studies have been performed, specifically in the striatum, the mesencephalic dopaminergic areas and the prefrontal cortex, as these brain areas are the classical ones for their critical role in sensitization and may play a role in the well-characterized anxiety response. In this study we analyzed, in RHA and RLA groups (N=5 each), the density of NeuN neurons counterstained with toluidine blue in the cingulate cortex (subdivision 1) and the hippocampus (CA1, CA2 and CA3). A statistical difference was found in the density of neurons of CA1 and CA2 (p=0.047 in both) and in the total density of the hippocampus (p=0.009). Contrary to our expectations, significant strain differences for the density of neurons in the cingulate cortex were not found. The relationship between those differences in the hippocampus and the between-strain differences in anxiety and in learning processes depending on anxiety are discussed., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Physiological and environmental variables, or covariates, can account for an important portion of the variability observed in behavioural/physiological results from different laboratories even when using the same type of animals and phenotyping procedures. We present the results of a behavioural study with a sample of 1456 genetically heterogeneous N/Nih-HS rats, including males and females, which are part of a larger genome-wide fine-mapping QTL (Quantitative Trait Loci) study. N/Nih-HS rats have been derived from 8 inbred strains and provide very small distance between genetic recombinations, which makes them a unique tool for fine-mapping QTL studies. The behavioural test battery comprised the elevated zero-maze test for anxiety, novel-cage (open-field like) activity, two-way active avoidance acquisition (related to conditioned anxiety) and context-conditioned freezing (i.e. classically conditioned fear). Using factorial analyses of variance (ANOVAs) we aimed to analyse sex differences in anxiety and fear in this N/Nih-HS rat sample, as well as to assess the effects of (and interactions with) other independent factors, such as batch, season, coat colour and experimenter. Body weight was taken as a quantitative covariate and analysed by covariance analysis (ANCOVA). Obliquely-rotated factor analyses were also performed separately for each sex, in order to evaluate associations among the most relevant variables from each behavioural test and the common dimensions (i.e. factors) underlying the different behavioural responses. ANOVA analyses showed a consistent pattern of sex effects, with females showing less signs of anxiety and fear than males across all tests. There were also significant main effects of batch, season, colour and experimenter on almost all behavioural variables, as well as "sex × batch", "sex × season" and "sex × experimenter" interactions. Body weight showed significant effects in the ANCOVAs of most behavioural measures, but sex effects were still present in spite of (and after controlling for) these "body weight" effects. Factor analyses of relevant variables from each test showed a two-fold factor structure in both sexes, with the first factor mainly representing anxiety and conditioned fear in males, while in females the first factor was dominated by loadings of activity measures. Thus, besides showing consistent sex differences in anxiety-, fear- and activity-related responses in N/Nih-HS rats, the present study shows that females' behaviour is predominantly influenced by activity while males are more influenced by anxiety. Moreover, the results point out that, besides "sex" effects, physiological variables such as colour and body weight, and environmental factors as batch/season or "experimenter", have to be taken into account in both behavioural and quantitative genetic studies because of their demonstrated influences on phenotypic outcomes.

