Neurol Sci DOI 10.1007/s10072-014-1775-8
ORIGINAL ARTICLE
A movable microelectrode array for chronic basal ganglia singleunit electrocorticogram co-recording in freely behaving rats Xiaobin Zheng • Jia Zeng • Ting Chen • Yuanxiang Lin • Lianghong Yu Ying Li • Zhangya Lin • Xiyue Wu • Fuyong Chen • Dezhi Kang • Shizhong Zhang
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Received: 8 December 2013 / Accepted: 20 February 2014 Ó Springer-Verlag Italia 2014
Abstract The basal ganglia–cortical circuits are important for information process to brain function. However, chronic recording of single-unit activities in the basal ganglia nucleus has not yet been well established. We present a movable bundled microwire array for chronic subthalamic nucleus (STN) single-unit electrocorticogram co-recording. The electrode assembly contains a screwadvanced microdrive and a microwire array. The array consists of a steel guide tube, five recording wires and one referenced wire which form the shape of a guiding hand, and one screw electrode for cortico-recording. The electrode can acquire stable cortex oscillation-driven STN
firing units in rats under different behaving conditions for 8 weeks. We achieved satisfying signal-to-noise ratio, portions of cells retaining viability, and spike waveform similarities across the recording sections. Using this method, we investigated neural correlations of the basal ganglia–cortical circuits in different behaving conditions. This method will become a powerful tool for multi-region recording to study normal statements or movement disorders.
X. Zheng Y. Lin L. Yu Z. Lin X. Wu F. Chen D. Kang (&) Department of Neurosurgery, Key Clinic Specialty Discipline of Fujian, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China e-mail: [email protected]
Introduction
J. Zeng Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA T. Chen Department of Radiotherapy, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China Y. Li Intensive Care Unit, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China S. Zhang (&) Department of Neurosurgery, Institute of Neurosurgery, National Key Clinic Department, Key Laboratory on Brain Function Repair and Regeneration of Guangdong, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China e-mail: [email protected]
Keywords Microwire bundle Basal ganglia Single unit Electrocorticogram
The basal ganglia (BG) are interconnected to the cortex and thalamus [1, 2]. The basal ganglia–thalamo-cortical circuits play a key role in processing information within the global brain, such as motor control, motor learning [3], as well as cognition and memory processing [4]. Abnormal pattern switch of synchronous oscillation and discharge are underlying mechanisms of movement disorders and mental disorders [5, 6]. However, techniques for electrophysiological co-recording in BG and cortex are not well established. In the past two decades, most functional studies in the interactions between the BG and cortex have mainly been focused on immobile animals. Researchers combined electrocorticogram (ECoG) with extracellular recordings of single-unit spikes using glass micropipettes, during acute recording in animals under anesthesia [7–9]. Others recorded single-unit spikes in non-anesthetized animals with their head restrained by a U-shaped piece under sleep condition [10, 11]. Using either technique, researchers need to keep animals immobile and limit the number of
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recording channels. Neural ensemble recording, a revolutionizing technique, achieves simultaneous multi-channel recording in groups of well-isolated single units in behaving and freely moving animals. But it is so far mainly used in animal cortex [12, 13]. It is rarely applied in deep brain region, especially the BG nuclei which are smaller and more irregular, such as subthalamic nucleus (STN) and external segment of globus pallidus (GPe). We designed and developed a movable microelectrode array for simultaneous recording between the BG singleunit spiking activities and cortex oscillations. Using this method, we detected the phase relationship between STN single-unit activities and M1 cortex synchronized oscillations in rats under different behaving conditions.
Fig. 1 Construction of the movable microwire array and the implantation procedure
Materials and methods
Microelectrode construction
Drive design
Most of the operations below were performed under a microscope.
The structural elements of the microdrive include: a 2-cmlong cylinder cut from an insulin syringe (3/10, 31ga, Becton Drive, USA), a 1.5-cm core bar with piston from the same syringe, a stainless steel screw (8 mm long, 1.75 mm in diameter, 0.25 mm pitch) with matching knurled nut (3.0 mm in external diameter) as the propeller screw, two micro-screws (1.4 mm in diameter, 3 mm long) as the locking screw, and a 28-ga hypodermic needle from an 1-ml syringe as a keyway needle to the syringe piston (Fig. 1). The microdrive was installed as follows: 1.
