Helplessness-like escape deficits of NIH-HS rats predict passive behavior in the forced swimming test: Relevance for the concurrent validity of rat models of depression

Marta Palència, Sira Díaz-Morán, Carme Mont-Cardona, Toni Cañete, Gloria Blázquez, Esther Martínez-Membrives, Regina López-Aumatell, Adolf Tobeña, Alberto Fernández-Teruel

Medical Psychology Unit, Department of Psychiatry & Forensic Medicine, Institute of Neurosciences, School of Medicine, Autonomous University of Barcelona, Barcelona, Spain.
Medical Psychology Unit, Department of Psychiatry & Forensic Medicine, Institute of Neurosciences, School of Medicine, Autonomous University of Barcelona, Barcelona, Spain; Welcome Trust Centre for Human Genetics, Oxford, UK.

DOI: 10.4236/wjns.2013.32012                

Abstract

The genetically heterogeneous NIH-HS rat stock has been characterized by its response to anxiety- and fear-inducing situations, thus leading to the conclusion that they are a rather anxious and passive coping type of rats. Taking advantage of these profiles, and knowing that they show very poor performance in the two-way active (shuttle box) escape/avoidance task, we have tested NIH-HS rats (n = 80) in the forced swimming test (FST) as well as we have studied escape response deficits (i.e. response failures) of the same animals in the two-way shuttle box task. They were also tested for anxiety in the elevated zero-maze. The goal of such a study was that of investigating whether there are associations or relationships among helplessness-like or passive coping responses between both models of depression, i.e. the FST and the helplessness-like escape deficits in the shuttle box task. The results for the first time show associations among responses from both depression models and that selecting rats for displaying extreme (active or passive) responses in one of the models predict in a coherent manner (according to the hypothesis) their behaviour in the other model. These findings are discussed in the context of the concurrent validity of both models of depression as well as concerning the possible relevance of NIH-HS rats as a tool for future studies on this field.

Keywords

Genetically Heterogeneous Rats; NIH-HS Stock; Forced Swimming Test; Helplessness; Passive Coping; Depression; Anxiety

1. INTRODUCTION

The forced swimming test (FST), first introduced by Porsolt et al. [1], is a behavioral procedure used to reproduce in rodents passive coping responses to stress that may model certain aspects of human depression. This test has been widely used to evaluate the clinical efficacy of antidepressant treatments. Thus, antidepressant drugs with different pharmcodynamic properties (see [2]), as well as electroconvulsive shock, transcranial magnetic stimulation, and vagus nerve stimulation [3,4] decrease the duration of immobility and increase active escapedirected behaviors (i.e. active swimming or struggling). The FST is also sensitive to a number of experimental models of predisposition to depression, including chronic mild stress and unpredictable vs. predictable acute stress [5,6], and has been used as a marker of depressive-like behavior in selectively bred lines or strains of rodents [7-11].  

The learned helplessness model of depression, first described by Overmier and Seligman [12], is based on the impairments of escape learning that follow exposure to inescapable (uncontrollable) electric shock. Such a cognitive impairment, jointly with several motivational/ emotional alterations (e.g. anhedonia), provides face validity to the model, while the syndrome reversibility by chronic antidepressant treatment lends predictive validity to it (for review see for instance [13]). While prior uncontrollable/unpredictable stressful (shock) experience is commonly used for induction of learned helplessness, some particular rodent strains/lines (or subpopulations within a given strain) provide models in which helplessness-like responses can be observed even without prior stress exposure, that is to say, animals selected according to a given behavioural criterion (related with emotionality/fearfulness; e.g. [14,15]), specific rat strains (e.g. the WKY rat strain; [7,10,13,16]) or selectively bred strains, i.e. psychogenetically or pharmacogenetically selected rodent strains (e.g. [9,11,17-21]), may provide “depresssion” models per se, without the need of using previous exposure to predisposing factors such as stressful experiences (see further in the “Discussion”).

The “National Institutes of Health -N/NIH-Genetically Heterogeneous Rat Stock” (hereafter, NIH-HS rats) was developed by Hansen and Spuhler [22] with the aim of having a more naturalistic, genetically heterogeneous rat stock which could yield a broad-range distribution of responses to experimental conditions and could serve as a base population for selection studies. With this aim, the NIH-HS rat stock was formed through an eight-way cross among eight inbred rat strains (see “Methods” below; Hansen and Spuhler [22]). Recent genetic studies have demonstrated that the NIH-HS rat stock is a unique animal model for the simultaneous identification and fine mapping of QTLs (Quantitative Trait Loci) even to a gene resolution level (for reviews see [23,24]). From the phenotypic stand point, our behavioural and hormonal studies of the heterogeneous rat stock clearly indicate that the NIH-HS rat colony (established in our laboratory, at Autonomous University of Barcelona, in 2004) exhibits a behavioural “defensive” profile indicating that these animals are rather fearful and anxious, presenting a predominantly passive/reactive coping style as well as stressprone (i.e. enhanced) hormone response profile. Thus, the anxiety/fear and stress hormone response profiles of NIH-HS rats are much closer to the (high anxious, stress prone) passive coper RLA-I rats than to the (low anxious, stress resistant) proactive coper RHA-I rats [23,25-30].