The double, fear-driven "passive avoidance/active avoidance" conflict appearing during acquisition of two-way active avoidance, involves high levels of anxiety and a dominant tendency for freezing responses, which in turn run against the appearance of active escape/avoidance behavior. In the present study, by using a large sample genetically heterogeneous (N/Nih-HS) rats, we have tested the hypothesis that rats showing relatively higher levels of context-conditioned freezing (during the initial trials of that task) should show lower efficiency to acquire two-way avoidance behavior, i.e. the prediction that the initial context-conditioned freezing in the shuttle box would be negatively related to avoidance acquisition efficiency. In agreement with such a hypothesis, the results from the three rat subsamples used show that context-conditioned freezing (during the first 5 inter-trial intervals of the 40-trial two-way avoidance session) is negatively correlated (r=-0.34 to r=-0.64, p<0.001) with two-way avoidance acquisition, in a way that subgroups of rats with extremely high or low levels of freezing markedly differ in their avoidance performance: "high freezer" rats show much worse avoidance acquisition than "low freezers". Moreover, the relationships of conditioned freezing and avoidance acquisition with baseline and fear-potentiated startle, as well as with unconditioned anxiety (in the elevated zero-maze test), have also been studied. Taken collectively, the results indicate that: (i) context conditioned freezing is a reliable (and negative) predictor of two-way avoidance acquisition; (ii) baseline and fear-potentiated startle responses show positive associations with avoidance responding, and (iii) unconditioned anxiety in the elevated zero-maze is also negatively associated with two-way avoidance acquisition. Such patterns of associations are considered to be very informative in regard to the search for (common or differential) neural and genetic mechanisms of different forms of (unlearned or learned) anxious or fear responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Anxiety-related behaviors were evaluated across five tests in a sample of 277 rats from a genetically heterogeneous stock (N/Nih-HS rats), derived from an eight-way cross of inbred strains, and compared with the performance of RLA-I (high anxious) and RHA-I (low anxious) rats in the same tests. These tests either evoke unlearned (novel-cage activity (NACT), elevated "zero" maze (ZM), baseline acoustic startle response (BAS)) or learned (fear-potentiated startle (FPS), two-way active-shuttle box-avoidance acquisition (SHAV)) anxious/fearful responses. The results overall showed that unlearned anxiety responses/behaviors were predictive of behavior in learned fear (i.e. fear-potentiated startle) and conflict (i.e. two-way active avoidance acquisition) situations. Moreover, it was found that N/Nih-HS rats either resemble RLA-I rat anxiety/fear scores or fall in between those of the RLA-I (high anxious) and the RHA-I (low anxious) rat strains. An additional regression analysis (of N/Nih-HS rat data) showed significant positive influences of (unlearned) baseline startle response, risk assessment (i.e. stretch-attend) behavior and activity (5min) in a novel cage on SHAV acquisition, while baseline startle and entries into the open section of the elevated 'zero' maze test of anxiety were the main variables influencing FPS. This indicates that startle responses may have a facilitating role in the rat's active responses in the two-way active (shuttlebox) avoidance acquisition. The results of this behavioral evaluation of N/Nih-HS rats show that unconditioned anxiety (e.g. in the ZM test) predicts learned fear-related responses (e.g. FPS and SHAV) to some extent, while a positive association is also observed between BAS and SHAV. These findings are discussed in terms of their potential usefulness for present and future neurobehavioral and genetic studies of fearfulness/anxiety.

The present experiment was designed with the goal of studying the partial reinforcement extinction effect (PREE) in female inbred Roman high- (RHA-I) and low-avoidance (RLA-I) rats. Two groups of RHA-I and two of RLA-I food-deprived animals were placed in a straight alley where they were partially or continuously reinforced. Once the animals reached the acquisition criterion, they were exposed to an extinction phase where the reinforcement was omitted. During the extinction phase RHA-I animals continuously reinforced during acquisition exhibited more resistance to extinction than their RLA-I counterparts, whereas only RLA-I rats partially reinforced during acquisition showed an increased resistance to extinction in comparison to continuously reinforced control RLA-I rats, this PREE being absent in RHA-I animals. These results are discussed within the framework of PREE theories that account for this effect by using emotional mechanisms, as pertains to the repeatedly observed RHA-RLA differences in emotional reactivity.

Several studies performed in outbred Roman high- and low-avoidance lines (RHA and RLA, respectively) have demonstrated that the more anxious line (RLA) is characterized by a higher hypothalamic-pituitary-adrenal (HPA) response to certain stressors than the less anxious one (RHA). However, inconsistent results have also been reported. Taking advantage of the generation of an inbred colony of RLA and RHA rats (RHA-I and RLA-I, respectively), we have characterized in the two strains not only resting and stress levels of peripheral HPA hormones but also central components of the HPA axis, including CRF gene expression in extra-hypothalamic areas. Whereas resting levels of ACTH and corticosterone did not differ between the strains, a greater response to a novel environment was found in RLA-I as compared to RHA-I rats. RLA-I rats showed enhanced CRF gene expression in the paraventricular nucleus (PVN) of the hypothalamus, with normal arginin-vasopressin gene expression in both parvocellular and magnocellular regions of the PVN. This enhanced CRF gene expression is not apparently related to altered negative corticosteroid feedback as similar levels of expression of brain glucorticoid and mineralocorticoid receptors were found in the two rat strains. CRF gene expression tended to be higher in the central amygdala and it was significantly higher in the dorsal region of the bed nucleus of stria terminalis (BNST) of RLA-I rats, while no differences appeared in the ventral region of BNST. Considering the involvement of CRF and the BNST in anxiety and stress-related behavioral alterations, the present data suggest that the CRF system may be a critical neurobiological substrate underlying differences between the two rat strains.