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The cylinder was threaded by the two micro-screws after symmetrically perforating it using a flaming needle. The knurled nut was rotated to the cap end of the propeller screw. Then the screw’s tail end was vertically injection molded into the core bar at its mesial part, and reinforced by Cyanoacrylate gel (Chemmer Enterprise Co., Ltd. Taipei, Taiwan). The core bar and propeller screw were pushed into the cylinder, then the knurled nut was molded to the inner wall of the cylinder. The 28-ga hypodermic needle was bent at 90° about 7 mm away from the tip. After lubricated with mineral oil, the needle tip was vertically penetrated throughout the syringe piston at outer edge point. The needle tip was rotated slightly to bring the mineral oil into the needle trajectory for lubrication. The cap of the insulin syringe was cut at about 7 mm to the closed end to cover the propeller screw as its protective cap.
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A 10- to 12-mm-long 29-ga hypodermic stainless steel tube (330.2 lm outer diameter, 165.1 lm inner diameter, A-M Systems, Carlsborg, USA) was used as the guide tube. A 4-cm full-annealed stainless steel (PFA insulated, 76.2 lm bare diameter, 139.7 lm coated diameter, A-M Systems, Carlsborg, USA) was first stripped 1 cm for insulation. The stainless steel was tightly wound on the proximal end of the tube and fixed with silver paint (Electrolube, H K Wentworth.ltd, UK), serving as the ground wire. Five 5-cm platiniridium wires (PFA insulated, 25.4 lm bare diameter, 38.01 lm coated diameter, A-M Systems, Carlsborg, USA) was used as the recording electrodes. Each was slotted nearly 10 mm throughout the guide tube. One 4-cm half annealed stainless steel (PFA insulated, 50.8 lm bare diameter, 114.3 lm coated diameter, A-M Systems, Carlsborg, USA) was insulation stripped by a brief burn at its end. The bare section was trimmed to 0.5 mm using a microscissor. The stainless steel was slotted though the guide tube, with the bare tip protruding out 2.0–2.5 mm, serving as the reference electrode. The platiniridium wires were adjusted to form a fanlike shape with about 250 lm intervals at about 3 mm away from the tube end using a fine forceps. Cyanoacrylate gel was siphoned into the guide tube through a plastic capillary. A thin layer of gel was left out of the tube but kept free from the bare tip of the reference wire (Fig. 2a). This helped to keep the microwire arrangement and increase the tip rigidity.
Neurol Sci Fig. 2 a The outward arrangement of microwire tips. b The inward arrangement of microwire tips. c The movable electrode consists of one microdrive with keyway needle (unfilled arrow), one microwire electrode array, and one 10-pin electrode connector with one 76.2-lm-diameter ECoG recording wire (solid arrow). d The intraoperative brain penetration with tiny dimples. It pushes away blood vessel without hemorrhage. e The side view of the electrode assembly implanted in the rat’s brain with a protective cap. f Right side view of the electrode assembly implanted in the rat’s brain
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The recording microwire tips were linearly trimmed at 35–45 horizontal degree from right to left (for left STN implantation) using a microscissors with 3.0–3.5 mm left out of the guide tube. So that the recording tips are 0.5–1.0 mm longer than the reference wire tip, and the arrangement of electrode tips formed a guiding-hand shape (Fig. 2a, b). The free ends of all the microwires were 5 mm insulation stripped and wound tightly to a 10-pin subminiature connector (DRP-10-vv-ESR, Omnetics Connector, Minneapolis, MN, USA). A 5-cm fullannealed stainless steel with the same diameter as the ground wire was 5 mm insulation stripped at both ends for connecting to the connector and the ECoG recording screw (Fig. 2c). Silver paste was applied to all the connection points. The pins and the base of the connector were sealed by thin layer epoxy glue (Chemmer Enterprise Co., Ltd.
Taipei, Taiwan). This stabilized the microwire connections after the silver paste solidified. The tip impedance of the recording electrodes was 0.5–1.0 MX at 1 kHz (TH2893 impedance instrument, Tonghui Electronics, China).
