Environmental bias? Effects of housing conditions, laboratory environment and experimenter on behavioral tests.

Behavioral testing does not always yield similar results when replicated in different laboratories, and it usually remains unclear whether the variability in results is caused by different laboratory environments or different experimenters conducting the tests. In our study, we applied a systematic variation of housing conditions, laboratories and experimenters in order to test the influence of these variables on the outcome of behavioral tests. We wanted to know whether known effects of different housing conditions on behavior can be demonstrated regardless of the respective laboratory and experimenters. In this study, we compared the behavior of mice kept under enriched housing conditions with mice kept in unstructured cages regarding their exploratory, locomotor and anxiety-related behavior in the barrier test, in the open-field test and in the elevated plus-maze test. Experiments were conducted by six different persons in two different laboratories. In spite of an extensive protocol standardizing laboratory environment, animal maintenance and testing procedures, significant differences in absolute values between different laboratories as well as between different experimenters were noticed in the barrier test and in the elevated plus-maze test but not in the open-field test. However, with regard to the differences between enriched and unstructured housing conditions, overall consistent results were achieved by different experimenters in both laboratories. We conclude that the reliability of behavioral phenotyping is not challenged seriously by experimenter and laboratory environment as long as appropriate standardizations are met and suitable controls are involved.

Adaptation of corticosterone-but not beta-endorphin-secretion to repeated blood sampling in rats.

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.

Effects of cage enrichment on territorial aggression and stress physiology in male laboratory mice.

The activation of different neuroendocrine subsystems depends on the individual perception and coping with the challenging situation, the formulation of these relations by J.P. Henry represents a most useful concept also for the assessment of welfare consequences of particular caging variables. We investigated effects of cage enrichment on behaviour and neuroendocrine activations of male laboratory mice. Mice in enriched cages behaved more aggressive, lacked stable dominance hierarchies and exhibited neuroendocrine alterations depending on their individual social position. Subdominant passive mice were characterized by an augmented adrenal capacity to synthesize epinephrine despite low activities of the tyrosine hydroxylase. Dominant mice showed elevated circulating corticosterone concentrations despite high tyrosine hydroxylase-activities. Findings showed a dissociation of neurosympathetic and adrenomedullary components in subdominant passive mice and a simultaneous activation of sympathetic adrenomedullary and hypothalamo-adrenocortical components in dominant mice. Within the conceptual framework of the Henry model this would suggest different deteriorations of welfare in dominant and subdominant passive mice. In the situation of intensified aggression in the enriched cages the increased epinephrine synthesis in subdominant mice reflect their more frequent receipt of attacks and the elevated corticosterone secretion in dominant mice reflect their hindered ability to control the dominant position.

Dissociation between adrenal tyrosinehydroxylase and phenylethanolamine N-methyltransferase activities following repeated experience of defeats in individually housed male DBA/2J mice.

Adrenal activities of tyrosinehydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were compared between individually housed defeated intruders and socially housed residents (SR) (Experiment 1), and between individually housed defeated intruders and individually housed victor residents (Experiment 2). In addition, corticosterone titers were determined and dominance status of SR (dominant, subdominant active, and subdominant passive) considered. Defeats were induced twice a week for 6 consecutive weeks in short resident-intruder encounters. The main findings were: 1) within the groups of SR, TH and PNMT activities were high in dominant and low in subdominant-passive mice; 2) in Experiment 1, TH activities of individually housed defeated intruders were low like those of subdominant-passive SR whereas their PNMT activities were high like those of dominant SR; 3) in Experiment 2, TH activities were low in both categories of individually kept mice whereas PNMT activities were significantly elevated in the defeated intruders; 4) in both experiments, PNMT activities of intruders correlated significantly with the accumulated number of attacks they had received throughout all resident-intruder tests. Findings show separate effects of permanent social stimulation and of episodic experience of defeats on adrenal catecholamine synthesis. Lack of permanent social stimulation decreased adrenal TH and PNMT activities whereas repeated episodic experience of defeats specifically increased PNMT but not TH activities in individually housed mice.

Effects of environmental enrichment on aggressive behavior, dominance hierarchies, and endocrine states in male DBA/2J mice.

