Exp Neurobiol 2011; 20(4): 189-196
Published online December 30, 2011
© The Korean Society for Brain and Neural Sciences
Hyun Joo Lee1,5#, Yunjun Nam2#, Chin Su Koh1, Changkyun Im1, In Seok Seo1, Seungjin Choi2,3,4 and Hyung-Cheul Shin1*
1Department of Physiology, College of Medicine, Hallym University, Chuncheon 200-702, 2School of Interdisciplinary Bioscience and Bioengineering, 3Division of IT Convergence Engineering and 4Department of Computer Science, POSTECH, Pohang 790-784, 5Department of Electrical Engineering, University of California, Los Angeles, CA, 90095, USA
Correspondence to: #These authors equally contributed to this work.
*To whom correspondence should be addressed.
TEL: 82-33-248-2585, FAX: 82-33-248-3426
In this study, we characterize the hemodynamic changes in the main olfactory bulb of anesthetized Sprague-Dawley (SD) rats with near-infrared spectroscopy (NIRS, ISS Imagent) during presentation of two different odorants. Odorants were presented for 10 seconds with clean air via an automatic odor stimulator. Odorants are: (i) plain air as a reference (Blank), (ii) 2-Heptanone (HEP), (iii) Isopropylbenzene (IB). Our results indicated that a plain air did not cause any change in the concentrations of oxygenated (Δ[HbO2]) and deoxygenated hemoglobin (Δ[Hbr]), but HEP and IB induced strong changes. Furthermore, these odor-specific changes had regional differences within the MOB. Our results suggest that NIRS technology might be a useful tool to identify of various odorants in a non-invasive manner using animals which has a superb olfactory system.
Keywords: near-infrared spectroscopy (NIRS), hemodynamic response, main olfactory bulb (MOB), non-invasive, odorant, brain-machine interface (BMI)
An animal's olfactory system is very important for finding food, mating and avoiding predators. These behaviors are initiated by detection and discrimination of odorants in the environment, which involves representation of odorants as unique patterns of neuronal activity in the olfactory system. Previous studies have shown that segregation of sets of functionally similar inputs from olfactory receptor neurons [1, 2] and the postsynaptic outputs of the dendrites of an exclusive set of mitral/tufted cells. The outputs delineate a mechanism for spatial coding of odors among the MOB neuron ensembles. Many studies have reported this concept of glomeruli as the functional units of spatial code [3-6]. In addition to spatial patterns, both glomerular and mitral/tufted cell activities demonstrate a stimulus-specific temporal structure .
Previously, we simultaneously recorded NIRS signals from many single neurons in the anesthetized rats while presenting five different odorants. We found odor-dependent neural responses in the same ensemble of MOB neurons and odor-specific localized differential activation in different regions of the MOB as in McNamara et al.'s study . We have also reported a method for odorant discrimination based on the maximum likelihood estimation . These results have given us the hope that we might develop a sensory brain-machine interface (BMI) for the discrimination of valuable chemicals that have odors.
A drawback of an invasive BMI is the problem of immune reaction and inflammation caused when we implant the recording micro-wire electrode in the brain. A potential alternative for an invasive BMI is a non-invasive BMI based on hemodynamic response measurement with near-infrared spectroscopy (NIRS). The NIRS is a method to measure hemodynamic changes due to different absorption of oxy- (HbO2) and deoxy-hemoglobin (Hbr) via light diffusion in the brain and is useful for investigating brain function for a number of reasons including reasonable cost, high temporal resolution (up to 100 Hz), and portability [10-12]. Therefore, in this study we carried out experiments to test the possibility of discriminating different kinds of smells by measuring hemodynamic responses with a NIRS system in the main olfactory bulb of anesthetized rats. We also tested the possible presence of odor-dependent regional differences of hemodynamic response changes in the MOB.
All protocols were approved by the Hallym University Animal Care and Use Committee, and were performed according to the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health as well as the guidelines of the Animal Welfare Act. Adult Sprague-Dawley (SD) rats (350~400 g, male, n=5) were used in this study. The rats where obtained from the experimental animal center of Orient Bio Co. (linked to the Charles River Lab.). The breeding room environment was maintained at a temperature of 23±2℃ and relative humidity of 55±10%. Artificial lighting was maintained for 12 hours per day. Two animals were housed in each cage with unlimited food and water. Subjects were anesthetized with i.p. injections of urethane (20%, 1.25 g/kg body weight) and they were transferred to a stereotaxic apparatus and fixed in a prone position. Prior to the surgery and optical recording, we checked their motor responses. After a one-inch middle incision and removal of the skin and soft tissue, the skull was exposed.
