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A high sensitive in vivo biosensing detection for odors by multiunit in rat olfactory bulb
Sensors and Actuators B 186 (2013) 308–314
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A high sensitive in vivo biosensing detection for odors by multiunit in rat olfactory bulb Liujing Zhuang, Ning Hu, Qi Dong, Qingjun Liu, Ping Wang ∗ Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China
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Article history: Received 16 April 2013 Received in revised form 31 May 2013 Accepted 9 June 2013 Available online 17 June 2013 Keywords: In vivo biosensing system Odorant detection Microelectrode array Olfactory bulb Multiunit
a b s t r a c t This study presents an in vivo biosensing system for odorant detection. Taking advantage of mammal’s extraordinarily sensitive olfactory systems, we extract the odorant information from conscious rats’ olfactory bulb (OB) using microelectrode array. By chronically implanting the microelectrode into OB, high-quality mitral/tufted (M/T) cell activity evoked by odorants could be obtained for at least three weeks. The responses of multiple M/T cells broadly represent information about the presented odorants. Six odorants (carvone, citral, isobutyl alcohol, diacetyl, anisole and isoamyl acetate) used in this study could be discriminated by analyzing the neuronal activity. What is more, we found the concentration detection limit of system is below 10−15 mol/L for carvone. These results demonstrate that in vivo biosensing system has characteristic of high sensitivity, continuous recording, specificity, which presents a promising platform for specific trace odorant detection in many fields. © 2013 Published by Elsevier B.V.
1. Introduction In mammalian olfactory system, the mechanism of olfactory information transmission is based on neuronal activity of sensory projections. Initial event of olfactory sensing takes place when the odor molecules are bound to the receptors of sensory neurons in olfactory epithelium [1]. Then the sensory neurons send olfactory information through their axon to olfactory bulb (OB) for coding and processing before transmitting to the olfactory cortex [2]. OB is considered as the major site of integration for olfactory information. When stimulated with odorants, the populations of neurons in OB exhibit distinct, widespread spatial–temporal activation patterns [3–5]. The odorant information is encoded in neuronal activity. With combination of cell culture and micro-fabrication technologies [6–10], we incorporated living olfactory cells [11–13] or tissues [14–16] as sensing element with neuron chips in vitro for biomimetic olfactory-based biosensor. These biosensors provide a suitable platform with advantages of sensitivity, specificity and rapid response for odorant detection. However, in vitro cultured environment leads to shortened cell/tissue survival, so the working life of biosensor is short. In vitro culture would also damage the intact nerve structure of olfactory system and the natural pattern of neuronal activity may be changed.
∗ Corresponding author. Tel.: +86 571 87952832; fax: +86 571 87952832. E-mail address: [email protected] (P. Wang). 0925-4005/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2013.06.032
Multiple microelectrode implant technology could overcome these limitations by investigating electrophysiological properties of neuronal populations in vivo [17–20]. Although this technology causes local tissue damage, it ensures integrity of biological nerve system. The propensity for multielectrode electrophysiological investigation of neuronal populations is well established in both sensory and motor systems. Some initial studies provide evidences that in conscious animals, odorant responses of OB are different from responses in anesthetized animals [21,22]. To obtain more natural neuronal activity, we prefer conscious rats. In this study, we present a biosensing system for odorant detection. 16-channel microwire array electrode was used to obtain simultaneous recordings from single or multiple M/T cells in conscious rats. Relevant signal processing methods were employed to address the representation of odorant information in neuronal activity. To examine the efficacy of developed biosensing system as a platform for odorant detection, we respectively explored its working life, specificity and sensitivity. Detailed work will be described in the following sections. 2. Materials and methods 2.1. Microwire array electrode and biosensing system In this study, simultaneous recordings from a number of OB sites were obtained through 16-channel home-made microwire (65 m in diameter, AM system, WA; #762000) array electrodes. The impedance of each microwire is about 100 k at 1 kHz. The
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Fig. 1. Elements of in vivo biosensing system. Microelectrode records neuronal signals from rat’s olfactory bulb that specifically sensitive to odorants. The electrophysiological signals are converted to electronic signals and amplified by data acquisition system, then sent for processing by computer software to be converted to voltage parameters which present the odorant information.
microelectrode contains two parallel rows of eight microwires each. The distance between microwires in a row varied from 100 to 200 m, and the distance between the rows varied from 200 to 300 m. We chronically implanted the microelectrode in the dorsal aspect of rat OB (surgical procedure is detailed described below). Simultaneous recordings could be obtained by attaching the connector of microelectrode to pre-amplifier with headstage cable connected to OmniPlex Data Acquisition System (Plexon, Inc., Dallas, TX). Fig. 1 shows the elements of in vivo odorant detection biosensing system. Neuronal signals from microelectrode were sampled at 40 kHz, amplified by 1500× gain, and filtered from 0.5 to 8 kHz. Raw data were saved for off-line analysis.
