Wireless transmission of fast-scan cyclic voltammetry at a carbon-fiber microelectrode: proof of principle

Journal of Neuroscience Methods 140 (2004) 103–115

Wireless transmission of fast-scan cyclic voltammetry at a carbon-fiber microelectrode: proof of principle Paul A. Garrisa,∗ , Robert Ensmanb,c , John Poehlmanc , Andy Alexanderc , Paul E. Langleyd , Stefan G. Sandberga , Phillip G. Grecoa , R. Mark Wightmane , George V. Rebecd a

Cellular and Integrative Physiology Section, Department of Biological Sciences, Illinois State University, 210 Julian Hall, Normal, IL 61791-4120, USA b Ensman Instrumentation, 1102 Berkshire Court, Bloomington, IN 47401, USA c Electronic Instrument Services, Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, IN 47405-7102, USA d Department of Psychology and Program in Neural Science, Indiana University, 1101 East Tenth Street, Bloomington, IN 47405-7007, USA e Department of Chemistry and Neuroscience Center, University of North Carolina, CB #3290, Venable Hall, Chapel Hill, NC 27599-3290, USA Received 19 November 2003; accepted 19 April 2004

Abstract Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM) provides exquisite temporal and spatial resolution for monitoring brain chemistry. The utility of this approach has recently been demonstrated by measuring sub-second dopamine changes associated with behavior. However, one drawback is the cable link between animal and recording equipment that restricts behavior and precludes monitoring in complex environments. As a first step towards developing new instrumentation to overcome this technical limitation, the goal of the present study was to establish proof of principle for the wireless transmission of FSCV at a CFM. Proof of principle was evaluated in terms of measurement stability, fidelity, and susceptibility to ambient electrical noise. Bluetooth digital telemetry provided bi-directional communication between remote and home-base units and stable, high-fidelity data transfer comparable to conventional, wired systems when tested using a dummy cell (i.e., a resistor and capacitor in series simulating electrical properties of a CFM), and dopamine measurements with flow injection analysis and in the anesthetized rat with electrical stimulation. The wireless system was also less susceptible to interference from ambient electrical noise. Taken together, the present findings establish proof of principle for the wireless transmission of FSCV at a CFM. © 2004 Elsevier B.V. All rights reserved. Keywords: Voltammetry; Carbon-fiber microelectrode; Electrical stimulation; Rat; Dopamine; Wireless; Telemetry; Bluetooth

1. Introduction Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM) has become a powerful tool for investigating the function and regulation of the central nervous system (Adams, 1990). Like other real-time electroanalytical techniques, such as amperometry (Suaud-Chagny et al., 1995) and high-speed chronoamperometry (Gerhardt, 1995), the primary advantage is millisecond temporal resolution at a micron-sized probe. The high sampling rate permits characterizing basic mechanisms of extracellular neuronal sig∗

Corresponding author. Tel.: +1 309 438 2664; fax: +1 309 437 3722. E-mail address: [email protected] (P.A. Garris).

0165-0270/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2004.04.043

naling (Stamford, 1995; Garris and Rebec, 2002) and subsecond changes in neurotransmitter levels associated with behavior (Wightman and Robinson, 2002). Additionally, the small probe size causes little tissue damage during implantation, thus affording placement near neurotransmitter release sites and minimizing diffusional distortion (Lu et al., 1998; Allen et al., 2001). A feature unique to FSCV is the voltammogram, which serves as a chemical signature to identify the origin of the measured response (Baur et al., 1988; Michael et al., 1998). Because of the plethora of easily oxidized species in the brain (Marsden et al., 1988) and sensitivity of voltammetry to changes in ion concentration (Jones et al., 1994; Rice and Nicholson, 1995), positive analyte identification is a critical concern.

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The first brain application of FSCV at a CFM monitored electrically evoked dopamine levels in the anesthetized animal (Millar et al., 1985). In the next decade, technology was advanced to record similar signals (Garris et al., 1997) and dopamine transients elicited by a novel sensory stimulus (Rebec et al., 1997) in ambulant animals. More recently, this approach measured sub-second changes in dopamine during sociosexual (Robinson et al., 2001, 2002) and cocaine-seeking (Phillips et al., 2003) behaviors. Despite these gains, one factor limiting FSCV at a CFM for studying brain-behavioral relationships is the cable link between animal and recording equipment. Not only is behavior affected as a result, but also important experimental paradigms, particularly involving complex or enriched environments, are precluded from study. It is therefore necessary to develop technology for use in awake animals that exploits the attractive analytical characteristics of FSCV at a CFM but overcomes limitations imposed by cable tethers. Because of these considerations, we are developing instrumentation for the next step in its evolution: wireless transmission. Several issues must be considered for developing a wireless instrument supporting FSCV at a CFM in behaving animals. For example, features of real-time electroanalytical techniques impose difficulties for wireless transmission, including fast acquisition of high-resolution data, susceptibility to noise artifacts, and low amplitude signals. Analog telemetry using amplitude and frequency modulation may be data-rate limited and lack the necessary fidelity. In theory, the recent development of digital telemetry (Weisman, 2002) holds great promise for this purpose. On a practical level, it is not known whether a commercially available package will suffice or whether one will need to be designed. Digital telemetry will additionally require an onboard microprocessor for digitizing analog signals collected at the CFM. Whether such a microprocessor can manage digitization, control FSCV, direct wireless transmission, and perhaps perform other tasks seamlessly, is a key question. Although adding another level of complexity, bidirectional communication between remote and home-base units will expand functionality by enabling wireless experimental control. Ideally, measurements collected wirelessly should also be insensitive to ambient electrical interference in order to support measurements outside a Faraday cage, which is required for conventional ‘wired’ systems and limits applications. The goal of the present study is to establish proof of principle for the wireless transmission of FSCV at a CFM. Because of the technical complexities described above, it is our belief that the first step towards realizing a wireless instrument is to establish proof of principle. Otherwise, if the design were flawed, the considerable effort required for instrument miniaturization would be wasted. To this end, a prototype system was developed and tested. The remote unit contained three modules: voltammetry analog front-end, microprocessor and transmitter–receiver. The home-base unit

