Fuqiang Zhao1, Xiaohai Wang2, Hatim A Zariwala2, Jason M. Uslaner2, Andrea K Houghton2, Jeffrey L Evelhoch1, Eric Hostetler1, and Catherine Diane Gard Hines1
1Imaging, Merck Co. & Inc., West Point, PA, United States, 2Neuroscience, Merck Co. & Inc., West Point, PA, United States
Synopsis
fMRI
offers an excellent opportunity to study olfactory processing in different
olfactory structures. Measurement of the magnitude of odor stimulation-induced
activations, and their suppression with agonist/antagonist of different neural
receptors can provide important information to understand the mechanism of
olfactory habituation. In this study, cerebral blood volume (CBV) fMRI with
USPIO was used to measure odorant-induced olfaction in different olfactory structures
of rats. The dynamics of habituation in different olfactory structures can be
robustly measured by fMRI. MK801 can reverse habituated olfactory responses in
all olfactory structures, suggesting that glutamate/NMDA receptor plays a major
role in olfactory habituation.INTRODUCTION
Olfactory habituation, characterized by attenuation of response to continuous or repeated stimulations is associated with learning and memory [1]. The mechanism that underlies habituation has not been fully elucidated [2]. Previous olfactory habituation studies are mainly performed by ex-vivo electrophysiology recordings [3, 4]. It is not clear if the findings from ex-vivo studies translate to in-vivo situations. Techniques which can directly measure neural activations in-vivo in the olfactory pathway are limited. fMRI has been used to study olfaction in the olfactory bulb (OB) of rats [5], but no fMRI activations in higher olfactory regions (HOR) of rodents have ever been reported. In this study, CBV fMRI with USPIO was used to study odorant-induced olfaction in rats. Effects of the NMDA receptor antagonist (MK801) on olfactory habituation in different structures were further studied.
METHODS
The animal protocol was approved by the IACUC of Merck Research Laboratories. Naive rats (n=8) which had not experienced any odor stimulations were used with dexmedetomidine anesthesia [6]. MRI: 7T Bruker Biospec system. Coils: 2-cm receive-only surface coil, and 72-mm transmit volume coil. T2*-weighted images by single-shot GE EPI; matrix size = 64 × 64; TE=11 ms; FOV = 2.5 × 2.5 cm. Sixteen consecutive coronal slices covering brain region from the rostral edge of olfactory bulb to the caudal edge of thalamus (Fig. 1A). Odorant: Isoamyl acetate (2870 ppm). Each fMRI measurement had a 40 s (baseline) + 40 s (stimulation) + 80 s (recovery) paradigm (Fig. 1B). Eighty fMRI measurements were made for each rat during a 4-h experimental session (Fig. 1C). A bolus of saline (3 ml/kg) was intraperitoneally (i.p.) injected at 1-h as a vehicle control, and MK801 was i.p. injected with the dose of 1 mg/kg at 2-h.
RESULTS
Fig. 1A shows activations in 3 slices before MK801 injection. Positive activations (red/yellow) indicating an increase of neural activities were observed in the OB and anterior olfactory nucleus (AON), while negative activations were observed in the piriform cortex (PC). In the OB, activations were wide-spread, with higher activations (yellow) in the parenchyma close to the OB surface.
Fig. 1B shows the activations after MK801 injection. MK801 increased activations in all regions. Interestingly, the orbital, medial prefrontal cortex (OmPFC) also showed robust activations. Response to odor stimulation in OmPFC has been observed by electrophysiology recording [7]. The MK801 effect on olfaction in OB has been observed by c-fos mRNA [8] [3].
Fig. 2 shows the temporal profiles of olfactory responses to repeated stimulations in 4 ROIs during the 4-h experiment session. The strength of the olfactory fMRI response to each stimulus was calculated by averaging the amplitudes of fMRI signals during the stimulation period. The first odor stimulation induced the strongest responses in all 4 ROIs, then the responses started to habituate. Saline injection did not alter the trends in any ROI. After MK801 injection, responses in all 4 ROIs recovered, indicating that MK801 can block habituation in all olfactory structures.
Fig. 3 shows the temporal pattern of olfactory responses to 40-s odor stimulation in different conditions. All 4 ROIs showed responses to the first stimulation when habituation to repeated stimulations had not been developed. Under the habituated condition (averaged fMRI signals from 11th to 40th fMRI measurements), PC showed negative response and OmPFC showed no response. After MK801 (averaged fMRI signals from 41st to 80th fMRI measurements), all 4 ROIs showed robust positive responses, further proving that MK801 can block olfactory habituation in all olfactory structures.
Fig. 4. shows OB depth-dependent habituation and recovery by MK801. The habituation trend and recovery trend were similar in different depth of OB, which is consistent with the observation by c-fos [3]. Discussion
The data shows that odor-induced fMRI activations can be robustly observed in the OB and some HOR before MK801 injection, but HOR habituate more significantly than the OB. Interestingly, robust negative responses (activations decrease) during odor stimulation were observed in the PC under habituated conditions, which can be explained by ‘sparse coding and global inhibition’ of the response to odor stimulation in the PC [9]. Furthermore, the NMDA receptor antagonist MK801 can reverse habituated olfactory responses in all olfactory structures. Previous ex-vivo study [3] has shown that glutamate/NMDA receptor plays a major role in olfactory habituation in OB. Our results demonstrate that ex-vivo observation reported in [3] can translate to in-vivo situation. More importantly, our data indicate that glutamate/NMDA receptor plays a major role not only in OB, but also in other olfactory structures.
Acknowledgements
We thank Drs. Donald S. Williams, Christopher Winkelmann, Richard Kennan, Darrell A. Henze and Mark Bowlby for insightful discussions and helpful suggestions.
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