Approach-avoidance Test Research Articles

Rats show aversion to argon-induced hypoxia

Laboratory rats and mice are commonly killed using carbon dioxide gas. However, recent studies have shown that rodents find this gas aversive. Argon is a tasteless and odourless gas that causes hypoxia by displacing air. The aim of the present study was to use approach–avoidance testing to evaluate rats’ responses to argon-induced hypoxia when argon was introduced over a range of flow rates. The experiment was run in two phases: Phase 1 tested low flow rates (40–120% of the test cage volume per min) and Phase 2 tested higher flow rates (120–239%). Rats were trained to enter the bottom cage of a two-cage apparatus for a reward of 20 Cheerios™. Argon (or air as a control) entered the cage at the assigned flow rate as soon as the rats started eating. During control trials with air, rats ate for an average (±standard deviation) of 252 ± 22 s in Phase 1 and 232 ± 51 s in Phase 2, with no effect of flow rate. When tested with argon, rats never remained in the test cage long enough to lose consciousness. They consumed fewer Cheerios™, stopped eating sooner, and left the test cage quicker than when tested with air. Rats ate for only 104 ± 31 s when tested at even the lowest argon flow rate, and this time decreased with increasing flow rates in both Phase 1 ( P < 0.0001) and Phase 2 ( P < 0.0001). The oxygen (O 2) concentration at which rats stopped eating ( P < 0.0001) and left the test cage ( P < 0.0009) decreased over the lower range of flow rates tested in Phase 1, but stabilized at about 7.7 and 6.8%, respectively, with the higher flow rates tested in Phase 2; these O 2 concentrations are too high to cause unconsciousness or death. Although humans exposed to hypoxia report only subtle symptoms, rats are more sensitive than humans to changes in O 2. We conclude that rats are averse to argon-induced hypoxia and that alternative methods of euthanasia are still required.

The validity of using an approach-avoidance test to measure the strength of aversion to carbon dioxide in rats

Approach-avoidance tests are often used to estimate the strength of aversion, by making food rewards available during exposure to a stimulus at a known level of food deprivation. It is reasoned that if subjects are unwilling to tolerate the stimulus even when severely food deprived, their aversion to the stimulus must be strong. However, this interpretation assumes that the effects of the competing motivations are additive. The present study tested the validity of this assumption by measuring whether rats’ tolerance of CO 2 increased monotonically with food deprivation. In two experiments, 21 male Wistar rats were exposed to a gradually increasing CO 2 concentration while eating sweet food items. Rats were tested at seven deprivation levels ranging from 0 to 24 h (Experiment 1), or from 0 to approximately 10 h (Experiment 2). Subjects always left the gas chamber, but the concentration at which they left did not increase monotonically with deprivation level, instead showing a bitonic response ( F 1,64 = 5.30, P = 0.025 when deprivation was defined in terms of food intake in the previous 24 h; F 1,61 = 6.07, P = 0.017 when defined as time since last eating). This result indicates an interaction between hunger and CO 2 aversion, contrary to the assumption of additivity. We conclude that the approach-avoidance method cannot be used to ‘titrate’ aversion to CO 2 against hunger, perhaps because hunger causes anxiety.

