Toward a mouse neuroethology in the laboratory environment

Abstract

In this report we demonstrate that differences in cage type brought unexpected effects on aggressive behavior and neuroanatomical features of the mouse olfactory bulb. A careful characterization of two cage types, including a comparison of the auditory and temperature environments, coupled with a demonstration that naris occlusion abolishes the neuroanatomical changes, lead us to conclude that a likely important factor mediating the phenotypic changes we find is the olfactory environment of the two cages. We infer that seemingly innocuous changes in cage environment can affect sensory input relevant to mice and elicit profound effects on neural output. Study of the neural mechanisms underlying animal behavior in the laboratory environment should be broadened to include neuroethological approaches to examine how the laboratory environment (beyond animal well-being and enrichment) influences neural systems and behavior.

Figure 1. Housing differences result in marked behavioral changes.

A. Picture of a high ventilation (HV) cage and B. a low ventilation (LV) cage. The duration (C) and frequency (D) of various types of aggressive behavior are significantly different depending on the type of cage (HV or LV) the mice were housed in. Resident males were exposed to an intruder male for five minutes. A mixed ANOVA revealed a significant effect of cage type on the latency to first fight (Latency) (F_1,12 = 7.09, P = 0.0027), the total time spent interacting (Sniff) (F_1,12 = 21.56, P = 0.0006), the total time spent fighting (Fight) (F_1,12 = 11.35, P = 0.0039), and on the number of fights (Fights) (F_1,12 = 13.33, P = 0.0022). When mice did not attack, the latency was set to 300 sec. The bars represent mean±SEM (n = 6 per group).

Figure 2. Cage environment affects the neuroanatomical characteristics of the urine volatile-responsive P2 glomerulus.

A. Fluorescent micrograph of a representative P2 glomerulus. P2 olfactory sensory neuron axons are in green, juxtaglomerular cell nuclei (labeled with DAPI) in blue, and outline of glomerulus used for volume measurement in red. B. Bar graph illustrating the effect of odor environment on P2 glomerular volume in the lateral and medial domains. A mixed effects ANOVA indicated significant effects on volume of cage type (F_1,12 = 6.32, P = 0.027) and of the interaction between cage type and domain (lateral vs. medial) (F_1,49 = 10.9, P = 0.0018). Post-hoc tests revealed a significant effect of environment on the lateral glomerular volume (P = 0.0013) whereas the medial glomerular volumes did not show any differences (P = 1.0). All bar graphs are mean ± SEM (n = 8 for LV and 6 for HV cages). Asterisks indicate post-hoc tests with P<0.05. C. Bar graph illustrating the effect of odor environment on the number of P2 glomeruli in the lateral and medial domains. A mixed ANOVA revealed a significant effect of the interaction between environment and domain on the number of P2 glomeruli (F_1,26 = 11.98, P = 0.0019). Post-hoc comparison of the number of lateral, but not medial, P2 glomeruli showed a significant effect of environment (P = 0.03). The data in the LV cages is reproduced with permission from a previous publication in the Journal of Comparative Neurology .

Figure 3. Naris occlusion abolishes the difference in glomerular volume and glomerular number between animals raised in different environments.

A and B. Glomerular volume: A (non-occluded naris) and B (occluded naris). Occlusion of one naris abolished the difference in glomerular volume between HV and LV cages. A mixed effects ANOVA showed no differences in glomerular volume between different cage types (F_1,37 = 0.37, P = 0.55). We did find significant differences in glomerular volume between the naris occluded and unoccluded sides (F_1,100 = 16.7, P<0.0001). C and D. Number of glomeruli: C (non-occluded naris) and D (occluded naris). A mixed effects ANOVA showed no differences in glomerular number between different cage types (F_1,20 = 1.82, P = 0.19) and between occluded and unoccluded sides. The data in the LV cages is reproduced with permission from a previous publication in the Journal of Comparative Neurology .

Figure 4. Sound levels are not affected by cage environment.

A. Sound levels (dB) computed in 1/3-octave bands (80 Hz–40.3 kHz) for the two cage conditions: LV (green), and HV (blue). An additional acoustical measurement was taken outside the cage (room, black). The audiogram (purple) of the mouse is replotted from . The shade region indicates the range of sound frequencies over which the broadband sound levels were computed in B-C. Broadband sound levels computed every 5 minutes over the range of frequencies indicated by the shading in A (1–40.3 kHz). D-E. Spectrograms showing sound level in 1/3-octave bands (color bar, right-hand side of panel E) measured every 5 minutes over a 24 hr period.