consisted of a transmitter–receiver and personal computer. Although too large for attaching to a rat, the size of the remote unit expedited circuit construction, modification and testing, consonant with the goal of proof of principle. A commercial, digital telemetry package called Bluetooth (Ericsson, Sweden) was used for bi-directional communication. Using a resistor–capacitor circuit mimicking the electrical properties of a CFM, the prototype wireless system was tested for fidelity, stability, susceptibility to ambient electrical interference, and transmission distance. Comparisons to conventional wired technology for FSCV at a CFM were also made for measuring dopamine in vitro with flow injection analysis and with electrical stimulation in vivo in the striatum of an anesthetized rat. Taken together, the results described herein establish proof of principle for the wireless transmission of FSCV at a CFM.

2. Materials and methods 2.1. Animals One adult male Sprague–Dawley rat (400 g) was purchased from Harlan Sprague Dawley (Indianapolis, IN). The animal was housed under standard conditions of lighting, temperature and humidity. Food and water were provided ad libitum. Care was in accordance with NIH guidelines (publication 86–23) and approved by the Institutional Animal Care and Use Committee of Indiana University. 2.2. Voltammetry experiments 2.2.1. In vivo voltammetry Electrically evoked levels of extracellular dopamine were monitored in the caudate-putamen of an anesthetized rat by FSCV at a CFM (Rebec et al., 1997; Bergstrom and Garris, 2003). Under chloral hydrate anesthesia (400 mg/kg i.p.), holes were drilled in the skull for placement of stimulating, reference and voltammetric electrodes. All stereotaxic coordinates were obtained from the atlas of Paxinos and Watson (Paxinos and Watson, 1986). Antereoposterior (AP) and mediolateral (ML) coordinates were referenced to bregma, and dorsoventral (DV) coordinates were referenced to dura. The stimulating electrode was positioned just dorsal to the medial forebrain bundle (−4.6 AP, +1.6 ML and −7.5 DV) and incrementally lowered until a robust signal for dopamine was obtained in the caudate-putamen measured voltammetrically. The reference electrode, a stainless steel screw, was placed in superficial cortex. The peak oxidation potential for dopamine measured with a stainless steel screw as a reference electrode is slightly more positive than that measured with a Ag/AgCl reference electrode (see next section). The CFM was positioned in the dorsomedial caudate-putamen (+1.2 AP and +3.0 ML, respectively). The DV coordinate of the CFM varied between −4.3 and −5.0.

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2.3. Flow injection analysis In vitro measurements using FSCV at a CFM were performed by flow injection analysis (Kristensen et al., 1986). The CFM was positioned in the center of outflow tubing emptying into a reservoir. Dopamine was injected into the flowing stream by a solenoid and pneumatic actuator (Rheodyne Model 7163, Solenoid Valve Kit, Alltech Associates, Deerfield, IL, USA) driving an injection port (Rheodyne Model 5712 Low Pressure Switching Valuve, Alltech Associates). Buffer composed of 150 mM sodium chloride and 25 mM Hepes at a pH 7.4, was pumped through the system at a rate of 4 ml/min (Pump II Pico Plus, Model 70-2213, Harvard Apparatus, Holliston, MA, USA). The Ag/AgCl reference electrode, a chloridized silver wire, was positioned under the buffer level in the reservoir. 2.4. Dummy cell A dummy cell, consisting of a 1500 pF capacitor and 100 k resistor, was used to mimic the electrical properties of the CFM during simulated measurements of FSCV. The dummy cell was connected between inputs for the reference

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electrode and CFM. Two-electrode mode is most commonly used in FSCV, as IR drop problems are minimal at the low currents encountered. 2.5. Electrochemistry Carbon-fiber microelectrodes (r = 2.5 ␮m) were prepared as previously described (Cahill et al., 1996). Two conventional systems performed electrochemistry in the present study. One wired voltammetry system was used for dummy cell measurements in the Faraday cage (Fig. 6) and flow injection analysis (Fig. 7). This system consisted of an EI400 bipotentiostat (Cypress Systems, Lawrence, KS, USA), which was computer controlled by software locally written in LabVIEW (National Instruments, Austin, TX, USA) (Michael et al., 1999). The second wired voltammetry system (Rebec et al., 1997), consisting of a locally built potentiostat under computer control by software locally written in assembly language (Microsoft Macro Assembler Ver. 5.1, Microsoft Corporation, Redmond, WA, USA), was used for in vivo voltammetry in the anesthetized rat (Fig. 8). All data were digitally stored for subsequent analysis. Details of electrochemistry are graphically shown in Fig. 1 and described in Section 3.1.