Effects of novelty on rats’ responses to CO 2 exposure

Laboratory rats are routinely killed using a gradually increasing concentration of carbon dioxide (CO 2) gas. However, CO 2 exposure has been shown to cause aversion and to increase exploratory and escape behaviours in rats. We examined whether these responses are due, in part, to novelty, using two different approaches. For the first experimental approach, we used approach-avoidance testing to examine gas aversion by pairing gas exposure with a food reward. Rats were tested with repeated exposure to a gradually increasing concentration of either CO 2 (Experiment 1) or a novel odour (Experiment 2) to determine whether these stimuli cause aversion, and to document whether rats show habituation to these stimuli. We reasoned that aversion associated with novelty would decline with repeated exposure, but that aversion due to other factors would not. During control sessions in Experiment 1, rats were tested with air and they ate for an average of 266 ± 4 s (mean±S.E.M.). However, on the four CO 2 test days they stopped eating after only 30 ± 2 s. Across the first three days of testing with CO 2, the latency for rats to stop eating and leave the test cage did not change, and the average CO 2 concentrations at these time points were 11 and 14%, respectively. On the final day of testing with CO 2, after eight additional days of CO 2 exposure, these latencies had increased slightly, but rats were still leaving the chamber at only 18% CO 2. In contrast, rats in Experiment 2 showed similar latencies to stop eating and leave the test cage with both air and a novel odour (232 versus 235 ± 14 s), and their responses did not change with repeated exposure. These results suggest that novelty has only a minor effect on rats’ aversion to CO 2. For the second experimental approach, we examined the behavioural responses of rats during baseline and during exposure to a gradually increasing concentration of either CO 2 or peppermint odour (Experiment 3) to determine which of the behavioural responses observed during CO 2 exposure can be attributed to novelty. In comparison to baseline, rats that underwent forced exposure to either CO 2 or a novel odour showed increased activity and rearing, and spent more time with the nose touching the cage lid. However, only one of 16 rats performed escape behaviours during exposure to the novel odour, while 10 of 16 rats performed escape behaviours during CO 2 exposure. These results indicate that exploratory behaviours observed during CO 2 exposure could be due to novelty, but that escape behaviours are not. Both of the experimental approaches indicate that novelty accounts for only a minor portion of rats’ responses to CO 2 exposure.

Social stress during adolescence in Wistar rats induces social anxiety in adulthood without affecting brain monoaminergic content and activity

Adolescence has been described as an important period to acquire social competences required for adult life. It has been suggested that early stress experiences could affect the development of the brain at different levels. These changes in the brain during adolescence may be related with the development of psychopathologies such as depression and social anxiety in adulthood. In the first experiment, we examined long-term effects of repeated social stress during adolescence on adult social approach–avoidance behavior. For that purpose, adolescent male Wistar rats were exposed twice at postnatal day (Pnd) 45 and Pnd48 to the resident–intruder paradigm followed by three times psychosocial threat with the same resident. Three weeks after the last psychosocial threat experience the animals were behaviorally tested in a social approach–avoidance test. Socially stressed animals spent less time in the interaction zone with an unfamiliar male adult rat. These data suggest that animals exposed to social stress during adolescence show a higher level of social anxiety in adulthood. In the second experiment, we investigated whether these long-term effects of social stress during adolescence on behavior draw a parallel with changes in brain monoamine content, biosynthesis and turnover. Using the same experimental design as in the first experiment, HPLC analysis of various brain regions showed that there were no differences in monoamine content, monoamine biosynthesis and monoamines activity in the prefrontal cortex, hippocampus, hypothalamus and striatum in adulthood. These results indicate that long-lasting changes in social behavior following social stress during adolescence are not accompanied by changes in brain monoamine content, biosynthesis and turnover.

Effect of flow rate on aversion to gradual-fill carbon dioxide exposure in rats

Laboratory rats are commonly killed by a gradually increasing concentration of carbon dioxide (CO2), but an optimal flow rate for minimizing distress during this procedure has not yet been identified. While fast flow rates minimize time to unconsciousness, slow flow rates may allow an animal to lose consciousness before CO2 concentrations become sufficient to cause pain and dyspnoea. We used an approach-avoidance test, where access to a food reward was contingent on entering a chamber that was gradually filling with CO2, in order to determine whether rats’ aversion to gradual-fill CO2 exposure varies with flow rate. During testing, the exposure chamber was filled with either air or CO2, and we examined CO2 flow rates ranging from 3% to 27% of the chamber volume/min. The rats left the chamber before loss of consciousness at all flow rates, and at higher flow rates they left the chamber more quickly. The maximum CO2 concentrations tolerated were at intermediate flow rates, but even at the optimal flow rate of 14%/min rats left the chamber once the CO2 concentration reached just 15.9%, and no rats remained in the chamber until they lost consciousness. Rats tolerated even lower concentrations when flow rates were higher and lower, but this response function was not steep. For example, at the highest flow rate of 27%/min rats left the chamber at a CO2 concentration of 13%. In conclusion rats are sensitive to CO2 flow rate and are most tolerant of CO2 when introduced at a flow rate of around 15%. Despite this modest effect of flow rate, gradual-fill CO2 exposure is still aversive to rats over all flow rates tested and research into alternative methods of euthanasia is now urgently required.