2.2. Surgical preparation and mitral/tufted cell search Surgical and experimental techniques performed in this study were in accordance with a protocol approved by the Zhejiang University Animal Care and Use Committee. During electrode implantation process, adult male Sprague–Dawley rat (200–230 g) was anesthetized with an intraperitoneal injection of chloral hydrate (4 mL/kg). The rat was held in a standard stereotaxic apparatus, and a craniotomy was performed to expose the dorsal aspect of OB. After craniotomy had been done, microelectrode touched down the surface of dorsal OB to M/T layer. Then it was chronically implanted using dental cement (Fig. 1). Mammalian OB has a simple cortical structure. The M/T layer is localized in a narrow band (Fig. 2(a)). The search for M/T cells was done by advancing the microelectrode into OB in microsized steps with an oil hydraulic micromanipulator (Narishige, Japan) (Fig. 2(b)). The identity of M/T cell was based on their spike firing rates and waveforms [21,22]. Combined with histological examination in our previous studies [23], we presumed the dorsal aspect of M/T cells located at the depth of 400–500 m (Fig. 2(c)). To obtain similar odorant response, we always record from a fixed position (∼8 mm anterior to bregma and ∼1 mm left lateral), which is always in the middle position of olfactory bulb.
Fig. 2. (a) Photomicrographs of a Nissl-stained coronal section of OB. Topmost rectangular area is at the dorsal surface of OB. GI, glomerular layer; EPI, external plexiform layer; Mi, mitral cell layer; IPI, internal plexiform layer; GrO, granular cell layer. D, dorsal; V, ventral; L, lateral; M, medial. (b) Microelectrode was implanted into the dorsal aspect of OB using micromanipulator. (c) Neuronal signals recorded at different depth of OB from anesthetized rat. The location of M/T layer is inferred at the depth of 400–500 m.
2.3. Odorant stimulation Six odorants were used in this study: (1) carvone (C10 H14 O), which smells like spearmint; (2) citral (C10 H16 O), with a strong lemon odor; (3) isobutyl alcohol (IBA, C4 H10 O), with a sweet, musty odor; (4) diacetyl (C4 H6 O2 ), which is added to some foods to impart a buttery flavor; (5) anisole (C7 H8 O), with a smell of anise seed; (6) isoamyl acetate (IAA, C7 H14 O), with fruit-like smell similar to banana and pear. Odorants were diluted with odorless mineral oil into different concentrations: from 10−1 to 10−15 mol/L. Odorant stimulation was conducted with rats placed in a custom-made polymethyl methacrylate chamber (30 cm long, 25 cm wide, 30 cm high). In each trial, we placed a glass dish containing a piece of filter paper on which was deposited 0.1 mL odorant in front of the rat’s nose (Fig. 1) and stayed for 5 s. Within this 5 s, the rat itself determined the duration (always about 1–3 s) of odorant sampling. After achieving odorant response, we would refresh the chamber with fresh air and waited for 5–15 min. Then, another trial will be started. 2.4. Data analysis The raw data were analyzed off-line by MATLAB (Mathworks, Inc.). We extracted spiking activity of neurons (250–2000 Hz) from the raw data by four-pole Butterworth filter. For a given epoch of signal, the distribution of spikes was achieved by peak value extraction method. The standard deviation (SD) value ‘SD’ of signals was set as the level of baseline noise, and 2.5 × SD was set as a threshold for spike registration. Every negative peak below this threshold was considered a spike. Spikes were clustered and displayed in two or three-dimensional coordinates. The clustered
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Fig. 3. Multiunit responses of M/T cells from the same channle over multiple days. (a) Spontaneous (control group) and odor-evoked (10−1 M carvone) neuronal activity recorded at different days. The upper two panels show the raw data of spike discharge. The lower panels show raster plots of different trials recorded at a single day (5 trials). Each sweep of the raster lasts 2 s. The bottom panels represent peristimulus time histograms (PSTHs, spike counts/bin, bin = 0.1 s). The dashed bars indicate the time of occurrence of first 200-ms bin that significantly differed from baseline during odorant application. (b) Mean firing rates by trial during odorant stimulation at a single day and different days. (c) Two single units were extracted from the signals in (a), and the percentage of spike counts of unit a/b per day was analyzed. Data were presented as mean ± standard deviation (SD) (n > 3).
spikes which had distinct waveform shape were considered as being generated from a single unit. While they were from multiunit if clusters were separable. The spike firing rate, amplitude, duration were applied to reveal the odorant information. Student’s t-test was used as an estimation for significance (P < 0.05). 3. Results and discussion In order to determine the efficacy of the developed biosensing system as a platform for odorant detection, we respectively explored its working life, specificity and sensitivity. By implanting 16-channel microelectrode in the dorsal aspect of ten rats, both spontaneous and odor-evoked responses from a number of M/T cells were obtained. According to researches about maps of odorant molecular features in the mammalian OB, temporally correlated M/T cells receive input from the same glomerulus in OB [24,25]. The activity of multiunit could provide highly specific odorant information to higher olfactory centers [26,27]. Thus the response of multiple M/T cells could represent odorants information more reliably than single unit [28]. In this study, we mainly analyzed the response of multiple M/T cells during odorant stimulation. Further, the contribution of single unit to the multiunit response was investigated. 3.1. Continuous recording from multiple M/T cells Researches in the mechanisms of olfactory system have promoted the development of olfactory-based biosensors to detect odorants. And much work has been done in developing these biosensors due to their emerging and promising potential in wide applications [29]. However, some significant shortcomings of these biosensors are limited stability and reproducibility [30]. To overcome the shortcomings caused by in vitro detecting, we turned to the biosensing system for in vivo detecting.