Anxiety-related behaviors were evaluated across various tests in a large sample (n=787, both sexes) of genetically heterogeneous (N/Nih-HS) rats, derived from an eight-way cross of inbred strains. These tests either evoke unlearned (black-white box, BWB-; novel-cage activity, NACT-; elevated "zero" maze, ZM-; baseline acoustic startle response, BAS-) or learned (fear-potentiated startle, FPS-; two-way active-shuttle box-avoidance acquisition, SHAV-) anxious/fearful responses. The results showed that, with the exception of fear-potentiated startle, almost all (unlearned and learned) behaviors assessed fit with a pattern of sex effects characterized by male rats as being more fearful than females. We applied factor analyses (oblique rotation) to each sex, with the final two-factor solution showing: (1) a first factor (labelled as "Timidity") comprising BWB, NACT and ZM variables in both sexes, plus SHAV responding in the case of males, and (2) a second factor (called "Defensive Flight") which grouped BAS, FPS, and SHAV responding in both sexes. An additional regression analysis showed significant influences of (unlearned) risk assessment (i.e. stretch-attendance) behavior on SHAV in males, while FPS was the main variable positively influencing SHAV (in the intermediate and advanced phases of acquisition) in females. This indicates, for the first time, that fear-potentiated startle may have a facilitating role in the rat's active responses (at least in females) to the cue in the intermediate to advanced phases (i.e. when the initial "passive avoidance/active avoidance" begins to fade) of shuttle box avoidance acquisition. The results of this first extensive behavioral evaluation of N/Nih-HS rats are discussed in terms of their potential usefulness for present and future neurobehavioral and genetic studies of fearfulness/anxiety.

It has been recently shown that Roman high- (RHA) and low- (RLA) avoidance rats show behavioural divergence in successive negative contrast (SNC) induced in one-way avoidance learning [Torres C, Cándido A, Escarabajal MD, de la Torre L, Maldonado A, Tobeña A, et al. Successive negative contrast effect in one-way avoidance learning in female roman rats. Physiol Behav 2005;85:377-82]. A 2-experiment study was conducted with the goal of analyzing whether these differences in SNC can also be extended to a different experimental paradigm. Food-deprived RHA and RLA female rats were exposed to a straight alley, recording the latency (DV) between leaving the start box and reaching the food available in the goal box at the end of the alley. To induce the SNC effect the amount of reinforcement received went from 12 pellets in the pre-shift phase to 1 pellet (Experiment 1) or 2 pellets (Experiment 2) in the postshift phase. The SNC effect appeared in both strains in Experiment 1, but only in RLA rats in Experiment 2. These results are discussed within the framework of SNC theories that account for this effect by using emotional mechanisms, as related to the differences in emotional reactivity seen between the RHA and RLA strains in a number of behavioural tests of fear/anxiety.

Behavioural assessment of mice lacking adenosine A1 receptors (A1Rs) showed reduced activity in some phases of the light-dark cycle, reduced exploratory behaviour in the open-field and in the hole-board, increased anxiety in the plus maze and dark-light box and increased aggressiveness in the resident-intruder test. No differences were found in spatial reference and working memory in several Morris water maze tasks. Both mutant mice had reduced muscle strength and survival rate. These results confirm the involvement of adenosine in motor activity, exploratory behaviour, anxiety and aggressiveness. A1Rs also appear to play a critical role in ageing-related deterioration.

A critical test for a gene that influences susceptibility to fear in animals is that it should have a consistent pattern of effects across a broad range of conditioned and unconditioned models of anxiety. Despite many years of research, definitive evidence that genetic effects operate in this way is lacking. The limited behavioral test regimes so far used in genetic mapping experiments and the lack of suitable multivariate methodologies have made it impossible to determine whether the quantitative trait loci (QTL) detected to date specifically influence fear-related traits. Here we report the first multivariate analysis to explore the genetic architecture of rodent behavior in a battery of animal models of anxiety. We have mapped QTLs in an F2 intercross of two rat strains, the Roman high and low avoidance rats, that have been selectively bred for differential response to fear. Multivariate analyses show that one locus, on rat chromosome 5, influences behavior in different models of anxiety. The QTL influences two-way active avoidance, conditioned fear, elevated plus maze, and open field activity but not acoustic startle response or defecation in a novel environment. The direction of effects of the QTL alleles and a coincidence between the behavioral profiles of anxiolytic drug and genetic action are consistent with the QTL containing at least one gene with a pleiotropic action on fear responses. As the neural basis of fear is conserved across species, we suggest that the QTL may have relevance to trait anxiety in humans.