Fig. 1. Fast-scan cyclic voltammetry at a carbon-fiber microelectrode. (A) A triangle wave is applied to the CFM at 100 ms intervals. Applied potential (E) is plotted vs. time. (B) The potential (E) of the triangle wave applied to the CFM (top panel) and the resulting current (I) recorded at the CFM (bottom panel) are plotted vs. time. The solid line in the bottom panel is current recorded in buffer alone. Dotted and dashed lines are current recorded in the presence of 5 and 10 ␮M, respectively. (C) The top panel re-plots the current vs. time traces shown in B. as current (I) vs. applied potential (E) to yield background voltammograms. The bottom panel shows background subtracted voltammograms calculated by subtracting the background voltammogram collected in buffer alone (solid line) from background voltammograms collected in the presence of 5 or 10 ␮M dopamine (dotted and dashed lines, respectively).

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2.6. Electrical stimulation The bipolar stimulating electrode was purchased from Plastics One (MS 303/2, Roanoke, VA, USA). The tips were separated by approximately 1.0 mm. Stimulus pulses were computer generated, biphasic and applied at a frequency of 60 Hz. Current and train duration are indicated in the text. Before application to the stimulating electrode, pulses were passed through a constant-current generator and optical isolator (NL 80, Neurolog, Medical Systems, Great Neck, NY, USA). 2.7. Drugs and chemicals All chemicals and drugs were used as received and purchased from Sigma Chemical Company (St. Louis, MO, USA). Aqueous solutions were prepared in doubly distilled, deionized water.

3. Results 3.1. General characteristics of FSCV at a CFM Fig. 1 describes the general characteristics of FSCV at a CFM used by all voltammetry systems described in the present study. The potential of the CFM was linearly scanned from a resting value of −400 to 1000 mV and back again at a rate of 300 V/s (Fig. 1A). The duration of the scan was approximately 9.3 ms, and scans were repeated at an interval of 100 ms. In between scans, the CFM rested at −400 mV. Current recorded at a CFM during one scan is shown in Fig. 1B. Both scan voltage (top panel) and measured current (bottom panel) are plotted versus time. A large background current was recorded at the CFM when immersed in buffer and in the absence of analyte (solid line). By conven-

tion, current is negative during the forward scan from −400 to 1000 mV and positive during the reverse scan from 1000 to −400 mV. Responses at the same CFM in the presence of two concentrations of dopamine, 5 and 10 ␮M, are also shown (dotted and dashed lines, respectively). In addition to background current, there was an additional feature during the forward scan representing current due to the oxidation of dopamine to a quionone and during the reverse scan representing the reduction of the electroformed quionone back to dopamine. As shown in Fig. 1C, current is more typically plotted against applied voltage to obtain a voltammogram. Traces shown in the top panel are background voltammograms and are the same current responses shown in Fig. 1B. Traces shown in the bottom panel are background subtracted voltammograms for the two concentrations of dopamine. Because background current is quite stable at a CFM, it can be subtracted from responses collected in the presence of analyte. The background subtracted voltammogram for dopamine clearly showed oxidation of dopamine near 600 mV and reduction of the electroformed quinone near −200 mV. In practice, several voltammograms are averaged together before subtraction. In addition to chemical information, FSCV also provides the temporal profile of analyte concentration. This profile is obtained by plotting current, averaged across a specified potential range for each voltammogram versus time. This range typically encompasses the peak oxidation potential for the analyte. Because scans are applied every 100 ms, the temporal profile is obtained with 10 S/s (samples per second) or 10 Hz resolution. The actual rate of data acquisition is, in fact, much higher. Data acquisition rates for the two conventional, wired voltammetry systems used in the present study were 40 kS/s (kilosamples per second) (Rebec et al., 1997) and 100 kS/s (Michael et al., 1999). These rates are determined by software and the analog-to-digital converter (ADC). Only current mea-

Fig. 2. Cartoon of instrumentation developed for the wireless transfer of fast-scan cyclic voltammetry at a carbon-fiber microelectrode. Abbreviations: Ref, reference electrode; WE, working electrode; ADC, analog-to-digital converter; DAC, digital-to-analog converter.

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Fig. 3. Simplified circuit diagram of remote unit. A two M resistor was used in the current-to-voltage circuit. Abbreviations: Ref, reference electrode; WE, working electrode; ADC, analog-to-digital; D I/O, digital input/output; S I/O, serial input/output.