To examine the stability of developed biosensing system for continuous recording, we analyzed the responses of M/T cells from the same recording channel over multiple days. In this experiment, odorants at high concentration were used to obtain robust responses. During odorant stimulation, the neuronal activity became bursty and was robust. While few spikes could be observed in the absence of odorants, which was regarded as control group (Fig. 3(a)). The mean firing rate of control group was about 2 Hz. The average value of noise level was about 20 V. Fig. 3 shows the results of response to 10−1 mol/L carvone recorded from the same channel over multiple days. Raster plots for single trials and peristimulus time histograms (PSTHs) were created to compare the odorant response over time. The shapes of PSTHs were found to be similar over a period of three weeks (Fig. 3(a)). The mean firing rates of each trial varied over the period, but maintained in a stable range of 50–55 Hz (Fig. 3(b)). Further, we investigated the contribution of individual unit to the odorant response. Two single units with uniquely discernable spikes (Fig. 3(c)) were extracted from the signals in Fig. 3(a). Each unit’s spike counts integrated during response. When the percentage of each unit’s spike counts was analyzed, the proportion of unit a (>50% of total spike counts) slightly exceed that of unit b (<50% of total spike counts). In fact, there was slight difference in the percentage of spike counts of each unit over three weeks (Fig. 3(c)). As the results show in Fig. 3, high-quality responses of M/T cells could be continuously recorded for at least three weeks. So the biosensing system has well stability and long working life. 3.2. Odorant specificity of biosensing system Mammals could discriminate and recognize a large variety of chemical signals in their environment. According to the recent
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Fig. 4. Multiunit responses of M/T cells to six odorants from the same channel. Spike discharges (top panels of each graph), raster plots (lower panels of each graph) and PSTHs (bottom panels of each graph, bin = 0.1 s) of M/T cells during stimulation of carvone, citral, isobutyl alcohol (IBA), diacetyl, anisole and isoamyl acetate (IAA). The dashed bars indicate the time of occurrence of first 200-ms bin that significantly differed from baseline during odorant application.
studies about spatial organization of odorant maps in mammalian OB, individual M/T cell selectively respond to a range of odorants that share a specific combination of molecular features. The molecular-feature M/T cells are located at stereotypical positions in the OB [31]. Once microelectrode was implanted into the OB, the electrode tips would contact with certain odor-specific M/T cells. To explore the specificity of developed biosensing system, we examined the responses of M/T cells to different odorants. Spike discharge and raster plots were created for carvone, citral, isobutyl alcohol (IBA), diacetyl, anisole and isoamyl acetate (IAA). For example, at concentration of 10−1 mol/L, the multiunit recorded from the same channel (Fig. 4) responded to all six odorants while the response patterns were not uniform based on spike discharge patterns and PSTHs. Further, we found a large proportion of examined units also responded to carvone (C10 H14 O), citral (C10 H16 O), IBA (C4 H10 O) and diacetyl (C4 H6 O2 ) with robust
discharge, indicating that M/T cells sensitive to these four odorants were distributed widely across the dorsal aspect of olfactory bulb. During the four odorants stimulation, the mean firing rates of multiunit discharge were about 50 Hz (Fig. 5(a)). However, there was no strong response to anisole (C7 H8 O) and IAA (C7 H14 O). The mean firing rates were below 20 Hz. We preliminarily found the odorants with same carbon atoms evoked similar discharge type. Further, we extracted individual units from multiunit and used the single units’ responses to investigate whether specific odorant information was more detail represented. Three single units with uniquely discernable spikes (Fig. 5(b)) were extracted from the signals in Fig. 4. The temporal characteristics of each unit’s spike waveforms were shown in Fig. 5(c). According to ‘maximum to minimum’ value of waveform, maximum value of waveform, duration, the three units were clustered into three regions in 3D space. We compared the distributions of unit spike counts in the presence
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Fig. 5. (a) Mean firing rates of multiunit response to six odorant were 52.80 ± 1.63 Hz, 56.93 ± 2.31 Hz, 50.93 ± 2.80 Hz, 50.47 ± 2.48 Hz, 13.07 ± 2.09 Hz and 16.20 ± 3.75 Hz, respectively. (b) Three single units were extracted from the signals in Fig. 4, and the percentage of spike counts of unit a/b/c for each odorant was analyzed. Carvone activated unit a (53% of total spike counts) and unit b (47% of total spike counts); citral activated unit a (55%) and unit b (45%); IBA activated unit a (42%) and unit b (58%); diacetyl only activated unit b (100%); anisole and IAA only activated unit c (100%). (c) According to ‘maximum to minimum’ value of spike (V, PC1), duration (ms, PC2), maximum value of spike (V, PC3), the three units were clustered into three regions in 3D space. Data were presented as mean ± SD (n > 10). Significant differences are indicated by **P < 0.01 and *P < 0.05 using student’s t-test.