Assembly of microelectrode system and preparation before implantation The microwires in guide tube were properly bent at the proximal end, and vertically anchored to the mesial part of the microdrive piston bottom with epoxy glue. After epoxy glue solidified, the microdrive piston was pulled about 5 mm back into the outer cylinder by counter-rotating the propeller screw. Before implanted, the microelectrode was first sterilized with ethylene epoxide. The end of the electrode was
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secured with a tiny spot of melted aseptic polyethylene glycol, which further reinforced the microwire tips (Fig. 2a). The gross weight of the microelectrode system was 13.8 g, including 6.6 g in the microdrive. Implant surgery Six Wistar rats (weight 280–300 g) were anesthetized with i.p. injection of 90 mg/kg ketamine and i.m. 5–9 mg/kg xylazine, with a supplementary dose (25 mg/kg, im) of ketamine if necessary. Each rat’s head was fixed to a stereotaxic frame with -3.3 mm incisor bar. After skull exposure, four stainless steel screws were secured into the skull, including one electrical screw (0.8 mm diameter, 1.6 mm long, Plasticsone, USA) at the dura above the M1 cortex (AP: ?2.00 mm, ML: ?2.80 mm, Paxinos and Watson 2005) as the ECoG recording electrode. One 3-mmdiameter hole was drilled just above the STN coordinate (AP: -3.70 mm, ML: ?2.20 mm, 7.90–8.10 mm below cortex, Paxinos and Watson 2005), and the dura was resected carefully using a hooked needle tip. Before implantation, the microdrive was held by a micromanipulator (MP-225, Sutter Instrument); the connector was held by a helping hand (A-M Systems, Carlsborg, USA). The ECoG microwire was wound onto the ECoG recording screw and fixed by silver paste. The row of electrode tips was adjusted to be parallel to the coronal section of the brain. The lowering speed was first set at 300 lm/min or manually controlled, following the dissolution of the polyethylene glycol. This facilitated the tips to penetrate the brain and push the blood vessels, without bending the microwire or generating big brain dimples (Fig. 2d). After the guide tube’s distal end touched the brain surface, the lowering speed
Fig. 3 a The illustration from the rat brain atlas [modified from Paxinos and Watson (2005)] with the tip trajectory from VPM, ZI to STN. b A 2 s duration representation of tracings of neuronal activities in the VPM, ZI and STN under ketamine anesthesia. c One track of
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was slowed down to 150 lm/min. The craniotomy was filled with gelfoam granule and then covered with tiny spot melted agar gel to avoid the leakage of the cerebrospinal fluid. Continuous extracellular recording was performed in the ventral posteromedial thalamic nucleus (VPM) and zona incerta (ZI) (Fig. 3a) along the microwire tip trajectory. The VPM neurons should show discrete spikes with irregular inter spike interval (ISI) and occasional short burst activity. ZI neurons had typical high amplitude spikes with longer regular ISI and lower background noise (Fig. 3b). Finally, more melted agar gel was applied to cover the skull hole and microdrive bottom for isolation. This avoided the influx of the dental cement into the microdrive bottom during electrode fixation. The electrode connector was placed at 45° to the microdrive. The whole assembly was fixed to the skull with dental cement (Fig. 2e, f). Rats were i.m. injected with 30 mg/kg lincomycin and 0.30 mg/kg buprenorphine for 3 days. Postoperative advancement and histology The electrodes were advanced 125 lm/day by rotating the propeller screw clockwise after the rats had recovered for 3 days. When the electrode tips were advanced into the STN, extracellular signal would show high cell density and background noise with occasional longer burst than VPM neurons (Fig. 3b). Before advancing the electrode tips, the locking screws were loosened. They were fastened again after the advancement. At the end of the recording period, the recording sites were electrolytic lesion marked. The brain coronal frozen sections were Nissl stained as Tseng et al. [14] described. Electrode tip tracts and lesion sites (Fig. 3c) were validated under a microscope (eclipse 80i, Nikon, Japan).