sured during the voltage scan is acquired. For calculating all current versus time profiles shown in the present study, current was averaged between 500 and 700 mV, which typically encompasses the peak oxidation potential for dopamine, and background subtracted. 3.2. Hardware development A cartoon of the hardware developed to acquire FSCV at a CFM wirelessly is shown in Fig. 2. The remote unit consisted of three components: voltammetry analog front-end, microprocessor and transmitter–receiver. A personal computer and transmitter–receiver made up the home-base unit. Fig. 3 shows a simplified circuit for the voltammetry analog front-end with connections to the microprocessor and transmitter–receiver. Three amplifiers are used in the FSCV circuit. The first, a low noise, high bandwidth operational amplifier (LMV751, National Semiconductor Corporation, Santa Clara, CA, USA), serves as a current-to-voltage converter. One half of a dual differential amplifier (INA2132, Texas Instruments Incorporated, Dallas, TX, USA) subtracts the applied triangle wave from the measured signal. In the present configuration, the voltage of the CFM or working electrode (WE) is scanned relative to the reference electrode (Ref), which is kept at circuit common. The second half of the dual differential amplifier (not shown) is used to generate potentials for the subtraction circuit and the ADC. The remote unit microprocessor (C8051F007DK, Silicon Laboratories, Austin, TX, USA) applies the voltage scan (i.e., triangle wave) generated at a digital-to-analog converter (DAC) and digitizes the signal collected at the CFM using an ADC. The microprocessor incorporates one 12-bit ADC with configurable gain and multiple inputs, each with 2.4 V of dynamic range, two 12-bit DACs, a high-speed serial interface, and internal RAM (2304 bytes) and FLASH (32 Kbytes) memory, which is easily programmed through a JTAG interface. The voltage scan is generated by software stored in FLASH memory. A 14.7456 MHz crystal enables an

ADC rate of 100 KS/s and 460 Kbaud serial communication with the third component of the remote unit, telemetry. Software controlling data acquisition and communication with the telemetry module is also stored in FLASH memory. A Bluetooth module (ROK 101 008/21, Ericsson, Stockholm, Sweden) performs telemetry for the remote and homebase units. This digital technology uses Gaussian Frequency Shift Keying, which is a form of frequency modulation, and supports data transfer at ∼700 Kbits per second (Kbps) with internal error correction at distances up to 9 m. Each Bluetooth module contains a transmitter–receiver for bidirectional communication, is 3.3 cm× 1.7 cm× 0.25 cm, and requires a 2.5 cm or shorter antenna. Acquired data is stored in RAM until transmission. Communication between the home-base telemetry module and computer utilizes a highspeed serial interface (232PCI1A, B&B Electronics, Ottawa, IL, USA). 3.3. Software development Programming of the remote-unit microprocessor was developed in assembly language (Cygnal IDE Ver. 1.3, Cygnal Integrated Products). The triangle wave applied to the CFM is software generated for each scan, as opposed to stored in memory and recalled. After wireless initiation, scan application is self-running under control of the remote-unit microprocessor. Data for each voltammogram is divided into nine, 229-byte packets, wirelessly transmitted separately during the time between scan applications. The 229-byte packet size is determined by Bluetooth but not required. Matching packet size makes the system more efficient and deterministic. Although data are acquired with 12-bit resolution, packet format provides space for 16-bit numbers, in anticipation of a remote-unit microprocessor with 16-bit ADC. Internal error correction of Bluetooth operates at the packet level, checking whether one has arrived before sending another. If not, the packet is re-sent. Each packet contains a header, identifying packet and scan number. This information is used at the

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home-base unit to verify proper reconstruction of the voltammogram. For the home-base unit, initial programming for Bluetooth control and terminal emulation software for serial port communication was written in Microsoft Visual Basic (Ver. 5.0, Microsoft Corporation). Software allowed full transfer rate (945 points per 100 ms) without data loss and bi-directional communication between remote and home-base units. The 945 points defined each voltammogram. Instructions originating from the home-base unit were used to initiate and terminate scanning, and initiate and terminate data collection. Alternative software was later developed for these purposes in LabVIEW (National Instruments). Software was also developed in LabVIEW (National Instruments) for analysis of all FSCV data formats used in the present study, whether collected wirelessly or by the two conventional, wired systems (Rebec et al., 1997; Michael et al., 1999). In addition to reconstructing voltammograms from data packets sent wirelessly, this software performed background subtraction, generated current versus time profiles, plotted data, and created text files for input into spreadsheet and graphics software.

3.4. Evaluation of stability and noise pick-up A sample triangle wave, generated by the remote unit and acquired wirelessly by the home-base unit, is shown in Fig. 4A. The triangle wave scans the appropriate voltages, from a bias of −400 mV to a peak of 1000 mV and back. Duration of the scan is approximately 9.3 ms, appropriate for this voltage range and a scan rate of 300 V/s. This result indicates that the remote unit is capable of providing a suitable triangle wave for FSCV. The resulting background current for one scan recorded at a dummy cell and also sent wirelessly is shown in Fig. 4B. Both recordings are composed of 945 points collected over a period of ∼9.3 ms and sent during the following ∼90.7 ms epoch. The background current recorded at a dummy cell is similar to that recorded at a CFM. Both show charging current expected for series resistance and capacitance. The CFM background additionally shows features representing electrolysis of surface groups on the carbon fiber. For all measurements described here forward, entire voltammograms were sent wirelessly. Background subtraction and generating current versus time profiles were performed at the home-base unit using raw voltammogram data.