of each of six odorant and found three odorant response patterns: (1) both unit a and unit b were activated during stimulation of carvone, citral and IBA, and the proportion of spike counts of unit a was about 53%, 55% and 42%, respectively; (2) only unit b was activated during stimulation of diacetyl; (3) only unit c was activated during stimulation of anisole and IAA (Fig. 5(b)). Then the analysis was combined with student’s t-test of mean firing rate to determine whether multiunit responses to each odorant could be discriminated. The results indicated that difference (P < 0.05) or significant difference (P < 0.01) exist in the mean firing rate between carvone, citral, IBA, and also exist between anisole, IAA (Fig. 5(a)). In our previous studies, we examined the odorant discrimination abilities of multiple M/T cells in anesthetized rats [32]. Now we represent the multiunit activity from conscious rats. The raw spike signals and 3D clusters (Figs. 5 and 6) in this study provided more visual information about odorant discrimination. The results indicate that activity of multiple M/T cells provided sufficient information to discriminate between odorants. Thus, the biosensing system has high specificity for odorant detection. 3.3. High sensitivity for odorant detection We obtained robust neuronal activity during some highconcentration odorant stimulation. To explore the concentration detection limit of developed biosensing system, we decreased the
odorant concentrations to extremely low level. In this experiment, we applied different concentrations from 10−15 to 10−1 mol/L to have a better control of odorant response. Fig. 6(a) shows the spike discharge from the same recording site evoked by different concentration of carvone. The mean firing rate of multiunit discharge evoked by each concentration of carvone was further analyzed in Fig. 6(b). We found that the discharge intensity changed slightly at concentration between 10−1 and 10−9 mol/L, which was considered as high concentration. Within this range, the multiunit response seemed to be independent of carvone concentrations. However, once the concentration decreased to 10−10 mol/L, we found abrupt changes in discharge intensity (Fig. 6). With decreasing concentration from 10−10 to 10−15 mol/L, the decreases in firing rate had linear relationship with the concentration, which may play a role in quantitative detection of low-concentration odorants in further study. Odorant concentration plays a critical role in determining the absolute detection threshold of an odorant [33,34]. Quantitative changes lead to concentration-dependent response patterns. Although this phenomenon may depend a lot on the collection of odorants and recording site, we found the developed system has detection limit to carvone odorant as low as 10−15 mol/L. The results suggest that the biosensing system may contribute to the screening and detection of trace odorants, such as exhaled-breath detection [35,36]. To achieve practical applications, further investigations and improvements are essential.
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Fig. 6. Analysis of concentration detection limit of developed biosensing system. (a) Spike discharges from the same recording site during stimulation of high (10−9 to 10−1 M)/low (10−15 to 10−9 M) concentration of carvone. We found abrupt changes in discharge intensity when the concentration decreased to 10−10 M. (b) The concentration–response curve to different concentrations of carvone. We found abrupt changes in firing rate when the concentration decreased to 10−10 M (48.00 ± 4.43 Hz at 10−9 M, 41.67 ± 3.49 Hz at 10−10 M, while 30.50 ± 3.52 Hz at 10−11 M). Data were presented as mean ± SD (n > 10). Significant differences are indicated by **P < 0.01 and *P < 0.05 using student’s t-test.
4. Conclusion Taking advantage of multiple microelectrode implant technology, we developed an in vivo biosensing system for odorant detection. Simultaneous recording from a number of M/T cells in conscious rats could be obtained by chronically implanting the 16-channel microwire array electrode into the dorsal aspect of OB. The results show high-quality neuronal activity from a rat could be continuously recorded for at least three weeks. Based on spike discharge patterns, activity of M/T cells provided sufficient information to discriminate between six odorants (carvone, citral, isobutyl alcohol, diacetyl, anisole and isoamyl acetate). Additionally, we found odorant concentration affect the response patterns, and the rats could detect the carvone as low as 10−15 mol/L. For the in vivo biosensing system has characteristic of high sensitivity, continuous recording, specificity, it presents a promising platform for specific trace odorant detection in real application. Acknowledgments This work was supported by National Natural Science Fund of China (No. 81027003), Research Fund for Doctoral Program of Education Ministry of China (No. 20120101130011) and National Public Welfare Project of China (No. 201305010). References [1] L. Buck, R. Axel, A novel multigene family may encode odorant receptors: a molecular basis for odor recognition, Cell 65 (1991) 175–187. [2] P.M. Lledo, G. Gheusi, J.D. Vincent, Information processing in the mammalian olfactory system, Physiological Reviews 85 (2005) 281–317. [3] E. Adrian, The electrical activity of the mammalian olfactory bulb, Electroencephalography and Clinical Neurophysiology 2 (1950) 377–388. [4] L.B. Buck, Information coding in the vertebrate olfactory system, Annual Review of Neuroscience 19 (1996) 517–544.
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Biographies Liujing Zhuang received her BE degree of biomedical engineering in Zhejiang University, PR China in 2011. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes research of olfaction, nerve electrophysiology, signal processing and biosensing system establishing. Ning Hu received his BE degree and MS degree of biomedical engineering in Zhejiang University, PR China in 2009 and 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes research of electrochemical sensors, cell-based biosensor, cell metabolic physiology, biosensor instrument establishing and signal processing.