We defined the coordinates of the main olfactory (MOB) regions (anterior to posterior: +4.0 mm~+12.0 mm, later to medial: 0~±4.0 mm) from bregma point on the bilateral hemisphere .
A commercial sixteen source-channel frequency-domain NIRS system (Imagent, ISS, IL., USA) was used to measure the cerebral hemodynamic responses to olfactory stimulation. This system uses two wavelengths (690 nm, 830 nm), and each channel contains two optical fibers with 400 µm core diameter (FT400EMT, Thorlabs, NJ, USA) for 690 nm and 830 nm. The optical fibers were arranged as shown in Fig. 1A and a picture of the actual experiment is shown in Fig. 1B. The NIRS channels, source and detector, were separated by 7 mm and the corresponding penetration depth is approximately 2 mm, which is sufficient to cover the cortex area . They encompass both left and right olfactory bulbs. After the optical fibers were placed at appropriate locations, dental cement (KetacCem, 3M, USA) was applied to the rat skull to maintain a constant boundary condition between optical fibers and skull [11, 12]. The sampling rate of the NIRS system was 28.4 Hz.
Odorants were diluted with mineral oil to the expected dilution rate (approximately 350 PPM) based on individual odorant vapor pressure. Following the study of Wilson (2008), mixtures were produced by odorant supplements to mineral oil in amounts that provided identical component dilution rates within the mixture . Fig. 2 shows the odor stimulatior sytem and the NIRS sytem where odorants were delivered by delivered by a motorized odor stimulator and presented 5 mm from the rat's nose for 10 seconds with plain air. The approximate dilution rate was 1 : 1 with plain air. Fig. 3 explains each stimulus was delivered at an interval of about 170 seconds and repeated 20 times for each experiment.
We changed silicone tubes and the bottles containing the odorant each time we used another odorant to prevent unexpected dilution rate. Odorants are: (i) plain air as a reference (Blank), (ii) 2-Heptanone (HEP), (iii) Isopropylbenzene (IB).
We used an analysis program of Boas and Huppert's  to calculate cerebral hemodynamic responses. This program provides calculation of ΔHbO2 and ΔHbr concentration, block average process and image reconstruction. For the recorded intensities from each channel, a modified Beer-Lambert law used to calculate the hemoglobin concentration.
where OD means optical density,
By using the above protocols, we collected NIRS signals for odor stimuli. Fig. 4 shows representative plots of hemodynamic responses on each channel. The scale of each plot is the "Hbr concentration (AU)"; horizontal axis, "Time (s)": verticalaxis and for the representation, while initial level of each signal is set to 0. As shown in the figure, stimuli induced an increase in HbO2 levels and a decrease in Hbr levels.