electrode tips (arrow) and electrolytic lesion (asterisk) within STN boundary (dashed line) in brain coronal section with Nissl staining. Scale bar 200 lm
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Data acquisition and analysis STN single units and ECoG activities were monitored during behaving conditions. Signals were headstage pre-amplified and transmitted via a light-connecting cable to two differential AC-coupled amplifiers (Model 1700, A-M Systems, Carlsborg, USA). They were analog/digital converted by the Powerlab data acquisition system (Model ML870, ADInstruments, USA), and recorded by the labChart software (professional version 7.3). The STN spike trains were filtered between 300 and 3,000 Hz and sampled at 20 kHz rate. The ECoG was filtered between 0.1 and 300 Hz, and sampled at 1 kHz rate. For the unit stability analysis, STN neuronal activities were recorded weekly for 8 weeks. STN spikes were template-matching sorted. ISI was another factor for verifying unit identities when spike amplitude changed largely during long term following recordings. Units were considered to be alive only when both coincident ISI and similar waveforms still remained, rather than being lost. Unit size was quantified by the peakto-peak amplitude of the averaged waveforms collected from the 10-min period in the conscious condition [13, 14]. Signal-to-noise ratio (SNR) was calculated for unit quantification as well [15]. STN units stabilities were evaluated by the similarity among spike waveforms, which was defined as the linear correlation (r) values between timeshifted average waveforms (r = 1 indicates identical spike waveforms) [13]. ECoG rhythms were detected using methods described in Zhang et al. paper [16]. The relationship between ECoG rhythms and STN unit activities was analyzed using two methods. The first method used cross-correlogram to show the oscillation of correlated activities [7, 17]. The second method used Hilbert transform to decompose the filtered ECoG to into instantaneous amplitude and phase components. The peak of the up state in ECoG was defined at 180° [18, 19] (Fig. 4). STN spike phase was determined by the angle of the analytic ECoG signal at its location. Finally, phase distribution and mean phase of STN spikes were showed by polar histogram. Rayleigh test was performed for circular uniformity (P \ 0.001 indicates significant phase locking between ECoG and STN spike trains) [16, 18, 20].
Results All the six rats survived longer than 9 weeks after implantation surgery. We successfully obtained STN single-unit recordings and ECoG. 90 % (27/30) microelectrode tips tract went through the STN boundary. We acquired 46 STN units. 67.4 % (31/46) STN units survived more than 1 week and were defined as the stable units. Nine STN new units appeared during the recordings.
Fig. 4 The steps for determining phase relationship between STN spiking activities and theta-ECoG oscillation. a, b The raw ECoG. We applied a band-pass filter to (a) at 4–12 Hz and got (b). c The instantaneous phase of filtered trace after Hilbert transformation. The 180° gray dashed line corresponds to the peak of the ECoG oscillation. d Shows the phases of STN spikes (red numbers in c) which are determined by the instantaneous phase of filtered ECoG trace (red dashed lines) (color figure online)
ECoG rhythm-driving STN single units Previous studies showed the relationship between cortex synchronous oscillation and the BG activities under physiological state [21–24]. We also found some STN neurons driven by ECoG rhythms. Figure 5 shows spiking activities of a STN unit and the coincident ECoG oscillations during different physiological states. Under active exploration, the real-time ECoG showed theta rhythm (Fig. 5a). In power analysis, the principal frequency shows theta rhythm; the frequency of sub-peaks shows gamma band oscillations (Fig. 5b). The STN unit showed rhythmic burst activities that tended to occur at the ascending phase close to the peak of ECoG activities (Fig. 5a). The spike autocorrelogram of the STN unit consistently characterized a high level of theta rhythmicity (Fig. 5c). The STN unit is prone to burst at about 18 ms before the peak of the ECoG theta oscillation, according to the cross-correlogram (Fig. 5d). Phase relationship analysis showed that the STN spike trains phase locked the ECoG theta oscillation, with a mean phase at 168.3 degree (Rayleigh test, P \ 0.001) (Fig. 5e). The ISI histogram also demonstrated that the prominent
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peak indicated the intraburst interval; the sub-peak at about 0.15 s indicated the interburst interval. This meant about 7 Hz burst occurrence of this STN unit during active exploration, which coincided with the simultaneous ECoG theta oscillation (Fig. 5g). Interestingly, during slow wave sleep (SWS), the STN unit turned into a tonic firing mode when slow wave (*1 Hz) oscillated in the coincident ECoG. Spindle-like background activities emerged in short-term burst activities of the STN unit intermittently. We observed simultaneous positive-going sharp waves (SPW) in the corresponding ECoG (Fig. 5f). In the fourth week, the spike peak-to-peak amplitude decreased. However, the spike shapes overlapped well and showed a similarity with the waveforms in previous periods, compared with superimposed spike waveforms in 10 s of different recording periods (Fig. 5h). This indicated a robust living state of this STN unit. STN multi-unit recordings Most multi-unit recordings had relatively low peak-to-peak amplitude and SNR, indicating high-density neurodendrites or synapses around the microwires. Thus, new units probably arose in the recording of such microwires. An example of STN multi-unit recording is shown in Fig. 6. This microwire captured two stable units in the first week, which lasted for more than 2 weeks. In the third week, the spike waveforms in one of the units had a low level of similarity and lower amplitude. This unit disappeared in the fourth week. However, in the subsequent days, a new unit emerged in the recording of the same microwire with different spike waveforms (Fig. 6c) and a smaller pulse width (Fig. 6d) compared with the disappeared unit. Tonic firing pattern was also different from the long burst firing pattern of the survived unit (Fig. 6b).