Qi Dong received the BE degree in biomedical engineering from Zhejiang University, Hangzhou, PR China in 2009. Now he is currently working toward the Ph.D. degree in the Department of Biomedical Engineering of Zhejiang University. His research interests include olfaction, neural signal processing and discrimination of odors. Qingjun Liu received his Ph.D. degree in biomedical engineering in Zhejiang University, PR China in 2006. He is currently an associate professor in Biosensors National Special Lab, Zhejiang University. He is also a visiting scholar in the Department of Health Technology and Informatics of the Hong Kong Polytechnic University. He published the book of Cell-based Biosensors: Principles and Applications, by Artech House Publishers, USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system. Ping Wang received his BS degree, MS degree and Ph.D. degree of electrical engineering in Harbin Institute of Technology, Harbin, PR China in 1984, 1987 and 1992, respectively. He is currently a professor of Biosensors National Special Lab, Department of Biomedical Engineering of Zhejiang University. His research interests include biomedical sensors, electrochemical sensors and measurement technique.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A high sensitive in vivo biosensing detection for odors by multiunit in rat olfactory bulb Liujing Zhuang, Ning Hu, Qi Dong, Qingjun Liu, Ping Wang ∗ Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China
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Article history: Received 16 April 2013 Received in revised form 31 May 2013 Accepted 9 June 2013 Available online 17 June 2013 Keywords: In vivo biosensing system Odorant detection Microelectrode array Olfactory bulb Multiunit
a b s t r a c t This study presents an in vivo biosensing system for odorant detection. Taking advantage of mammal’s extraordinarily sensitive olfactory systems, we extract the odorant information from conscious rats’ olfactory bulb (OB) using microelectrode array. By chronically implanting the microelectrode into OB, high-quality mitral/tufted (M/T) cell activity evoked by odorants could be obtained for at least three weeks. The responses of multiple M/T cells broadly represent information about the presented odorants. Six odorants (carvone, citral, isobutyl alcohol, diacetyl, anisole and isoamyl acetate) used in this study could be discriminated by analyzing the neuronal activity. What is more, we found the concentration detection limit of system is below 10−15 mol/L for carvone. These results demonstrate that in vivo biosensing system has characteristic of high sensitivity, continuous recording, specificity, which presents a promising platform for specific trace odorant detection in many fields. © 2013 Published by Elsevier B.V.
1. Introduction In mammalian olfactory system, the mechanism of olfactory information transmission is based on neuronal activity of sensory projections. Initial event of olfactory sensing takes place when the odor molecules are bound to the receptors of sensory neurons in olfactory epithelium [1]. Then the sensory neurons send olfactory information through their axon to olfactory bulb (OB) for coding and processing before transmitting to the olfactory cortex [2]. OB is considered as the major site of integration for olfactory information. When stimulated with odorants, the populations of neurons in OB exhibit distinct, widespread spatial–temporal activation patterns [3–5]. The odorant information is encoded in neuronal activity. With combination of cell culture and micro-fabrication technologies [6–10], we incorporated living olfactory cells [11–13] or tissues [14–16] as sensing element with neuron chips in vitro for biomimetic olfactory-based biosensor. These biosensors provide a suitable platform with advantages of sensitivity, specificity and rapid response for odorant detection. However, in vitro cultured environment leads to shortened cell/tissue survival, so the working life of biosensor is short. In vitro culture would also damage the intact nerve structure of olfactory system and the natural pattern of neuronal activity may be changed.
∗ Corresponding author. Tel.: +86 571 87952832; fax: +86 571 87952832. E-mail address: [email protected] (P. Wang). 0925-4005/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2013.06.032
Multiple microelectrode implant technology could overcome these limitations by investigating electrophysiological properties of neuronal populations in vivo [17–20]. Although this technology causes local tissue damage, it ensures integrity of biological nerve system. The propensity for multielectrode electrophysiological investigation of neuronal populations is well established in both sensory and motor systems. Some initial studies provide evidences that in conscious animals, odorant responses of OB are different from responses in anesthetized animals [21,22]. To obtain more natural neuronal activity, we prefer conscious rats. In this study, we present a biosensing system for odorant detection. 16-channel microwire array electrode was used to obtain simultaneous recordings from single or multiple M/T cells in conscious rats. Relevant signal processing methods were employed to address the representation of odorant information in neuronal activity. To examine the efficacy of developed biosensing system as a platform for odorant detection, we respectively explored its working life, specificity and sensitivity. Detailed work will be described in the following sections. 2. Materials and methods 2.1. Microwire array electrode and biosensing system In this study, simultaneous recordings from a number of OB sites were obtained through 16-channel home-made microwire (65 m in diameter, AM system, WA; #762000) array electrodes. The impedance of each microwire is about 100 k at 1 kHz. The
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Fig. 1. Elements of in vivo biosensing system. Microelectrode records neuronal signals from rat’s olfactory bulb that specifically sensitive to odorants. The electrophysiological signals are converted to electronic signals and amplified by data acquisition system, then sent for processing by computer software to be converted to voltage parameters which present the odorant information.
microelectrode contains two parallel rows of eight microwires each. The distance between microwires in a row varied from 100 to 200 m, and the distance between the rows varied from 200 to 300 m. We chronically implanted the microelectrode in the dorsal aspect of rat OB (surgical procedure is detailed described below). Simultaneous recordings could be obtained by attaching the connector of microelectrode to pre-amplifier with headstage cable connected to OmniPlex Data Acquisition System (Plexon, Inc., Dallas, TX). Fig. 1 shows the elements of in vivo odorant detection biosensing system. Neuronal signals from microelectrode were sampled at 40 kHz, amplified by 1500× gain, and filtered from 0.5 to 8 kHz. Raw data were saved for off-line analysis.
2.2. Surgical preparation and mitral/tufted cell search Surgical and experimental techniques performed in this study were in accordance with a protocol approved by the Zhejiang University Animal Care and Use Committee. During electrode implantation process, adult male Sprague–Dawley rat (200–230 g) was anesthetized with an intraperitoneal injection of chloral hydrate (4 mL/kg). The rat was held in a standard stereotaxic apparatus, and a craniotomy was performed to expose the dorsal aspect of OB. After craniotomy had been done, microelectrode touched down the surface of dorsal OB to M/T layer. Then it was chronically implanted using dental cement (Fig. 1). Mammalian OB has a simple cortical structure. The M/T layer is localized in a narrow band (Fig. 2(a)). The search for M/T cells was done by advancing the microelectrode into OB in microsized steps with an oil hydraulic micromanipulator (Narishige, Japan) (Fig. 2(b)). The identity of M/T cell was based on their spike firing rates and waveforms [21,22]. Combined with histological examination in our previous studies [23], we presumed the dorsal aspect of M/T cells located at the depth of 400–500 m (Fig. 2(c)). To obtain similar odorant response, we always record from a fixed position (∼8 mm anterior to bregma and ∼1 mm left lateral), which is always in the middle position of olfactory bulb.
