Introduction

Olfaction is an important perception involving brain regions. Mice have a well-developed olfactory system. In mice, an odorant substance binds to olfactory receptors on the olfactory epithelium. The signal is transduced to the olfactory bulb, and then to higher-order brain regions, leading to specific behaviors, emotions, and physiological changes (Fig. 1). The odor-evoked neural pathways vary, depending on the type of odorant substance, which determines the resulting behavior of the mouse.¹ To identify the activation pathways, the olfactory bulb of mice stimulated with odorant substances has been investigated by the BOLD-fMRI method.² However, fMRI studies to detect odor-evoked responses over higher-order brain regions remain rare.³ The mouse brain is small, and thus the BOLD analysis is more likely to suffer from peripheral hemodynamic changes.⁴ We previously introduced independent component analysis (ICA) to the BOLD-fMRI method.⁵ In the BOLD-ICA method, stimulations are applied at constant intervals, and responses are detected by the ICA method.⁵ This method was applied to analyze olfactory responses in mice.^5,6 In our odor stimulation experiment for mice, a syringe pump is used to infuse small amounts of odorant substances. However, the syringe pump was manually operated, thus raising concerns about the accuracy of the timing and duration of the odor administration. In this study, we implemented an automated odor stimulation system that controls the syringe pump without manual operation.

Materials and Methods

We have been using a syringe pump to infuse odorant substances. A drop of the odorant substance solution was placed into the syringe. The solution odorant substance became vaporized to form a saturated vapor in the syringe. The saturated vapor was infused into the air line by operating the syringe pump. In this study, an automatic odor stimulation machine (in collaboration with ARCO SYSTEM, Inc.) was constructed. The operating software controls the syringe pumps and the electromagnetic valve at scheduled times (Fig. 2). Muscone, a major musk compound, was used as an odorant substance. MRI experiments were performed with a 7.0 Tesla Bruker BioSpec 70/20 scanner and a mouse brain 2-channel phased array surface cryogenic coil (Bruker BioSpin). Mice (male C57BL/6, 8–10 weeks old) were anesthetized with medetomidine (i.p. 0.3 mg/kg initial; 0.1 mg/kg/hr supplemental). GRE-EPI images were acquired: TR/TE = 2000/21.4 ms; FOV = 1.92×1.44 cm²; matrix = 96×72; resolution = 200×200 µm²; slice thickness = 400 µm; number of slices = 20; NEX = 1; flip angle = 70°. At 1 min intervals, muscone vapor was applied for 5 sec, by either manually or automatically operating the syringe pump, and this task was repeated 24 times. The scanned data were analyzed by the ICA method, using the FSL program. The data obtained from 3 mice in the automated operation were combined and analyzed by group ICA with the FSL program. The MR signal transition components with the frequency of 16.6 mHz were selected. The peak intensity profiles were created by summing the intensity profiles of the 24 tasks over 3 mice.

Results

Upon stimulation with muscone using the automated system, the brain responses were detected in the piriform cortex (Fig. 3A) and amygdala (Fig. 3D). A significant intensity increase was detected over the time courses of the signals in the two regions (Fig. 3B, E). In contrast, with manual operation, no significant increase was detected in the corresponding brain regions (Fig. 3C, F). Next, we performed a group analysis by combining the data obtained from three mice in automated operations. With the accumulated data, noise-less activation maps were obtained and the signal-to-noise was improved in the time course of the signal (Fig. 4A, C). In both the piriform cortex and amygdala, the increase of the signal occurred 13 sec after the odor stimulation (Fig. 4B, D).

Discussion

Muscone-evoked responses were detected in the piriform cortex and amygdala, consistent with a previous report based on an immunohistochemical assay.⁷ While the activation in these regions was detected in our previous study by manual operation, the detection required combined data from multiple mice.⁶ By virtue of the automated system, the activation could be detected using data from a single mouse. The increased accuracy of the timing of odor administration allowed the evaluation of the time lag between the stimulation and the occurrence of the BOLD signal, which may be associated with the identification of odorant substances by mice.

Conclusion

The implementation of the automatic odor stimulation system significantly improved the detectability of the BOLD-ICA method.

Acknowledgements

No acknowledgement found.

References

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