Abstract
Olfaction has adaptive value for rodents as it is essential for feeding and mating, the establishment of social and territorial relationships, or the detection of potential predators, among others (Apfelbach et al., 2005). Sensory input from the olfactory mucosa is first processed in the main olfactory bulb (MOB), a telencephalic structure that exhibits neurogenesis during the lifespan of the animal. Changes in MOB circuitry due to neuronal dysfunction or changes in interneuron turnover rate affect olfactory performance in different ways (Fleming et al., 2008; Breton-Provencher et al., 2009; Attems et al., 2014). Alterations in adult MOB neurogenesis, in particular, result in changes in odorant discrimination which can be assayed in habituation-dishabituation behavioral paradigms (Mouret et al., 2009; Delgado et al., 2014). Here, we present a simple protocol for the quantitative assessment of two olfactory tasks that can be used to detect neurogenic alterations in the MOB (Delgado et al., 2014). The procedure has been optimized to require little time and can, therefore, be used to analyze genetically modified mice that are housed in an isolated specific pathogen-free (SPF) mouse facility.
Materials and Reagents
Equipment
Olfactory experiments are performed in a dedicated lab room with positive pressure (10-15 gauges) and frequent air reposition (no less than 20 times per hour) for fast removal of odors. Indirect dim lighting is recommended for testing. Essence dilutions should be prepared in a different, not connected room. The materials used (see Figure 1) are:
Procedure
Data analysis
In both types of experiment, the total time spent by each mouse performing any type of olfactory exploration action is assessed for every trial in the video recordings after the experiment is finished (Figure 2B and Video 1). Actions evaluated as olfactory exploration include touching the cotton or smelling, sniffing or heading the nose towards the stick at a close distance. The Smart Junior® software (Panlab, Spain) can be used to track all behaviors and to measure the time each mouse spends exploring the stick. The measurements can also be done manually using the video recording. The variables to compare statistically are “exploration time” and “trial” as detailed in Figure 3. For the statistical analyses, a mixed-model ANOVA of repeated measures (“trial” factor) is used, followed by post hoc comparisons (e.g. Tukey or Bonferroni tests) to determine the occurrence of habituation and dishabituation. Finally, a “between-subjects” factor can be added to the ANOVA for comparison of two experimental groups. Statistical differences (p ˂ 0.05) are represented by *(dishabituation) and # (habituation). Figure 1. Equipment and materials. Photograph of the testing box and some of the required materials listed in the same order as they appear in the text. Arrow head points to the hole through which the scented stick is introduced into the exploratory box. Figure 2. Evaluation scene. Illustrative image of a mouse during the test. Actions evaluated as olfactory exploration include touching the cotton or smelling, sniffing or heading the nose towards the stick at a close distance. For this, the image of a square with a side length of 6 cm centered at the swab tip is overlapped by the software in the video image. Computer-assisted evaluation may not be necessary if spatial references are set-up manually by the observer and kept standardized across sessions. Figure 3. Representative results. A. Threshold detection test. Histogram showing the mean exploration time (in seconds) ± s.e.m. of a number of mice in trials with different concentrations of citralva interspersed by mineral oil sticks. For the analysis, a Student´s t-test comparing the time of exploration of the odorant stick vs. the non-odorant stick at each concentration can be used to analyze differences in odor detection. Threshold detection is established as the minimum scent concentration at which a significant difference is found (1:160 in the example). Thresholds for control and experimental groups can be compared (see Delgado et al., 2014). B. Habituation-dishabituation test. Graph showing the mean exploration time (in seconds) ± s.e.m. of a number of mice in successive trials of mineral oil, geraniol (1:20), and citralva (1:20) in the indicated order. Habituation is reflected as less time sniffing successive same-odor trials. Habituation is generally accelerated when mice get familiarized with the task and, therefore, it occurs more rapidly during the testing of a second odorant. Dishabituation is reflected as more time sniffing a new smell and is analyzed by comparing “trial 5” with “new-smell trial 1”. Eventually, a rise in olfactory exploration in trial 1 and 2, or even trial 3 may occur, usually during exposure of naïve freely exploring mice to the first odorant (see geraniol vs. citralva responses).
Acknowledgments
We thank the Servicios Centrales de Soporte a la Investigación Experimental and I. Noguera for technical assistance and animal care. I.F is supported by Fundación Botín and by Banco Santander through its Santander Universities Global Division, and by grants from Generalitat Valenciana (Programa Prometeo 2013/020and ISIC) and Ministerio de Economía y Competitividad (SAF2011-13332, CIBERNED CB06/05/0086, and RETIC TerCel RD12/0019/0008). In memoriam of our beloved friend Nicholas J. Mackintosh, Emeritus Professor of Experimental Psychology (University of Cambridge).
References
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