Fig. 2. (a) Photomicrographs of a Nissl-stained coronal section of OB. Topmost rectangular area is at the dorsal surface of OB. GI, glomerular layer; EPI, external plexiform layer; Mi, mitral cell layer; IPI, internal plexiform layer; GrO, granular cell layer. D, dorsal; V, ventral; L, lateral; M, medial. (b) Microelectrode was implanted into the dorsal aspect of OB using micromanipulator. (c) Neuronal signals recorded at different depth of OB from anesthetized rat. The location of M/T layer is inferred at the depth of 400–500 m.
2.3. Odorant stimulation Six odorants were used in this study: (1) carvone (C10 H14 O), which smells like spearmint; (2) citral (C10 H16 O), with a strong lemon odor; (3) isobutyl alcohol (IBA, C4 H10 O), with a sweet, musty odor; (4) diacetyl (C4 H6 O2 ), which is added to some foods to impart a buttery flavor; (5) anisole (C7 H8 O), with a smell of anise seed; (6) isoamyl acetate (IAA, C7 H14 O), with fruit-like smell similar to banana and pear. Odorants were diluted with odorless mineral oil into different concentrations: from 10−1 to 10−15 mol/L. Odorant stimulation was conducted with rats placed in a custom-made polymethyl methacrylate chamber (30 cm long, 25 cm wide, 30 cm high). In each trial, we placed a glass dish containing a piece of filter paper on which was deposited 0.1 mL odorant in front of the rat’s nose (Fig. 1) and stayed for 5 s. Within this 5 s, the rat itself determined the duration (always about 1–3 s) of odorant sampling. After achieving odorant response, we would refresh the chamber with fresh air and waited for 5–15 min. Then, another trial will be started. 2.4. Data analysis The raw data were analyzed off-line by MATLAB (Mathworks, Inc.). We extracted spiking activity of neurons (250–2000 Hz) from the raw data by four-pole Butterworth filter. For a given epoch of signal, the distribution of spikes was achieved by peak value extraction method. The standard deviation (SD) value ‘SD’ of signals was set as the level of baseline noise, and 2.5 × SD was set as a threshold for spike registration. Every negative peak below this threshold was considered a spike. Spikes were clustered and displayed in two or three-dimensional coordinates. The clustered
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Fig. 3. Multiunit responses of M/T cells from the same channle over multiple days. (a) Spontaneous (control group) and odor-evoked (10−1 M carvone) neuronal activity recorded at different days. The upper two panels show the raw data of spike discharge. The lower panels show raster plots of different trials recorded at a single day (5 trials). Each sweep of the raster lasts 2 s. The bottom panels represent peristimulus time histograms (PSTHs, spike counts/bin, bin = 0.1 s). The dashed bars indicate the time of occurrence of first 200-ms bin that significantly differed from baseline during odorant application. (b) Mean firing rates by trial during odorant stimulation at a single day and different days. (c) Two single units were extracted from the signals in (a), and the percentage of spike counts of unit a/b per day was analyzed. Data were presented as mean ± standard deviation (SD) (n > 3).
spikes which had distinct waveform shape were considered as being generated from a single unit. While they were from multiunit if clusters were separable. The spike firing rate, amplitude, duration were applied to reveal the odorant information. Student’s t-test was used as an estimation for significance (P < 0.05). 3. Results and discussion In order to determine the efficacy of the developed biosensing system as a platform for odorant detection, we respectively explored its working life, specificity and sensitivity. By implanting 16-channel microelectrode in the dorsal aspect of ten rats, both spontaneous and odor-evoked responses from a number of M/T cells were obtained. According to researches about maps of odorant molecular features in the mammalian OB, temporally correlated M/T cells receive input from the same glomerulus in OB [24,25]. The activity of multiunit could provide highly specific odorant information to higher olfactory centers [26,27]. Thus the response of multiple M/T cells could represent odorants information more reliably than single unit [28]. In this study, we mainly analyzed the response of multiple M/T cells during odorant stimulation. Further, the contribution of single unit to the multiunit response was investigated. 3.1. Continuous recording from multiple M/T cells Researches in the mechanisms of olfactory system have promoted the development of olfactory-based biosensors to detect odorants. And much work has been done in developing these biosensors due to their emerging and promising potential in wide applications [29]. However, some significant shortcomings of these biosensors are limited stability and reproducibility [30]. To overcome the shortcomings caused by in vitro detecting, we turned to the biosensing system for in vivo detecting.