Fig. 4. Evaluation of stability and noise pick-up. (A) The triangle wave was generated by the digital-to-analog converter of the remote unit and sent wirelessly. (B) Background current recorded at the remote unit and sent wirelessly is plotted vs. time. Anodic current is negative. The corresponding applied voltage is found in Panel A. (C) The four different recordings were collected and sent wirelessly from the same fixed location. (D) The recordings were collected and sent wirelessly from four different sites surrounding the home-base unit. In all cases, the remote unit was approximately 2 m from the home-base unit. Scales are identical in Panels C and D. (E) The recording was collected at one, fixed location and sent wirelessly during various perturbations of the remote and home-base units. Each perturbation was for 20 s and was preceded by 20 s of baseline measurements. Triangle symbols indicate the beginning of each perturbation. The same three perturbations were applied in the same order to the remote unit first followed by the home-base unit. The perturbations were as follows: waving of a metal coat rack near but not touching the unit; finger touching the unit; and waving a live surge protector near but not touching the unit. All voltammetric recordings were collected at a dummy cell.

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Recordings collected at the dummy cell were also used to assess the stability of wireless measurements. Fig. 4C shows four individual recordings collected with the remote unit at one fixed location, approximately 2 m from the home-base unit. The unit was not held by investigators. Recordings are current versus time and background subtracted. Data indicate that the signal is stable at one fixed location. As shown in Fig. 4D, current versus time recordings were then collected at four different, fixed locations for the remote unit. Each location was approximately 2 m from the home-base unit, together bounding it on all four sides. Again, the remote unit was not held by investigators. The bottom recording was collected at the same location as those in Fig. 4C. As can be seen, recordings were similar but not identical. In particular, the second trace from the bottom exhibited some distortion. This distortion could reflect interference from ambient electrical noise, because this recording was collected when the remote unit was positioned near a wall outlet and power cords. The distortion is presumably aliasing of 60 Hz line noise. To evaluate susceptibly of the wireless instrument to ambient electrical interference and sites of noise pick-up, the remote unit was placed in one fixed location, the same location as that for Fig. 4C, and evaluated. The unit was not held by investigators. Results are shown in Fig. 4E. Perturbations, the start of which is demarcated by the triangle symbols, lasted for 20 s and were preceded by 20 s of baseline recording. The first perturbation was a metal coat rack, acting as an antenna, moved around in close proximity to, but not touching, the remote unit. No change was recorded in the signal. Finger touching components of the remote unit (e.g., the exposed portion of dummy cell leads), the second perturbation, clearly resulted in noise spikes, as did waving a live surge protector near but not touching the remote unite, the third perturbation. In contrast, none of these perturbations altered the signal when applied to the Bluetooth transmitter–receiver module of the home-base unit. These results suggest that the wireless instrument is susceptible to some, albeit minimal, ambient electrical interference, especially in close proximity to power lines and electrical equipment, but that the Bluetooth telemetry is not a site of noise pick-up.

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directions. There was small distortion of the recorded signal while the remote unit was mobile. Maximal transmission distance was assessed by monitoring the dummy cell recording while walking the remote unit away from the home-base unit in one direction. As shown in Fig. 5B, wireless transmission was successful at distances beyond the specified 9 m, but failed at distances farther than ∼16 m. Similar to panel Fig. 5A, recordings are current versus time but plotted as current versus distance. 3.6. Comparisons to conventional, wired FSCV systems Experiments using FSCV at a CFM are typically performed in a Faraday cage to limit noise pick-up, particularly 60 Hz interference from power lines. Because the wireless instrument demonstrated some susceptibly to noise pick-up in previous tests, we evaluated how well it performed compared to a conventional FSCV system in a Faraday cage. For wireless measurements, the home-base transmitter–receiver was also placed inside the cage at all times. This telemetry module is connected to the external home-base computer by a serial cable. The home-base transmitter–receiver was placed inside the cage, because Bluetooth signals cannot easily pass through the wire screening. This phenomenon is shown in the Fig. 6, inset, where the wireless dummy cell signal degraded quickly after closing the Faraday cage door at 20 s (inverted triangle symbol). The recording is current versus time.

3.5. Evaluation of transmission distance To test the Bluetooth specification for transmission distance, the remote unit was walked 9 m in one direction and the same distance in the opposite direction while recording FSCV at a dummy cell. Results are shown in Fig. 5A. The top two traces are repetitions and are continuous recordings as the remote unit is walked to the described distance along the x-axis. Hence, recordings are current versus time but plotting as current versus distance. For comparison, the remote unit was immobile, approximately 0.5 m from the home-base unit, for the duration of the bottom recording. The remote unit was held by one of the investigators for all measurements in this experiment. Bluetooth was clearly able to transmit simulated FSCV data over the specified distance of 9 m in both

Fig. 5. Evaluation of transmission distance. (A) The top two recordings were collected and sent wirelessly as the remote unit was walked 9 m away on both sides of the home-base unit. The bottom recording, collected and sent wirelessly from a fixed location approximately 0.5 m from the homebase unit, serves as the control. (B) The recording was collected and sent wirelessly as the remote unit was walked in one direction away from the home-base unit. All recordings were collected at a dummy cell.

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Fig. 6. Comparison of wireless and conventional FSCV systems in a Faraday cage. Recordings were collected at a dummy cell by the wireless (A) and conventional (B) systems during 20 s perturbations. The beginning of each perturbation is shown by the diamond symbol. For the first 20 s of the recording, the Faraday cage door was shut. The perturbations were as follows: 20 s, cage door opened; 40 s, hand inserted into the cage and waved; 60 s, hand removed and cage door closed; 80 s, ground to the Faraday cage disconnected; 100 s, ground to the Faraday cage re-connected. Because signals collected by the conventional FSCV system were filtered at 2 KHz by the potentiostat, a comparable amount of filtering was applied to the wireless signals offline during processing. Inset: the recording was collected from a dummy cell by the wireless system. The Faraday cage door was closed at 20 s (triangle symbol).

