Behavioral testing constitutes the primary method to measure the emotional states of nonhuman animals in preclinical research. Emerging as the characteristic tool of the behaviorist school of psychology, behavioral testing of animals, particularly rodents, is employed to understand the complex cognitive and affective symptoms of neuropsychiatric disorders. Following the symptom-based diagnosis model of the DSM, rodent models and tests of depression and anxiety focus on behavioral patterns that resemble the superficial symptoms of these disorders. While these practices provided researchers with a platform to screen novel antidepressant and anxiolytic drug candidates, their construct validity-involving relevant underlying mechanisms-has been questioned. In this review, we present the laboratory procedures used to assess depressive- and anxiety-like behaviors in rats and mice. These include constructs that rely on stress-triggered responses, such as behavioral despair, and those that emerge with nonaversive training, such as cognitive bias. We describe the specific behavioral tests that are used to assess these constructs and discuss the criticisms on their theoretical background. We review specific concerns about the construct validity and translational relevance of individual behavioral tests, outline the limitations of the traditional, symptom-based interpretation, and introduce novel, ethologically relevant frameworks that emphasize simple behavioral patterns. Finally, we explore behavioral monitoring and morphological analysis methods that can be integrated into behavioral testing and discuss how they can enhance the construct validity of these tests.

Deficits in motor impulsivity, that is, the inability to inhibit a prepotent response, are frequently observed in psychiatric conditions. Several studies suggest that stress often correlates with higher impulsivity. Among the brain areas affected by stress, the orbitofrontal cortex (OFC) is notable because of its role in impulse control. OFC subregions with unique afferent and efferent circuitry play distinct roles in impulse control, yet it is not clear what OFC subregions are engaged during motor impulsivity tasks.

In this study we used a rodent test of motor impulsivity, the 1-choice serial reaction time test, to explore activation of OFC subregions either during a well-learned motor impulsivity task or in a challenge task with a longer wait time that increases premature responding. We also examined the effects of acute inescapable stress, chronic intermittent cold stress and chronic unpredictable stress on motor impulsivity.

Fos expression increased in the lateral OFC and agranular insular cortex during performance in both the mastered and challenge conditions. In the ventral OFC, Fos expression increased only during challenge, and within the medial OFC, Fos was not induced in either condition. Inescapable stress produced a transient effect on premature responses in the mastered task, whereas chronic intermittent cold stress and chronic unpredictable stress altered premature responses in both conditions in ways specific to each stressor.

These results suggest that different OFC subregions have different roles in motor impulse control, and the effects of stress vary depending on the nature and duration of the stressor.

The issue of whether the decrease in food intake induced by inescapable shock is due to the uncontrollability of the stressor or the shock per se has not yet been settled. Besides, whether food intake is differentially affected by an uncontrollable chronic stressor has been explored only by a few studies. Thus, we evaluated the effects of chronic escapable or inescapable electric shocks on eating behavior. Rats were exposed to shock sessions for 20 days in two occasions separated by baseline sessions with no shock in an ABAB design. Results showed a reduction in food and water intake and body weight gain during stress periods, especially with inescapable shocks. The findings support a close link between learned helplessness, chronic stress, and eating behavior.

Psychiatric disorders are characterized by sex differences in their prevalence, symptomatology and treatment response. Animal models have been widely employed for the investigation of the neurobiology of such disorders and the discovery of new treatments. However, mostly male animals have been used in preclinical pharmacological studies. In this review, we highlight the need for the inclusion of both male and female animals in experimental studies aiming at gender-oriented prevention, diagnosis and treatment of psychiatric disorders. We present behavioural findings on sex differences from animal models of depression, anxiety, post-traumatic stress disorder, substance-related disorders, obsessive-compulsive disorder, schizophrenia, bipolar disorder and autism. Moreover, when available, we include studies conducted across different stages of the oestrous cycle. By inspection of the relevant literature, it is obvious that robust sex differences exist in models of all psychiatric disorders. However, many times results are conflicting, and no clear conclusion regarding the direction of sex differences and the effect of the oestrous cycle is drawn. Moreover, there is a lack of considerable amount of studies using psychiatric drugs in both male and female animals, in order to evaluate the differential response between the two sexes. Notably, while in most cases animal models successfully mimic drug response in both sexes, test parameters and treatment-sensitive behavioural indices are not always the same for male and female rodents. Thus, there is an increasing need to validate animal models for both sexes and use standard procedures across different laboratories.