Rats avoid exposure to carbon dioxide and argon

Abstract A common method for euthanizing laboratory rats is exposure to carbon dioxide (CO 2 ) gas, either in a pre-filled chamber or using a gradually increasing concentration. Physiological and human self-report data suggest that this procedure has the potential to cause pain and distress, but behavioural responses by rats during exposure have been variable. We used an approach-avoidance test to examine rat aversion to CO 2 exposure, pairing a food reward with gas exposure. First, we examined rats’ responses to air and to static CO 2 concentrations of 5, 10, 15 and 20%. Second, we tested rats with a gradually increasing concentration of CO 2 at a rate of 17% of the chamber volume per minute. Finally, we examined rats’ responses to 90% argon gas, which has been suggested as a suitable replacement for CO 2 euthanasia, and compared it to air. Rats tolerated prolonged exposure to 5 and 10% CO 2 , but the amount eaten and the latency to leave the test cage decreased significantly when the test cage contained 15% CO 2 . At 20% CO 2 , the majority of animals refused to eat and left the test cage immediately. During gradual-fill exposure, animals stopped eating and left the chamber when CO 2 concentrations reached on average 17.3 ± 2.1% and 18.4 ± 2.0%, respectively. Rats did not remain in the test cage for long enough to lose consciousness with any of the static or gradual-fill conditions. When the test cage contained 90% argon, no rats ate and they either refused to enter or left the test cage immediately. These results suggest that both CO 2 and argon euthanasia cause aversion in rats prior to loss of consciousness.

Social approach–avoidance behavior of a high-anxiety strain of rats: effects of benzodiazepine receptor ligands

To better understand anxiety disorders with social impairments as well as to identify new treatments, it is important to develop preclinical procedures involving social components of anxiety. Recently, a novel procedure was reported: the rat Social Approach-Avoidance (SAA) test. In this test, the time spent by a test rat in a large social compartment containing an unfamiliar stimulus rat reflects the anxiety state of the animal. It was shown that pre-stressing the test rat increased the avoidance of the social compartment as characterized by an increase in the time spent in the nonsocial compartment. (1) To use a high-anxiety strain, F-344 rats, instead of pre-stressed animals, (2) to automate the test by using video tracking to measure time spent in both compartments and their subdivisions, and (3) to validate this modified test with known benzodiazepine receptor ligands. F-344 rats were treated with either chlordiazepoxide or diazepam (benzodiazepine receptor agonists), or RO 19-4603 or FG 7142 (benzodiazepine receptor inverse agonists). The agonists produced anxiolytic-like effects, whereas the inverse agonists produced anxiogenic-like effects. These effects were most marked in the two extreme zones in terms of distance to the stimulus rat. F-344 rats display spontaneous avoidance behavior in the modified SAA procedure, and approach-avoidance behavior in these rats is sensitive to benzodiazepine agonists and inverse agonists in a bidirectional manner. The finding that the assessment of time spent into two virtual zones in each of the compartments markedly increased the sensitivity to both anxiolytics and anxiogenics will be discussed using the concept of physical defensive distance.