Adult male mice (DBA/2J) siblings were housed three per cage for 6 wk, either in standard cages (SC) or in enriched cages (EC). Both attacks among group members and attacks against strange intruders were monitored once a week within each of 22 experimental groups. According to its attacking behavior, each mouse was categorized into one of three dominance categories: dominant, subdominant active, subdominant passive. Aggressive behavior and social organization were compared between the two types of housing conditions, and the effects of housing condition and dominance category on endocrinological and some organometrical parameters were analysed. The main findings were: (a) Mice in EC attacked intruders significantly more frequently compared to mice in SC; (b) In EC groups the position of the dominant male was less stable than in SC groups; (c) Plasma corticosterone titers (PCT) were significantly elevated in EC. Activities of tyrosinehydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) did not differ significantly between the housing conditions; (d) TH and PNMT activity were significantly enhanced in the dominant males of SC groups compared to subdominant passive males. Intermediate activities for both enzymes were determined for subdominant active males; and (e) PCT were significantly elevated in dominant males of the EC groups compared to subdominant active and subdominant passive males and also compared to the dominant males in the SC groups. Findings suggest that keeping adult male mice in structured cages can result in increased aggression towards intruders, a change in the social organization, and altered endocrine states, depending on the individual dominance position.

The cage design affects intermale aggression in small groups of male laboratory mice: strain specific consequences on social organization, and endocrine activations in two inbred strains (DBA/2J and CBA/J).

Effects of the enrichment of conventional laboratory housing cages with an additional labyrinth on intermale aggression, social organization, and activations of the pituitary-adrenocortical and sympathetic-adrenomedullary neuroendocrine subsystems are compared between adult male DBA/2J and CBA/J mice, differing genetically in their intermale aggression. Mice of both strains were kept three per cage for six weeks either in standard laboratory cages (SC) or in enriched cages (EC). Intermale aggression against a strange intruder and between cagemates was monitored once a week within each group. Dominance relations were derived from the distribution of attacks within a group. Plasma corticosterone titers (PCT) and activities of the tyrosinehydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were determined for each mouse at the end of the study. The main findings were: 1. Intermale aggression increased in both strains in groups kept in EC. 2. In the more aggressive DBA/2J the pattern of social organization shifted from groups with a single permanent dominant mouse in SC to groups with a frequently changing dominant mouse in EC. 3. In CBA interchanges of the dominant mouse was prevailing and did not differ between the two housing conditions. 4. In DBA/2J mice PCT were significantly elevated in EC. 5. In CBA/J mice activities of TH and PNMT were significantly elevated in EC. 6. Body weight gain was significantly delayed in mice kept in EC in both strains. Findings revealed strainspecific environmental effects on both social organization and endocrine states. The PCT increase obtained only in DBA/2J indicate that alterations of the social relations between cagemates are more likely to induce states of stress than an increase in intermale aggression alone.

Social isolation modifies the response of mice to experimental Mengo virus infection.

To investigate the effects of social isolation on host resistance male mice were housed either individually (IH) or in groups of four or five (GH). All animals were infected with MengoM,L virus. Incubation time (INCUB), duration of illness (ILL), death rate (DR), histopathological changes, and serum corticosterone levels (CORT) were recorded. First, the effect of IH starting 4 days prior to infection was studied in 5 different inbred strains. Next, the effect of different IH length was examined, and the role of T-cells was investigated by comparing euthymic (+/+) and athymic (nu/nu) NMRI mice. Finally, the effects of the infection on CORT in IH and GH mice were compared in C57BL/6 mice. The major findings were: 1. IH significantly increased ILL in all but the DBA/2 strain, whereas DR was not affected except in C57BL/6. 2. Longer IH (starting 35 [DBA/2] or 10 [NMRI] days prior to virus inoculation) significantly shortened INCUB and prolonged ILL, but IH starting on the day of virus inoculation [DBA/2] significantly prolonged INCUB and shortened ILL. 3. NMRI nude mice exhibited an unaltered DR accompanied by a tremendously prolonged INCUB. 4. Investigations in C57BL/6 mice revealed a significant rise of CORT after infection. This increase was higher in IH compared to GH mice. It is suggested that IH attenuates T-cell mediated inflammatory processes and/or increases macrophage activation, which in turn results in a prolonged course of the disease.

Coping with social conflict, and short-term changes of plasma cortisol titers in familiar and unfamiliar environments.

Each of 27 male guinea pigs was confronted with a strange opponent a) in its familiar home cage (CHC) and b) in the unfamiliar cage of the opponent (CSC) and was c) exposed to an unfamiliar open-field area without a conspecific present (OFA). The behavior displayed during confrontations was recorded in detail and plasma-cortisol-titers (PCT) were measured immediately before and after each test. The main findings were: 1) PCT increased significantly in response to the tests carried out in unfamiliar environments (OFA and CSC) compared with the response to the home-cage confrontation. 2) In both confrontation tests "offensive" males could be distinguished from "defensive" males according to marked differences in their agonistic behavior. Offensive males approached and threatened their opponents. Defensive males retreated and did not perform elements of threat behavior. 3) Offensive males showed significantly lower increases in PCT than defensive males in the home-cage confrontations. Increases in PCT did not differ significantly between the two categories of male in the strange-cage confrontations. The results show a) that different intensities of adrenocortical responses depend on the behavioral coping pattern and b) that this behavioral effect can be masked by environmental factors.