Thus, taking advantage of the fact that we know the NIH-HS rats are, as a population, passive coping rats with very poor ability for the acquisition of the twoway—shuttle box-active escape/avoidance task (see [23, 25,27, 28]), we made the hypothesis that at least a subsample of the heterogeneous rat stock could display a kind of “spontaneous” (i.e. as we are not using any prior stress) helplessness in the shuttle box task, i.e. a relatively elevated number of escape failures (hereafter, response failures or “R failures”), and that such a “helpless” profile would show some associations with passive coping strategies in the forced swimming test (i.e. relatively high levels of immobility and/or low levels of struggling -escape-directedbehaviour).

It is important to notice here that no previous work has addressed the issue of whether helplessness-like responses (i.e. response failures) in the shuttle box escape task and passive/depressive-like behaviour in the FST (i.e. high levels of immobility and/or low levels of struggling) are related or associated to each other in heterogeneous (outbred, unselected) rats, an issue that is important for the concurrent validity of both procedures as models of depressive-like symptoms.

2. METHODS

2.1. Animals

Subjects were 80 male NIH-HS rats, coming from 40 different litters and being 3 months old at the beginning of the experiment. They were housed in pairs in macrolon cages (50 cm × 25 cm × 14 cm), maintained with food and tap water available ad libitum, under conditions of controlled temperature (22˚C ± 2˚C; 50% - 70% humidity) and a 12-h light/12-h dark cycle (lights on at 08:00 h).  

2.2. Procedure and Apparatus

Experiments were performed during the light cycle, between 09:00 and 19:00 h., and in accordance with the Spanish legislation on “Protection of Animals Used for Experimental and Other Scientific Purposes” and the European Communities Council Directive (86/609/EEC) on this subject. The experimental protocol was approved by the Autonomous University of Barcelona Ethics committee. Three behavioral tests were administered (in order: FST, elevated zero-maze and two-way escape/ avoidance-shuttle box-task), with 1 week elapsing between each two consecutive tests. The sequence and the characteristics of the tests were as follows:

2.2.1. Forced Swimming Test (FST)

The procedure used was very similar to that described by Porsolt et al. [1] and modified by Detke et al. [31] (see also [32]). Swim sessions were conducted by plunging the rats in individual vertical plastic cylinders (60 cm tall X 19 cm diameter), containing 40 cm of water at 24˚C - 26˚C. The experimental procedure consisted of two sessions: an initial 15-min “pretest” followed 24 h later by a 5-min test. Following swim sessions, rats were removed from the cylinders, gently dried with paper towels and returned to their home cages. Water was changed for each rat. The apparatus was situated in a black-painted testing room dimly illuminated with white fluorescent light (36 W) situated in the ceiling (2.5 height), and the behavior was videotaped and measured outside the testing room. Variables recorded during two sessions, were: 1) Immobility: rat was making only those movements necessary to keep its head above water; 2) Struggling: rat was making active swimming motions, thus displaying vigorous (escape-directed) activity with its four legs and keeping a vertical position [31].

2.2.2. Elevated Zero-Maze (ZM)

The maze, similar to that described by Shepherd et al. [33], comprised an annular platform (105 cm diameter; 10 cm width) made of black plywood and elevated to 65 cm above the ground level. It had two open sections (quadrants) and two enclosed ones (with walls 40 cm height). The subject was placed in an enclosed section facing the wall. The apparatus was situated in a black testing room, dimly illuminated with red fluorescent light, and the behavior was videotaped and measured outside the testing room. Time spent in the open sections (Time), number of entries in the open sections (Entries), number of stretched attend postures (SAP) were measured for 5 minutes (for details of the validity of the test see references in [26-29,33].  

2.2.3. Two-Way Active, Shuttle Box Escape/Avoidance Task (SH)

The experiment was carried out with 2 identical shuttle boxes (Letica, Panlab, Barcelona, Spain), each placed within independent, sound-attenuating boxes constructed of plywood. A dim and diffuse illumination was provided by a fluorescent bulb placed behind the opaque wall of the shuttle boxes. The experimental room was kept dark. The shuttle boxes consisted of two equally sized compartments (25 cm × 25 cm × 28 cm), connected by an opening (8 cm × 10 cm). A 2400-Hz, 63-dB tone plus a light (from a small, 7-W lamp) functioned as the CS (conditioned stimulus). The US (unconditioned stimulus), which commenced at the end of the CS, was a scrambled electric shock of 0.7 mA delivered through the grid floor. Once the rats were placed into the shuttle box, a 4-min familiarization period elapsed before training commenced. Each training trial consisted of a 10-s CS, followed by a 20-s US. The CS or US was terminated when the animal crossed to the other compartment, with crossing during the CS being considered as an avoidance response and during the US as an escape response. Once a crossing had been made or the shock (US) discontinued, a 60-s inter-trial interval (ITI) was presented during which crossings (ITC) were also scored. Training consisted of a single 50-trial session.