The dummy cell recording in Fig. 6A is from the wireless instrument, whereas that in Fig. 6B is from a conventional system. Both recordings were collected with the dummy cell inside the same Faraday cage and are current versus time. The diamond symbols demarcate the beginning of perturbations lasting 20 s. The door to the Faraday cage was closed for the beginning of data collection. The first perturbation, opening the Faraday cage door for 20 s, caused small but perceptible artifacts in the recording collected by the conventional system. These artifacts were enhanced by the second perturbation at 40 s, when a hand was placed inside the Faraday cage and waved around the dummy cell but not touching it. The recording returned to normal when the hand was removed, and the cage door was closed at 60 s. However, a large artifact was produced when the ground to the Faraday cage was disconnected for the fourth perturbation at 80 s. The signal normalized only when ground was re-connected at 100 s. Interestingly, none of these perturbations affected the recording collected wirelessly. These results indicate that the wireless instrument is much less susceptible to noise pick-up than the conventional FSCV system. Fig. 7 compares wireless and conventional FSCV systems using flow injection analysis. Signals in the top three panels (A, B and C) were collected by the conventional system, whereas those in the bottom three panels (D, E and F) were collected wirelessly. All signals were collected at the same CFM and are current versus time. Scales in the top and

bottom sets of panels are identical and identified in the top panels. Panels A and D show the temporal response to a bolus injection of 10 ␮M dopamine. The beginning of the 10 s injection is demarcated by the inverted triangle in both panels. The slight delay until signal increase is due to the distance the bolus travels between the injector port and CFM. Panels B and E show background cyclic voltammograms collected prior to dopamine injection (no DA, solid line) and in the presence of dopamine (DA, dashed line). Panels C and F show background subtracted cyclic voltammograms characteristic for dopamine, as peaks for the oxidation of dopamine (downward) and reduction of the electroformed quinone (upward) are clearly evident. Background subtracted voltammograms were calculated by subtracted background current collected in the absence of dopamine from current collected in the presence of dopamine (solid and dashed lines, respectively in Panels B and E). Overall, signals were largely indistinguishable between wireless and wired systems, suggesting that the two systems performed similarly. Fig. 8A compares wireless and conventional FSCV systems for monitoring electrically evoked levels of extracellular dopamine in the brain of an anesthetized rat. Data described by the solid circles (WIRELESS) were collected wirelessly whereas open circles (WIRED) were collected by the conventional system. Recordings are current versus time, and the arrow demarcates the initiation of 2 s, 60 Hz (±300 ␮A) stimulation to the medial forebrain bundle. Similar to previous descriptions of evoked signals recorded voltametrically in vivo (Garris and Rebec, 2002; Bergstrom and Garris, 2003), extracellular dopamine levels increased during application of the stimulus train, due to neurotransmitter release, and decreased quickly after cessation of the train, due to neurotransmitter uptake. The three wired traces were collected at depths of 4.5, 4.7 and 4.9 mm. The depth of 4.5 mm produced the largest amplitude recording. The smallest amplitude trace, recorded at 4.9 mm, was collected just before switching from the wired to wireless system. Due to concern that the stereotaxic apparatus may have been jarred during switching, the CFM was lowered to 5.0 mm before wireless monitoring. The amplitude of the wireless trace was in between those collected with the wired system. Because all traces were recorded at the same CFM but at different depths, the most likely reason for the observed variation in amplitude is micro-heterogeneity in the density of striatal dopaminergic terminals (Stamford et al., 1986). Fig. 8B shows two recordings collected wirelessly at shorter train durations (0.4 and 1.0 s) and a lower stimulus current (±150 ␮A). The depth of the CFM was 5.0 mm. Both signals are well resolved from baseline. The recording for the 0.4 s train is notable, because its amplitude and duration is comparable to observed dopamine transients associated with behavior (Robinson et al., 2001, 2002). This result suggests that the wireless FSCV system exhibits the requisite sensitivity for monitoring physiological changes in extracellular dopamine.

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Fig. 7. Comparison of wireless and conventional FSCV systems using flow injection analysis. Top panels were recorded using the conventional system, whereas bottom panels were recorded wirelessly. All measurements were collected at the same CFM inside a Faraday cage. The scales are identical for each corresponding set of top and bottom panels. (A and D) Recordings were collected during injection of a bolus of 10 ␮M dopamine beginning at the triangle. Current was measured for a 50 mV range surrounding the peak oxidation potential for dopamine (550 mV). Anodic current is positive. (B and E) The solid line (no DA) is the background cyclic voltammogram recorded prior to dopamine injection, whereas the dashed line (DA) is the background cyclic voltammogram during dopamine injection. (C and F) The background cyclic voltammogram collected in the absence of dopamine was subtracted from that collected in the presence of dopamine, revealing a background-subtracted cyclic voltammogram characteristic for dopamine. Anodic current is negative for Panels B, C, E and F. Similar to Fig. 4, wireless signals were filtered for comparison to signals obtained with the conventional FSCV system.