Habituation and extinction in an approach–avoidance test: An example with sheep

During a behavioural test, the test animal's perception of the test situation may change, e.g. as a result of habituation. This change can occur within a test or between repetitions of a test. It is particularly important in the arena test because in an approach–avoidance situation, the negative stimulus may not be followed by the negative consequences it is initially associated with (tested ‘in extinction’). In this experiment, we tested single sheep for 10 min in a situation of conflict between either being too close to a negative stimulus (human being) or too far away from a positive stimulus (conspecifics). The test was carried out over four consecutive days, each sheep being tested once a day. We recorded the distance between the test sheep and the conflicting stimuli, the occurrence of urination and rumination and the duration of sniffing during the test. Principal components analysis (PCA) revealed three main components of distance; the average (76%), a linear (11%) and a quadratic change across time (6%). Distances decreased within and across tests and the occurrence of urination decreased, while rumination and sniffing increased across tests. These changes of behaviour across time suggest that extinction of the conditioned response occurred within as well as across tests. These results highlight the importance of choosing the appropriate duration for a test, particularly when the test is carried out in extinction.

Assessment of the aversion of hens to different gas atmospheres using an approach-avoidance test

Approach-avoidance tests were conducted to evaluate the responses of laying hens to different gas atmospheres. Twelve hens were trained in a test apparatus consisting of a raised entry chamber connected by a descending chute to a lower chamber into which gas could be injected. Feed was removed from hens overnight before training sessions, and layer feed was presented in the lower chamber as the stimulus to motivate hens to proceed through the apparatus. Once hens were trained to a stable minimum time taken to eat after being placed in the apparatus, approach-avoidance tests were initiated. These resembled training sessions except that they were conducted in mid-afternoon, with feed having been removed from the hens in the morning, and one of six gas treatments was injected into the lower chamber. The gas atmospheres evaluated were: air, 30% CO 2-in-air, 45% CO 2-in-air, 60% CO 2-in-air, 70% Ar/30% CO 2, and Ar. Each hen was tested once in random order with each atmosphere. Days of testing were alternated with days of retraining to minimize extinction of the learned response and the chance that exposure to a given atmosphere might affect a hen in its next test. Exposure to 45% CO 2-in-air and Ar appeared to elevate the time taken by hens to leave the upper chamber of the test apparatus in the subsequent retraining session, but the retraining appears to have extinguished any carryover effect because latencies to leave the upper chamber were small and not different between treatments during tests. Hens were evidently able to detect all the atmospheres enriched with CO 2, and the number of stops and retreats made during the approach to the lower chamber did not differ between these atmospheres. The control and Ar treatments had the lowest incidences of stops and retreats. Every hen but one was stunned on its first encounter with a CO 2-enriched atmosphere. Similarly, every hen but one was stunned in Ar. Overall, there were no significant differences among the stunning gas treatments in the percentages of hens stunned. Hens in the 30% CO 2-in-air treatment entered the lower chamber at almost the same frequency as in the Ar treatment. Most hens became stunned in the corridor when exposed to 60% CO 2-in-air, while the percentages of hens entering the lower chamber before being stunned were intermediate for 45% CO 2-in-air and 70% Ar/30% CO 2. From a behavioral standpoint, there does not appear to be a welfare disadvantage to the use of mixtures of up to 60% CO 2-in-air to stun/kill chickens compared to 70% Ar/30% CO 2. The results do suggest a modest welfare advantage for Ar, but not one so substantial that it could not be readily balanced by other welfare related considerations.