The variables recorded were 1) “context-conditioned freezing”, i.e. the time spent freezing (Freezing) during the first five inter-trial intervals of the training session (when no rat had made any avoidance response); 2) the number of escape response failures during training (“R failures”, i.e. the rat does not cross to the opposite compartment neither during CS nor US presentation; this is the first “helplessness”-like variable); 3) the average response latency for the first ten trials (“10Latency”, because this is the phase of training where most “R failures” are present, about 90% of them; this is the second “helplessness”-like variable); and 4) the number of avoidances (Avoidances) during the whole 50-trial training session. Context-conditioned freezing, was measured by two trained observers (between-observer reliability, r > 0.95) as the time a rat spent completely motionless except for breathing movements (see details and references on the validity of this task in [26-30,34]).

2.3. Statistical Analysis

A correlation matrix and obliquely-rotated (oblimin direct) factor analysis were performed to study the associations among the different and most relevant dependent variables (10 variables) of the three behavioral tests (these 10 representative variables were selected according to—and to be consistent with—the same criteria we have used in previous studies; see [26-30]). Finally, we constituted extreme subgroups of the NIH-HS rats by selecting animals with values ± 1 SD for (see Tables 1 and 4): 1) “Immobility5” and “Struggling5” combined (thus an extreme “Passive coping” subgroup—i.e. low “Struggling5” and high “Immobility5”, n = 8—and an extreme “Active coping” subgroup—i.e. high “Struggling5” and low “Immobility5”, n = 13—were constituted); 2) extreme scores in “R failures” (i.e. response failures), thus forming a “No R failures” (n = 30) sub-

Table 1. Descriptive statistics of the most relevant behavioral variables from the elevated zero-maze test, the forced swimming test and the shuttle box training session.

group and a “High R failures” (n = 10) subgroup; and 3) extreme scores in “10Latency” (the average response latency for the first 10 trials of shuttle box escape training), thus forming a “Short 10Latency” (n = 15) subgroup and a “Long 10Latency” (n = 14) subgroup (see Table 1 for descriptive statistics—mean ± 1 SD—of the whole sample, and Table 4 for descriptions and comparisons between those extreme subgroups).

Student’s t-tests for independent samples were used to analyze the differences between the values of each pair of extreme subgroups.

3. RESULTS

Table 1 shows the descriptive statistics of the whole NIH-HS sample for variables from the ZM, FST and the shuttle box training session.

Concerning the main hypothesis of the present study, the correlation matrix (Table 2) mainly shows: 1) significantly moderate to high correlations among FST measures (r = 0.68 between Struggling15' and Struggling5'; r = 0.43 between Immobility15' and Immobility5', and negative correlation—r = −0.56—between Struggling5' and Immobility5'); 2) a low but significant negative correlation between Struggling5' and “10Latency” (r = −0.25; and, 3) as expected, there are positive correlations among shuttle box variables (e.g. r = 0.31, between Freezing and “R failures”, and r = 0.64 between “10Latency” and “R failures”) (see Table 2).  

Obliquely-rotated factor analysis (Direct oblimin), to make more conceptual sense to those correlational patterns, was then applied to the 10 target variables (Table 3). A four-factor solution was obtained in which the first factor was dominated by FST variables, the second factor was especially represented by “Immobility” variables, the third is essentially a ZM factor and the fourth is exclusively a shuttle box task factor (see Table 3(a)). This first test-related 4-factor structure was then reduced to a twofactor solution after applying the Catell’s Scree test (Table 3(b); see the criteria for this test in [25,26]) according to the eigenvalues of the first two factors. This solution (Direct Oblimin) showed two independent factors which explained 42.5% of the variance (correlation between factors = 0.078; Table 3(b)). Factor 1, tentatively named “Coping style”, grouped FST variables (“Struggling5'” and “Struggling15'”, loading 0.74 and 0.85 respectively, and “Immobility15'”, loading −0.66) in association with the two helplessness-like measures from the shuttle box escape task (“10Latency” and “R failures”, loadings of −0.29 to −0.38, respectively) and also with low loadings of ZM measures (SAP and “Entries”, loading 0.31 and 0.26 respectively). Factor 2 mainly represented conditioned conflict (thus, temptatively named “Conflict solving”; [26,27]), as indicated by the loadings of shuttle box task variables (loadings of −0.70, −0.63, −0.44 for “R failures”, “10Latency” and “Freezing”, and 0.62 for “Avoidances”) (Table 3(b)).

Table 4(a) shows that selection for displaying extreme (combined) “Immobility5'” and “Struggling5'” scores, thus obtaining a “Passive coping” subgroup and an “Active coping” subgroup, leads to differences in helplessness-related responses, as the “Passive coping” group shows higher number of “R failures” (t(19) = 2.1, p < 0.05) and longer “10Latency” (t(19) = 3.1, p < 0.01) than the “Active coping” subgroup (Table 4(a)). Most outstandingly, when selecting subgroups of rats with relatively high helplessness-like behaviour (i.e. “High R

Conflicts of Interest

The authors declare no conflicts of interest.

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