In this study, we investigated whether (a) animals demonstrating the learned helplessness effect during an escape contingency also show learning deficits under positive reinforcement contingencies involving stimulus control and (b) the exposure to positive reinforcement contingencies eliminates the learned helplessness effect under an escape contingency. Rats were initially exposed to controllable (C), uncontrollable (U) or no (N) shocks. After 24h, they were exposed to 60 escapable shocks delivered in a shuttlebox. In the following phase, we selected from each group the four subjects that presented the most typical group pattern: no escape learning (learned helplessness effect) in Group U and escape learning in Groups C and N. All subjects were then exposed to two phases, the (1) positive reinforcement for lever pressing under a multiple FR/Extinction schedule and (2) a re-test under negative reinforcement (escape). A fourth group (n=4) was exposed only to the positive reinforcement sessions. All subjects showed discrimination learning under multiple schedule. In the escape re-test, the learned helplessness effect was maintained for three of the animals in Group U. These results suggest that the learned helplessness effect did not extend to discriminative behavior that is positively reinforced and that the learned helplessness effect did not revert for most subjects after exposure to positive reinforcement. We discuss some theoretical implications as related to learned helplessness as an effect restricted to aversive contingencies and to the absence of reversion after positive reinforcement. This article is part of a Special Issue entitled: insert SI title.

We tested the hypothesis that rats consuming bovine lactoferrin (bLf) during postnatal development would show better performance of stressful tasks during adolescence. In the first study, we orally administered bLf (750 mg/kg) once daily between postnatal days 16-34. Rats then underwent a battery of behavioral tests: open field (forced exploration of risky environment), light-dark emergence (voluntary exploration of risky environment), baited holeboard (working and reference memory), food neophobia (preference for familiar versus novel food), forced swim (test for antidepressant efficacy), and shuttle-box escape (learning to escape footshock). bLf-supplemented rats showed less exploration of the risky environment, greater preference for the familiar food odor, and faster escape responses. The effect of bLf on forced-swim behavior depended on sex: immobility increased for males and decreased for females. In the next study, we replaced the forced-swim test with an escape-swim test in which rats learned to use a visual cue to locate an escape platform, and we tested the dose response of bLf on this and the shuttle-box escape test, with subjects receiving vehicle or bLf at 500, 1,000, or 2,000 mg/kg. Under this modified testing battery, improvement of escape from footshock was not observed at any dose. However, males, but not females, showed a significant dose-dependent effect of bLf on acquisition of the water-escape task. On average, males receiving a higher dose mastered the task 20-25 % sooner than rats receiving a lower dose or vehicle. These results offer preliminary evidence that bLf supplementation during development can improve subsequent cognitive performance during stress.

This is the first metabolic mapping study of the effects of fluoxetine after learned helplessness training. Antidepressants are the most commonly prescribed medications, but the regions underlying treatment effects in affectively disordered brains are poorly understood. We hypothesized the antidepressant action of fluoxetine would produce adaptations in mesolimbic regions after 2 weeks of treatment. We used Holtzman rats, a genetic strain showing susceptibility to novelty-evoked hyperactivity and stress-evoked helplessness, to map regional brain metabolic effects caused by fluoxetine treatment. Animals underwent learned helplessness, and subsequently immobility time was scored in the forced swim test (FST). On the next day, animals began receiving 2 weeks of fluoxetine (5mg/kg/day) or vehicle and were retested in the FST at the end of drug treatment. Antidepressant behavioral effects of fluoxetine were analyzed using a ratio of immobility during pre- and post-treatment FST sessions. Brains were analyzed for regional metabolic activity using quantitative cytochrome oxidase histochemistry as in our previous study using congenitally helpless rats. Fluoxetine exerted a protective effect against FST-induced immobility behavior in Holtzman rats. Fluoxetine also caused a significant reduction in the mean regional metabolism of the nucleus accumbens shell and the ventral hippocampus as compared to vehicle-treated subjects. Additional networks affected by fluoxetine treatment included the prefrontal-cingulate cortex and brainstem nuclei linked to depression (e.g., habenula, dorsal raphe and interpeduncular nucleus). We concluded that corticolimbic regions such as the prefrontal-cingulate cortex, nucleus accumbens, ventral hippocampus and key brainstem nuclei represent important contributors to the neural network mediating fluoxetine antidepressant action.