Fig. 8. Electrically evoked dopamine levels in the anesthetized rat. (A) A 2 s, 60 Hz pulse train (±300 ␮A) was applied at the arrow. Data collected by the wireless system (WIRELESS) are described by the closed circles, whereas data collected by the conventional system (WIRED) are described by the open circles. Both recordings were collected at the same CFM, but its dorsoventral position was slightly different. (B) Both recordings were collected by the wireless system. The higher amplitude recording was evoked by a 1 s train, whereas the other recording was evoked by a 0.4 s train. Stimulation current and frequency were ±150 ␮A and 60 Hz, respectively. Both trains were applied at the arrow. In both panels, oxidative current is positive.

4. Discussion The present study describes new technology developed for the wireless transmission of FSCV at a CFM. The remote unit of the new instrument was composed of an analog front-end potentiostat, a microprocessor for data acquisition, triangle wave generation, and telemetry control, and a transmitter–receiver for bi-directional communication with the home-base unit. Simulated measurements using a dummy

cell were wirelessly transmitted at distances up to ∼16 m in real time with high fidelity and stability. The performance of the wireless instrument was comparable to conventional wired systems for measuring dopamine with flow injection analysis and with electrical stimulation in the brain of the anesthetized rat. Moreover, the wireless instrument was less susceptible to interference from ambient electrical noise. Taken together, these results establish proof of principle for the wireless transmission of FSCV at a CFM.

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4.1. Comparison to conventional, wired systems The wireless instrument used FSCV parameters (i.e., 300 V/s scan rate, −0.4 V bias potential, 1.4 V scan peakto-peak voltage, and 100 ms scan interval), which are standard for in vitro and in vivo measurements of dopamine in brain tissue (Garris and Wightman, 1995). It seems likely, in view of similar hardware and software requirements, that our instrument could eventually be applied to other voltammetry techniques. For example, alternative FSCV waveforms have been developed for increasing sensitivity to dopamine (Hafizi et al., 1990; Phillips et al., 2003), measuring histamine (Travis et al., 2000), serotonin (Jackson et al., 1995) and oxygen (Venton et al., 2003), and resolving norepinephrine and epinephrine (Cooper et al., 1992). It may also be possible to extend functionality to other real-time measurements such as amperometry (i.e., constant potential) (Suaud-Chagny et al., 1995) and high-speed chronoamperometry (i.e., square-wave pulses) (Gerhardt, 1995), and to enzyme-linked microsensors, which often amperometrically monitor hydrogen peroxide generated by an oxidase (Lowry et al., 1998; Kulagina et al., 1999; Burmeister and Gerhardt, 2001). The rate of data acquisition for the wireless instrument was ∼100 KS/s with 12-bit resolution. These ADC specifications are comparable to conventional, wired systems for FSCV at a CFM (∼16 to 100 KS/s and 12- and 16-bit resolution) (Wiedemann et al., 1991; Rebec et al., 1997; Lu et al., 1998; Michael et al., 1999). Availability of an alternative microprocessor for the remote-unit with 16-bit ADC is anticipated. Such high-resolution data acquisition would enable viewing all voltammograms in a three-dimensional graph (i.e., current versus voltage versus time) (Michael et al., 1998). Viewing all data is superior to plotting a single background subtracted cyclic voltammogram representing the difference between two time points (e.g., pre- and post-dopamine administration during flow injection analysis). Individual voltammograms were acquired wirelessly every 100 ms, the rate of collection at the dummy cell or CFM. These data demonstrate that the wireless instrument is capable of transmitting FSCV at a CFM in real time. The wireless system compared favorably to conventional FSCV systems for measuring dopamine during flow injection analysis and in the brain of the anesthetized rat during electrical stimulation. These signals were transmitted wirelessly without data loss or noise addition. In fact, the wireless system exhibited less noise pick-up in the Faraday cage experiment. Rather amazingly, perturbations generating marked artifacts in the wired signal, especially disconnecting ground, had no effect on the wireless signal. This advantage is most likely due to the close proximity of digitization to signal collection on the remote unit. The wireless instrument was not completely immune from noise pick-up, because some interference, perhaps generated by proximity to electrical lines, was observed. Noise was not introduced at the Bluetooth module or during wireless transmission, however, but was added at some other component(s). The origin of distortion in the wireless signal