To examine the stability of developed biosensing system for continuous recording, we analyzed the responses of M/T cells from the same recording channel over multiple days. In this experiment, odorants at high concentration were used to obtain robust responses. During odorant stimulation, the neuronal activity became bursty and was robust. While few spikes could be observed in the absence of odorants, which was regarded as control group (Fig. 3(a)). The mean firing rate of control group was about 2 Hz. The average value of noise level was about 20 V. Fig. 3 shows the results of response to 10−1 mol/L carvone recorded from the same channel over multiple days. Raster plots for single trials and peristimulus time histograms (PSTHs) were created to compare the odorant response over time. The shapes of PSTHs were found to be similar over a period of three weeks (Fig. 3(a)). The mean firing rates of each trial varied over the period, but maintained in a stable range of 50–55 Hz (Fig. 3(b)). Further, we investigated the contribution of individual unit to the odorant response. Two single units with uniquely discernable spikes (Fig. 3(c)) were extracted from the signals in Fig. 3(a). Each unit’s spike counts integrated during response. When the percentage of each unit’s spike counts was analyzed, the proportion of unit a (>50% of total spike counts) slightly exceed that of unit b (<50% of total spike counts). In fact, there was slight difference in the percentage of spike counts of each unit over three weeks (Fig. 3(c)). As the results show in Fig. 3, high-quality responses of M/T cells could be continuously recorded for at least three weeks. So the biosensing system has well stability and long working life. 3.2. Odorant specificity of biosensing system Mammals could discriminate and recognize a large variety of chemical signals in their environment. According to the recent
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Fig. 4. Multiunit responses of M/T cells to six odorants from the same channel. Spike discharges (top panels of each graph), raster plots (lower panels of each graph) and PSTHs (bottom panels of each graph, bin = 0.1 s) of M/T cells during stimulation of carvone, citral, isobutyl alcohol (IBA), diacetyl, anisole and isoamyl acetate (IAA). The dashed bars indicate the time of occurrence of first 200-ms bin that significantly differed from baseline during odorant application.
studies about spatial organization of odorant maps in mammalian OB, individual M/T cell selectively respond to a range of odorants that share a specific combination of molecular features. The molecular-feature M/T cells are located at stereotypical positions in the OB [31]. Once microelectrode was implanted into the OB, the electrode tips would contact with certain odor-specific M/T cells. To explore the specificity of developed biosensing system, we examined the responses of M/T cells to different odorants. Spike discharge and raster plots were created for carvone, citral, isobutyl alcohol (IBA), diacetyl, anisole and isoamyl acetate (IAA). For example, at concentration of 10−1 mol/L, the multiunit recorded from the same channel (Fig. 4) responded to all six odorants while the response patterns were not uniform based on spike discharge patterns and PSTHs. Further, we found a large proportion of examined units also responded to carvone (C10 H14 O), citral (C10 H16 O), IBA (C4 H10 O) and diacetyl (C4 H6 O2 ) with robust
discharge, indicating that M/T cells sensitive to these four odorants were distributed widely across the dorsal aspect of olfactory bulb. During the four odorants stimulation, the mean firing rates of multiunit discharge were about 50 Hz (Fig. 5(a)). However, there was no strong response to anisole (C7 H8 O) and IAA (C7 H14 O). The mean firing rates were below 20 Hz. We preliminarily found the odorants with same carbon atoms evoked similar discharge type. Further, we extracted individual units from multiunit and used the single units’ responses to investigate whether specific odorant information was more detail represented. Three single units with uniquely discernable spikes (Fig. 5(b)) were extracted from the signals in Fig. 4. The temporal characteristics of each unit’s spike waveforms were shown in Fig. 5(c). According to ‘maximum to minimum’ value of waveform, maximum value of waveform, duration, the three units were clustered into three regions in 3D space. We compared the distributions of unit spike counts in the presence
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Fig. 5. (a) Mean firing rates of multiunit response to six odorant were 52.80 ± 1.63 Hz, 56.93 ± 2.31 Hz, 50.93 ± 2.80 Hz, 50.47 ± 2.48 Hz, 13.07 ± 2.09 Hz and 16.20 ± 3.75 Hz, respectively. (b) Three single units were extracted from the signals in Fig. 4, and the percentage of spike counts of unit a/b/c for each odorant was analyzed. Carvone activated unit a (53% of total spike counts) and unit b (47% of total spike counts); citral activated unit a (55%) and unit b (45%); IBA activated unit a (42%) and unit b (58%); diacetyl only activated unit b (100%); anisole and IAA only activated unit c (100%). (c) According to ‘maximum to minimum’ value of spike (V, PC1), duration (ms, PC2), maximum value of spike (V, PC3), the three units were clustered into three regions in 3D space. Data were presented as mean ± SD (n > 10). Significant differences are indicated by **P < 0.01 and *P < 0.05 using student’s t-test.