Anxiety-related behaviours were evaluated across various tests in a 800 F(2)-intercross of the Roman high- and low-avoidance inbred rats. These tests either evoke unlearned (open field [OF]; plus-maze [PM]; hole-board [HB]; spontaneous activity [A]; and acoustic startle reflex [ASR]) or learned (classical fear conditioning [CFC]; and shuttlebox avoidance conditioning [SAC]), anxious/fearful responses. Using factor analysis (oblique rotation), we obtained a six-fold solution with 14 variables derived from all tests. These six factors represented SAC, CFC, PM anxiety, PM and OF activity, ASR anxiety, plus a mixed whole of anxious and activity variables (from OF and A), respectively. In searching for a smaller number of meaningful factors, we applied a three-factor solution that coherently corresponded with differentiated facets of fearfulness, rather than with the tests. Results showed that (1) measures of SAC and CFC strongly loaded onto Factor 1, labelled as "Learned Fear"; (2) a blend of almost all variables loaded onto Factor 2, called "Emotional Reactivity"; and (3) open arm behaviour in the PM loaded onto Factor 3, called "Fear of Heights." After discussing limitations of this apparently consistent behavioural map of anxiety, we advance some connections between those factors with quantitative trait loci candidates (genetic markers) as detected in the same sample.

In the present study, male inbred animals (from the 10th generation of an inbreeding program that has been carried out in parallel to that of the outbred Roman high- and low-avoidance rat lines), were compared for emotionality in different testing situations, exploratory behavior in the holeboard and two-way, active-avoidance acquisition. Compared to the inbred Roman high-avoidance (RHA-I/Verh) rats, inbred Roman low-avoidance (RLA-I-Verh) rats showed higher emotionality in the open field (reduced distance travelled and number of rearings, and increased self-grooming behavior), in the elevated plus-maze test (increased number of total and open-arm entries, reduced distance travelled in the open arms, and increased self-grooming behavior), and during the habituation period in the shuttle box (decreased number of crossings, increased self-grooming behavior and defecations). Results from the hyponeophagia test were not conclusive, probably due to the test-dependent hyperactivity shown by RHA-I/Verh rats. In the holeboard apparatus, RHA-I/Verh rats explored more than RLA-I/Verh rats, especially when novel objects were located beneath the holes. Finally, RHA-I/Verh animals rapidly acquired active, two-way (shuttlebox) avoidance, whereas RLA-I/Verh animals required four 50-trial sessions to achieve an assymptotic level of 30-40% avoidance. Thus, the behavioral patterns of the Roman inbred strains were very similar to those previously reported for the RHA/Verh outbred lines. Differences in locomotor activity, exploratory, and self-grooming behavior were actually greater between the inbred strains than between the outbred lines. Differences in defecation, however, although still significant, were not so pronounced as those noted previously at this laboratory with the outbred lines.

The present study investigated the effects of perinatal flumazenil (Ro 15-1788, a benzodiazepine receptor antagonist), given from gestational day 15 to the 14th day after giving birth, at two doses (3.5 and 6.3 mg/kg/ day) on the behavior of female RLA/Verh rats. This rat line has been selectively bred for non-acquisition of two-way active avoidance. Offspring of treated/non-treated dams were tested when adults for two-way active (shuttle box) avoidance acquisition. For comparison reasons additional offspring coming from the same treatments were used in an hyponeophagia test and in both an open field and a plus maze tests. The results show that perinatal flumazenil, specially the lower dose (and also the highest dose, although only in the final phases of the shuttle box training) was able to improve two-way active avoidance acquisition, as it has been previously found with males, whereas it failed to show any effect in the hyponeophagia task, the open field and the plus-maze tests. As two-way active avoidance acquisition can be enhanced both by reducing emotionality/anxiety or by improving memory, it is suggested that flumazenil treatment could have affected either one or both processes.

The Swiss sublines of Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats have been selected and bred for rapid (RHA/Verh) vs. extremely poor (RLA/Verh) acquisition of two-way active avoidance. Behavioral and physiological measures of emotionality, or reactivity to stress, appear to be among the most prominent characteristics differentiating both rat lines. The present study shows that RLA/Verh rats are more sensitive, as compared to their RHA/Verh counterparts, to the conflict involved in the shock-induced suppression of drinking paradigm, as well as in a hyponeophagia test. RLA/Verh rats also showed higher defecation values which were significantly correlated with the main hyponeophagia test variables. Likewise, self-grooming was more frequent in RLA/Verh rats than in their RHA/Verh counterparts and showed significant correlations with conflict-related behaviors (i.e., latency to start eating and time spent eating) from the hyponeophagia test. These results give additional support to the contention that RLA/Verh rats present higher anxiety (emotionality) than their RHA/Verh counterparts.