Learned helplessness in animals has been used to model disorders such as depression and post-traumatic stress disorder (PTSD), but there is a lack of knowledge concerning which individual behavioral characteristics at baseline can predict helpless behavior after exposure to inescapable stress. The first aim of this study was to determine behavioral predictors of helplessness using the novel and familiar open-field tests, sucrose consumption, and passive harm-avoidance tasks before learned helplessness training and testing. Individual differences in physiologic responses to restraint stress were also assessed. A cluster analysis of escape latencies from helplessness testing supported the division of the sample population of Holtzman rats into approximately 50% helpless and 50% non-helpless. Linear regression analyses further revealed that increased reactivity to the novel environment, but not general activity or habituation, predicted susceptibility to learned helplessness. During restraint stress there were no mean differences in heart rate, heart rate variability, and plasma corticosterone between helpless and non-helpless rats; however, a lower heart rate during stress was associated with higher activity levels during exploration. Our most important finding was that by using an innocuous screening tool such as the novel and familiar open-field tests, it was possible to identify subjects that were susceptible to learned helplessness.

Three experiments investigated learned helplessness in rats manipulating response topography within-subject and different intervals between treatment and tests among groups. In Experiment 1, rats previously exposed to inescapable shocks were tested under an escape contingency where either jumping or nose poking was required to terminate shocks; tests were run either 1, 14 or 28 days after treatment. Most rats failed to jump, as expected, but learned to nose poke, regardless of the interval between treatment and tests and order of testing. The same results were observed in male and female rats from a different laboratory (Experiment 2) and despite increased exposure to the escape contingencies using a within-subject design (Experiment 3). Furthermore, no evidence of helplessness reversal was observed, since animals failed to jump even after having learned to nose-poke in a previous test session. These results are not consistent with a learned helplessness hypothesis, which claims that shock (un)controllability is the key variable responsible for the effect. They are nonetheless consistent with the view that inescapable shocks enhance control by irrelevant features of the relationship between the environment and behavior.

Learned helplessness represents a failure to escape after exposure to inescapable stress and may model human psychiatric disorders related to stress. Previous work has demonstrated individual differences in susceptibility to learned helplessness. In this study, we assessed different factors associated with this susceptibility, including strain, sex, and open-field behavior. Testing of three rat strains (Holtzman, Long-Evans, and Sprague-Dawley) revealed that Holtzman rats were the most susceptible to helplessness. Holtzman rats not only had the longest escape latencies following inescapable shock, but also showed spontaneous escape deficits in the absence of prior shock when tested with a fixed-ratio 2 (FR2) running response. Moreover, when tested with fixed-ratio 1 (FR1) running - an easy response normally unaffected by helplessness training in rats - inescapable shock significantly increased the escape latencies of Holtzman rats. Within the Holtzman strain, we confirmed recent findings that females showed superior escape performance and therefore appeared more resistant to helplessness than males. However, regression and covariance analyses suggest that this sex difference may be explained by more baseline ambulatory activity among females. In addition, some indices of novelty reactivity (greater exploration of novel vs. familiar open-field) predicted subsequent helpless behavior. In conclusion, Holtzman rats, and especially male Holtzman rats, have a strong predisposition to become immobile when stressed which interferes with their ability to learn active escape responses. The Holtzman strain therefore appears to be a commercially available model for studying susceptibility to helplessness in males, and novelty-seeking may be a marker of this susceptibility.

The transient behavioral deficit produced in rodents by typical learned helplessness (LH) procedures limits the utility of LH in identifying the therapeutic mechanisms associated with chronic antidepressant administration. In addition, LH procedures do not differentiate between different antidepressant classes as observed in the forced swim test.

To produce both a long lasting and antidepressant reversible behavioral deficit in a modified LH procedure that administers inescapable shock (IS) in the same operant chamber used for shuttle box escape testing.

A single IS session produced a robust increase in the number of escape failures (FR-2 escape contingency) that endured for at least 21 days. This escape deficit was reversed by desipramine (24 mg/kg/day, 6 days) at the first shuttle box session. Fluoxetine (5 mg/kg/day, 6 and 21 days) improved escape performance only after repeated test sessions. In contrast, fluoxetine (5 mg/kg/day, 21 days) completely reversed the first shuttle box test escape deficit induced by exposure to a chronic unpredictable stress procedure devoid of shocks or exposure to operant chambers. These differential drug effects may be due to the presence or absence of contextual cues during escape testing. Repeated re-exposure to the IS context enhanced the FR-2 escape deficit.

These data suggest that performing escape testing and IS in the same environment improves the preclinical modeling of the time-dependency and behavioral pattern of antidepressant response observed clinically. Additionally, contextual information associated with the IS environment modulates escape performance and may interact differentially with discrete antidepressant classes.