of each of six odorant and found three odorant response patterns: (1) both unit a and unit b were activated during stimulation of carvone, citral and IBA, and the proportion of spike counts of unit a was about 53%, 55% and 42%, respectively; (2) only unit b was activated during stimulation of diacetyl; (3) only unit c was activated during stimulation of anisole and IAA (Fig. 5(b)). Then the analysis was combined with student’s t-test of mean firing rate to determine whether multiunit responses to each odorant could be discriminated. The results indicated that difference (P < 0.05) or significant difference (P < 0.01) exist in the mean firing rate between carvone, citral, IBA, and also exist between anisole, IAA (Fig. 5(a)). In our previous studies, we examined the odorant discrimination abilities of multiple M/T cells in anesthetized rats [32]. Now we represent the multiunit activity from conscious rats. The raw spike signals and 3D clusters (Figs. 5 and 6) in this study provided more visual information about odorant discrimination. The results indicate that activity of multiple M/T cells provided sufficient information to discriminate between odorants. Thus, the biosensing system has high specificity for odorant detection. 3.3. High sensitivity for odorant detection We obtained robust neuronal activity during some highconcentration odorant stimulation. To explore the concentration detection limit of developed biosensing system, we decreased the
odorant concentrations to extremely low level. In this experiment, we applied different concentrations from 10−15 to 10−1 mol/L to have a better control of odorant response. Fig. 6(a) shows the spike discharge from the same recording site evoked by different concentration of carvone. The mean firing rate of multiunit discharge evoked by each concentration of carvone was further analyzed in Fig. 6(b). We found that the discharge intensity changed slightly at concentration between 10−1 and 10−9 mol/L, which was considered as high concentration. Within this range, the multiunit response seemed to be independent of carvone concentrations. However, once the concentration decreased to 10−10 mol/L, we found abrupt changes in discharge intensity (Fig. 6). With decreasing concentration from 10−10 to 10−15 mol/L, the decreases in firing rate had linear relationship with the concentration, which may play a role in quantitative detection of low-concentration odorants in further study. Odorant concentration plays a critical role in determining the absolute detection threshold of an odorant [33,34]. Quantitative changes lead to concentration-dependent response patterns. Although this phenomenon may depend a lot on the collection of odorants and recording site, we found the developed system has detection limit to carvone odorant as low as 10−15 mol/L. The results suggest that the biosensing system may contribute to the screening and detection of trace odorants, such as exhaled-breath detection [35,36]. To achieve practical applications, further investigations and improvements are essential.
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Fig. 6. Analysis of concentration detection limit of developed biosensing system. (a) Spike discharges from the same recording site during stimulation of high (10−9 to 10−1 M)/low (10−15 to 10−9 M) concentration of carvone. We found abrupt changes in discharge intensity when the concentration decreased to 10−10 M. (b) The concentration–response curve to different concentrations of carvone. We found abrupt changes in firing rate when the concentration decreased to 10−10 M (48.00 ± 4.43 Hz at 10−9 M, 41.67 ± 3.49 Hz at 10−10 M, while 30.50 ± 3.52 Hz at 10−11 M). Data were presented as mean ± SD (n > 10). Significant differences are indicated by **P < 0.01 and *P < 0.05 using student’s t-test.
4. Conclusion Taking advantage of multiple microelectrode implant technology, we developed an in vivo biosensing system for odorant detection. Simultaneous recording from a number of M/T cells in conscious rats could be obtained by chronically implanting the 16-channel microwire array electrode into the dorsal aspect of OB. The results show high-quality neuronal activity from a rat could be continuously recorded for at least three weeks. Based on spike discharge patterns, activity of M/T cells provided sufficient information to discriminate between six odorants (carvone, citral, isobutyl alcohol, diacetyl, anisole and isoamyl acetate). Additionally, we found odorant concentration affect the response patterns, and the rats could detect the carvone as low as 10−15 mol/L. For the in vivo biosensing system has characteristic of high sensitivity, continuous recording, specificity, it presents a promising platform for specific trace odorant detection in real application. Acknowledgments This work was supported by National Natural Science Fund of China (No. 81027003), Research Fund for Doctoral Program of Education Ministry of China (No. 20120101130011) and National Public Welfare Project of China (No. 201305010). References [1] L. Buck, R. Axel, A novel multigene family may encode odorant receptors: a molecular basis for odor recognition, Cell 65 (1991) 175–187. [2] P.M. Lledo, G. Gheusi, J.D. Vincent, Information processing in the mammalian olfactory system, Physiological Reviews 85 (2005) 281–317. [3] E. Adrian, The electrical activity of the mammalian olfactory bulb, Electroencephalography and Clinical Neurophysiology 2 (1950) 377–388. [4] L.B. Buck, Information coding in the vertebrate olfactory system, Annual Review of Neuroscience 19 (1996) 517–544.
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Biographies Liujing Zhuang received her BE degree of biomedical engineering in Zhejiang University, PR China in 2011. Now she is a Ph.D. student of biomedical engineering of Zhejiang University. Her work includes research of olfaction, nerve electrophysiology, signal processing and biosensing system establishing. Ning Hu received his BE degree and MS degree of biomedical engineering in Zhejiang University, PR China in 2009 and 2011. Now he is a Ph.D. student of biomedical engineering of Zhejiang University. His work includes research of electrochemical sensors, cell-based biosensor, cell metabolic physiology, biosensor instrument establishing and signal processing.
Qi Dong received the BE degree in biomedical engineering from Zhejiang University, Hangzhou, PR China in 2009. Now he is currently working toward the Ph.D. degree in the Department of Biomedical Engineering of Zhejiang University. His research interests include olfaction, neural signal processing and discrimination of odors. Qingjun Liu received his Ph.D. degree in biomedical engineering in Zhejiang University, PR China in 2006. He is currently an associate professor in Biosensors National Special Lab, Zhejiang University. He is also a visiting scholar in the Department of Health Technology and Informatics of the Hong Kong Polytechnic University. He published the book of Cell-based Biosensors: Principles and Applications, by Artech House Publishers, USA in October 2009. His research interests concentrate on the biosensors (e.g. living cell sensor, DNA sensor and protein sensor) and BioMEMS system. Ping Wang received his BS degree, MS degree and Ph.D. degree of electrical engineering in Harbin Institute of Technology, Harbin, PR China in 1984, 1987 and 1992, respectively. He is currently a professor of Biosensors National Special Lab, Department of Biomedical Engineering of Zhejiang University. His research interests include biomedical sensors, electrochemical sensors and measurement technique.