The present studies evaluated the short- and long-lasting effects of postnatal handling (administered during the first 21 days of life) on the emotional behavior of female Sprague-Dawley rats. The performance of postnatally handled (H) and control nonhandled (NH) animals was compared in two different situations: an emotionality rating (ER) test (when they were 40 days or 4 months old), and a hyponeophagia (neophobia) test of anxiety, at the age of 4 months. The results showed that postnatal handling induced both short-term and long-term reductions of spontaneous emotional reactivity in the ER test, although the effects on some measures disappeared in 4-month-old rats. Postnatal handling also induced enduring decreases of anxiety as measured by the hyponeophagia test. None of the observed effects were attributable to changes in basal locomotor activity. ER measures were significantly related to hyponeophagia, because animals showing the highest emotionality scores in the ER test (preferentially NH animals) were those that showed the highest eating latencies and spent less time eating in the neophobic situation (i.e., hyponeophagia test).

The possible involvement of anxiety and learning/memory processes in escape-directed (struggling) behavior in a two-trial swimming test was investigated in mice, as well as the differential effects that low doses of flumazenil (a benzodiazepine receptor antagonist) could display depending on the animals' anxiety levels. Mice showing less anxiety in the plus-maze test exhibited less struggling behavior in the first swimming trial than the more anxious animals, suggesting a relationship between anxiety and struggling behavior in the swimming test. Flumazenil (5 mg/kg) given before the first swimming trial displayed differential effects depending upon the animals' anxiety levels. Thus, it increased struggling behavior in the first swimming trial in 'low-anxiety' mice whereas the opposite tendency was observed in 'high-anxiety' animals. Struggling decreased in the second swimming trial in all the animals, giving support to the involvement of learning/memory processes in the two-trial swimming test. That reduction in escape-directed behavior was greater in animals treated with flumazenil before the first swimming session, thus indicating a slight enhancement of retention.

The effects of low (2 g/kg) and high (4 g/kg) doses of ethanol and their interaction with the imidazobenzodiazepine Ro 15-4513 (a partial inverse agonist of the benzodiazepine receptor) were studied in two different models of anxiety in rats: the "elevated plus-maze" test and the early acquisition of two-way (shuttlebox) avoidance. In the elevated plus-maze, ethanol (2 g/kg) increased the percentage of entries into the open arms (%EOA) and both ethanol doses increased the percentage of time spent into the open arms (%TOA), thus indicating an anxiolytic action which was reversed by Ro 15-4513 (5 mg/kg). By contrast, Ro 15-4513 did not counteract the anxiolytic effect of the low dose of ethanol in the acquisition of shuttlebox avoidance. Thus, the treatment with 2 g/kg of ethanol (plus either vehicle or Ro 15-4513) significantly increased the total number of avoidances. Conversely, animals treated with 4 g/kg of ethanol showed impaired shuttlebox avoidance acquisition, and this effect was completely reversed by Ro 15-4513. Ro 15-4513 was without effect on its own in any of the anxiety-related parameters (i.e., %EOA, %TOA, and avoidance acquisition). The results indicate a different pattern of ethanol effects and Ethanol x Ro 15-4513 interactions depending upon the task used.

Several lines of evidence have established that performance during the initial steps of acquisition on a shuttle-box avoidance task is an anxiety-mediated behavior (i.e., the differences between strains selectivity bred for emotionality; the effects of postnatal handling; the course of the corticosterone response and behavioral measures of fear during acquisition). The present study was carried out to add pharmacological evidence to that view by testing the action of anxiogenic and anxiolytic drugs. Single 40-trial sessions with mild shocks (0.4 mA-0.6 mA) were used. In the first experiments the action of sodium pentobarbital (1.25, 2.5 and 5 mg/kg) and three benzodiazepines (diazepam, 2 and 4 mg/kg; alprazolam, 1, 1.25 and 1.5 mg/kg and adinazolam, 1, 2, 4 and 6 mg/kg) were tested. The last two experiments tested a possible proanxiety action of Ro 15-4513 (2, 5 and 10 mg/kg) and FG 7142 (5, 10 and 15 mg/kg), two partial inverse agonists of benzodiazepine receptors, which previous data had suggested to be anxiogenic. The results showed that the measure of acquisition of a two-way active avoidance is a sensitive mean for detecting either anxiolytic or anxiogenic effects of drugs, independently of their effects on locomotor activity, thus suggesting that such test could be a valid model of anxiety in animals.

Both postnatal handling of rat pups and perinatal treatment with Ro 15-1788 have been reported to reduce the emotional reactivity of the rats in adulthood, as measured by open field behavior, by the corticosterone response to stress, and by labyrinth and novelty-induced behaviors. We now report results obtained with a well-validated animal model of anxiety (i.e. the elevated plus-maze) that support and extend these previous findings. The results show the clearest reduction of anxiety-related behavior when postnatal handling and perinatal Ro 15-1788 administration were simultaneous.