Use of Statistical Tests of Equivalence (Bioequivalence Tests) in Plant Pathology

Hypothesis tests currently used in plant pathology are almost always based on a null hypothesis of equal means. In this framework, the experimenter determines whether or not there is evidence that the means are, in fact, different. This framework makes sense for many common questions such as whether a new management technique gives an increase in yield over existing management techniques. But suppose, for example, that a disease management technique is so effective that an experimenter is interested in whether its use in the presence of disease achieves the same yield as in the absence of disease. In this case, a more appropriate null hypothesis would be that mean yields are different. Examples of questions in plant pathology for which a null hypothesis of equal treatment means is not suitable include (corresponding phrasing for one-sided questions is in parentheses as appropriate):

Lesions of parvocellular subdivisions of the hypothalamic paraventricular nucleus alter open field behavior and acquisition of sensory and spatial discrimination

Rats with ibotenic acid (IBO) lesions of the hypothalamic paraventricular nucleus (PVN) were compared with operated control animals over a battery of tests designed to assess memory- and arousal-related behavioral processes. At the dose employed in these experiments, ibotenic acid selectively destroys parvocellular elements of the PVN, leaving magnocellular subdivisions relatively intact, allowing for experimental dissection of the influence of parvocellular and magnocellular PVN neuronal populations on the behavioral parameters measured. IBO-treated rats showed a greater incidence ofrearing behavior and exhibited greater levels of total and central ambulation in an open field than control rats. Acquisition of both sensory and spatial reward contingencies were retarded in the IBO-lesion group; however, no differences were evident between IBO-treated and control groups in an approach-avoidance test, nor in the ability to perform the spatial and sensory discrimination tasks to a criterion level of accuracy. Histological examination verified that bilateral IBO lesion destroyed parvocellular elements of the PVN, while sparing the majority of magnecellular neurons. Results suggest that parvocellular PVN lesion alter behavioral performance via interactions with physiological systems overning arousal level.

Naturalistic behavioral assessment of anxiolytic properties of benzodiazepines and ethanol in mice

Effects of chlordiazepoxide (CDZ), flurazepam (FLU) and diazepam (DZP) were assessed in mice by observation of digging behavior in an escape task. Reliable effects on escape latencies and number of exploratory head pokes were detected at 2 mg/kg for CDZ and FLU. DZP produced dose-dependent effects at 0.2, 0.4 and 0.6 mg/kg. Ro 15-1788 antagonized the effects of DZP (0.6 mg/kg). Ethanol (0.75 and 1.5 g/kg) produced effects similar to those of the benzodiazepines. The dynamics of this naturalistic behavior avoid the need for direct aversive stimulation or food/water deprivation to set up an approach avoidance or conflict test. Combined with a multivariate (discriminant) analysis, the approach provides a sensitive assessment of anxiolytic drug activity.

Toward an animal model of depression : A study of separation behavior in dogs

Six dogs were raised in an experimental setting by the author who was their sole consistent object. The animals were ranked daily in an approach-avoidance test and their temperament characterized in approach-avoidance terms. Eleven independent observers scored the animals on object seeking, object avoiding and aggressive behavior in each of three conditions (1) a pre-separation period in which the animals' relationship with the author was undisturbed (2) a separation condition in which the animals had no contact with the author and (3) a reunion period. Body weight and gross motor activity were also measured. Separation was associated with increases in object seeking for animals of the approach temperament and increases in object avoidance and aggressive behavior for animals of the avoidance temperament. Activity was significantly decreased in those animals for whom reliable data was obtained. Reunion was associated with further deviations from pre-separation behavior patterns. These results tentatively indicate that models of separation and depression can be constructed in experimental animals.

Acquisition of a passive avoidance response as determined by variations in prior aversive stimulation

The effects of various forms of prior enforced sleep Td (SD) and of pre-shock (PS), as presumed traumatizing agents, were studied in rats 15 days following treatment using an approach-avoidance test procedure involving successive response to food, mild punishment, food, and weak punishment. PS Ss and Ss whose SD treatment involved prolonged walking on an abrasive surface exhibited greater avoidance of the food area to mild punishment than controls, while Ss whose SD treatment consisted of prolonged walking on a smooth surface or prolonged standing in deep water exhibited significantly less conflict. With one exception, similar results were obtained under the weak test punishment.