To extract clear patterns about hemodynamic responses, signals were low pass-filtered with 0.5 Hz cut-off frequency. Then signals were segmented into two groups: the response state and the relax sate. The relax state indicates 20 seconds of signals before chemical stimuli, and the response state indicates of 20 seconds of signals after stimuli. The recorded signals are presented on Fig. 5A. To remove the differences of sensitivity on each channel, the signals were normalized by fixing their variances to 1. Black vertical lines indicate the temporal location of stimuli. When the stimuli is given on time point
Using these responses, we tried to detect an exact time point of the odor stimuli (
From the previous analysis, we could extract temporal locations of the stimuli. Our next object was classifying the kinds of odor stimuli. For the classification, we analyzed the spatial patterns of hemodynamic responses. From each trial (a single odor stimuli), we calculated each channel's mean values for relaxes and response states, and measured their differences. So, from a single trial, we obtained a 16-channel data vector. Odor stimuli were repeated 20 times for each kind of odor, and three kinds of odors (HEP, IB and Blank) were continuously given to the rat, as the sequence of Blank, HEP and IB. Then 5 sessions of the above experiment were performed with 5 individual rats. As a result, we collected 300 trials of the difference data vector (20 trials * 3 classes * 5 sessions) and made the difference data matrix X∈ℝ16×300. To fulfill our object, the classification of odorants, we re-segmented
Fig. 6 shows the analysis results for the experiment of 4th session of the experiment. From
Fig. 6A shows a comparison results between
To investigate the possibility of the classification, we performed a
The same kind of analysis performed between
From the above two analyses, we observed that the hemodynamic alteration caused by HEP and IB share similar patterns but their magnitude is different. In the third step of analysis, we compared
The results in the table were calculated by mean (
We extended the above analysis to the entire data set
By the above analysis, we confirmed that the classification of each odor stimuli could be possible. As the final step of the analysis, we tried classification of signals. At first, we attempt the classification between relax signals (
Secondly, we performed classification between odorant responses for two different stimuli. As already explained in the previous subsection, the difference between
For the classification of each session, we tried both linear kernel and RBF kernel-based classification approaches, and we show best results in Table 2. In the first session and the third session, RBF kernel was used and in other sessions, linear kernel was used. The mean accuracy of the classifying stimuli was 77.9%, while the mean accuracy of odor classifications was 75.0%. 77.9% and 75.0% may not be satisfactory scores from the viewpoint of interface research. However, we confirmed that the magnitude of hemodynamic response from two different odor stimuli is significantly different, and sometimes it enables plausible accuracy of the classification as shown in the result of the session 4. We also observed subtle differences of spatial patterns between two responses on the
The main goal of this study was to use the NIRS system to record activity in the olfactory bulb. As mirrored by changes in blood flow during presentation of chemical odorants as well as spicy fruit (HEP), aromatic smell (IB) and plain air (Blank), respectively.
The NIRS system has several advantages specifically it is noninvasive, not painful and confers no exposure to harmful light sources. Previously studies with functional magnetic resonance imaging (fMRI) in adults have shown that olfactory stimulation activates a number of different regions of the brain [15-17]. Previously NIRs studies just focused on results of fMRI or PET studies during motor, visual and cognitive experiments [18-21]. However, in recent years, NIRs has become useful systems in mapping the brain during dynamic experiments. We have obtained a significant result by investigating the change of hemodynamic responses in the olfactory regions of an animal's brain.
Our study confirmed that NIRS were able to measure the specific hemodynamic responses of the brain during the presentation of various odors. The responses were from the MOB of anesthetized rats in experiments. Here we wish to point out that the method of similar study that was previously carried out the difference pattern of neuron spikes from MOB via invasive method to the odor discrimination.
The alterations of hemodynamic responses caused by HEP, aromatic smells (perceived by adults as a spicy fruity smell) were 2.2 times greater than IB (perceived by adults as an aromatic smell) but those alterations were stronger than the response the plain air. It might be tempting to explain this in terms of quantitative differences in emitted molecules and hence in the number of odor receptors engaged or the intensity of the inactivation .
We showed topoplots that reflected the regional basic activation patterns of olfactory bulbs during presentation of various odors in rodents. And through recorded raw data we can inferred the time when stimuli presented during the experiment session. Each of these results is very interesting as a scientific research.
Hence further studies are needed to delve into the influence of other experimental parameters, such as the number of stimuli, rate of dilution, order of stimuli, kinds of odorant on hemodynamic responses on olfactory bulbs. In this study, we presented from 18 to 20 times odor stimuli (total n; 5 rats, 90~100 times odor stimuli/each rat) to measure hemodynamic responses. Further studies should also investigate the number of stimuli and the discrimination accuracy with multivariate autoregressive (MVAR) modeling method called "Granger causality". According to Schlogl's study , the accuracy of the MVAR estimation is somewhat dependent on the number of time samples and the variance of the prediction error becomes smaller than 10% of the signal variance if the number of samples is larger than 70. The topoplots could provide more chance for the understanding brain activities such as hemodynamic responses that in odor stimuli tasks using NIRs. This is also of interest in connection with the specific brain region while presenting various stimuli such as music, decision making problems, or video games.
This experiment is intended to yield results that can be applied to develop a prototype BMI system for olfactory discrimination from brain blood changes, recorded either electro-physiological or optical (NIRS) methods. And practical applications of the olfactory sensory BMI could be used for various scent industries.