obtained during evaluation of transmission distance is less clear. Nevertheless, the small magnitude of interference when observed suggests that it may be possible to apply FSCV at a CFM without a Faraday cage using wireless technology. 4.2. Wireless monitoring of neural activity Analog telemetry systems have been developed to monitor electrophysiology and electrochemistry in freely behaving animals. FM-based systems have suitable data transmission rates for capturing single unit electrophysiology and have been applied to several animals including the rat (Eichenbaum et al., 1977), rabbit (Eichenbaum et al., 1977), toad (Pinkwart and Borchers, 1987), monkey (O’Byrne et al., 1991), song bird (Nieder and Klump, 1999) and owl (Nieder, 2000). Stereo-FM has also been used to send electrophysiological recordings collected at two implanted electrodes simultaneously (Nieder, 2000). A slow scan electrochemical technique called differential normal pulse voltammetry, which samples electroactive neurotransmitter metabolites with minute resolution, has been coupled to optoelectronic telemetry (Annovazzi-Lodi and Donati, 1988). In addition to wireless measurements of neurochemistry, this infrared transmission technique can be combined with electroencephalographic, electrooculographic and electromyographic hardware using conventional wired set-ups for simultaneous measurements without electrical cross talk between circuits (De Simoni et al., 1990; Imeri et al., 1994, 1999). Digital telemetry systems using a higher carrier frequency will support applications requiring faster data transmission. Several digital technologies have now been developed to operate in the internationally allocated industrial, scientific and medical (ISM) band of 2.4 GHz, including ‘Wi Fi’, ‘Home RF’, and Bluetooth (Weisman, 2002). Of these, Wi Fi, used for wireless local area networks, transmits the fastest (11 Mbps for IEEE 802.11b and 54 Mbps for IEEE 802.11 g) and farthest (90 m). Home RF is intermediate between Bluetooth and Wi Fi. The fast transmission rates of Wi Fi are compatible with state-of-the-art electrophysiology systems, which monitor the electrical activity of several neurons simultaneously via multiwire array electrodes (Hollerman and Schultz, 1998; Carelli et al., 1999; Chang et al., 2000; Gulley et al., 2002; Nicolelis et al., 2003). For example, Wi FI (IEEE 802.11b) has been used to record single unit activity in the monkey from 16 electrodes sampled at 30 kS/s with 12-bit resolution (Obeid et al., 2002). This system weighed 260 g and measured 14 cm × 8.3 cm × 4 cm, about the size of a young adult rat. Bluetooth was selected for our application for several reasons, such as fidelity, small size, low power requirements, low costs and potential for becoming an industry standard (Weisman, 2002). To operate in the unlicensed 2.4 GHz band without interference from other telemetry signals, Bluetooth uses Frequency Hopping Spread Spectrum (FHSS) modulation at a rate of 1600 hops per second. While slower than the Direct Sequence Spread Spectrum used by Wi Fi, FHSS is

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Fig. 9. Future directions: miniaturization of instrument for wireless transmission of fast-scan cyclic voltammetry at a carbon-fiber microelectrode. The photograph shows the prototype wireless instrument tested in the present study and a smaller, printed circuit board (3.9 cm × 2.0 cm) that will be used to miniaturize the instrument. The prototype instrument contains a Bluetooth module board (bottom), microprocessor board (middle), FSCV circuit (top), and two batteries. The small board is designed to support FSCV and electrophysiology using surface mount components and a smaller microprocessor (0.7 cm × 0.7 cm) than that (1.0 cm × 1.0 cm) used in the prototype instrument.

more resistant to mulitpath and better suited for crowded radio frequency environments. The high rate of frequency hops and internal error correction, although limiting data rate as well, render the technology further immune to interference. Regardless, Bluetooth supported high-fidelity wireless transmission of FSCV at a CFM in real time at distances exceeding specifications. 4.3. Future directions Wireless transmission of FSCV at a CFM will confer important advantages over conventional, wired technology by reducing movement artifacts and noise interference and allowing freer and more natural movement. Two immediate directions for developing our wireless instrument beyond proof of principle are miniaturization and expanding functionality. Small size is absolutely critical for use with ‘free-roaming’ animals. Our immediate goal is an instrument suitably sized for the laboratory rat, which widely serves as an animal model for neuroscience research. Functionality will be expanded by the addition of electrical stimulation and electrophysiology. Coupling electrical stimulation to FSCV at a CFM confers several advantages for dopamine monitoring (Robinson et al., 2001; Garris et al., 2003; Phillips et al., 2003). Not

only can electrical stimulation be used to position the CFM near regions of high dopamine release and provide an evoked background-subtracted cyclic voltammogram for dopamine to compare with those associated with behavior, but also it can be used to determine whether the CFM is working properly. Electrophysiology is an excellent companion to FSCV at a CFM for integrated study of the central nervous system. The photograph in Fig. 9 demonstrates the current status of miniaturization. Shown next to the prototype system developed in the present study to establish proof of principle is a small, printed circuit board. This board was designed to house analog front-end circuits for FSCV and electrophysiology using surface mount components and a microprocessor with identical functionality as that used in the prototype system but in a smaller format. The miniaturized remote unit will consist of four modules, roughly the same size and sandwiched together. In addition to the analog front-end/microprocessor board, there will be the Bluetooth transmitter–receiver, battery and electrical stimulation module. Expected dimensions of the remote unit are 4.0 cm (L) × 2.0 cm (W) × 1.5 cm (H) with a weight of approximately 17 g. This size would be suitable for an adult rat when attached using a backpack. A similar backpack-arrangement is offered commercially by CleveMed (Cleveland, OH) to

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support wireless monitoring of electroencephalography and electrocardiography.

Acknowledgements This work supported by NSF (DBI-0138011 to PAG and GVR) and NIH (NS 15841 to RMW). We kindly thank David Felker (Information Technology Group, Department of Chemistry, Indiana University) for programming the LabVIEW serial link between the home-base computer and Bluetooth module. We also thank Dr. Alfred Strickholm for helpful discussion, and Jahnavi Mithyantha and Chris Leesman for technical support. Circuit diagrams and software are available upon request.

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