from the recorded neurons would hinder long-term stable recording. This was also supported by the fact that SNR of survived units accumulated at 5–10 (Fig. 7c). The mean SNR of stable units was 7.848 ± 2.282, which was larger than new units (5.550 ± 1.138). It indicated that new units tended to emerge at the further region from the electrode tips. Both stable units and new units showed significant linear correlations between peak-to-peak amplitude and SNR (stable units, r = 0.8892, P \ 1e-4; new units, r = 0.5265, P = 0.0118) (Fig. 8a). This illustrated high quality and stability of recordings in STN. We calculated the weekly cell lost rate of all the acquired STN units. Units tended to be lost in the first week after we advanced electrodes, at a lost rate of 32.6 % (15/ 46). This rate fell to 20.9 % in the second week and remained at the same level until the fifth week when it reached 9.2 %. This was because quite a small amount of units survived longer than 5 weeks (Fig. 8b). We also predicted the proportion of cells that would be kept retained on each recording period, by cumulating the loss rates in the following weeks. This prediction (dashed line in Fig. 8c) suggested that nearly half (46.4 %) of the units on the initially acquisition week would survive more than 2 weeks. However, the actual proportion of retained cells was higher than the predicted, which was opposite to the result of Jackson et al. paper [13]. It is because new units arose intermittently and were counted (Fig. 8c). Similarity between spike shapes of the cells over consecutive recording sessions was characterized by the maximum linear correlation coefficient between averaged waveforms in Fig. 8d. 84.6 % of waveforms in the same STN units were similar (r [ 0.95) in different recording weeks. Only 29.5 % of waveforms were similar in different units. We concluded that the waveforms stayed stable when we tracked the activities of each individual cell using this electrode assembly.
Stability of STN single-unit recordings Discussion Average peak-to-peak amplitudes of STN stable and new units across different recording periods were shown in Fig. 7a and b. Stable units had larger peak-to-peak amplitudes than new units (152.4 ± 48.63 versus 115.5 ± 19.55 lV). Average peak-to-peak amplitudes did not correlate with the recording periods following the electrode implantation in both stable units (Pearson’s r = -0.13, two tailed, P = 0.11) and new units (r = -0.14, P = 0.5377). At the end of the recording, we still observed amplitudes at around 184.7 lV in stable units and up to 146.3 lV in new units. Most units that survived less than 1 week (Fig. 7a, unfilled circles) had peak-to-peak amplitude either larger than 250 lV or less than 100 lV. It indicated that positioning the wire tip too close to or too far
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So far, long-term neural ensemble recording has mainly been used in rodents or primates cortex using microarray electrodes [12, 15, 25–28]. Researchers have made great progress in seeking more well-isolated and stable single units of interest after implantation [29]. Recently, movable microelectrode with tether [26] or wireless neurochip [13, 30, 31] becomes one of the prevailing techniques in awake animals. However, this advanced neural ensemble recording technique above has not yet been applied in deep brain regions, especially in the BG. It may be because traditional microarray electrodes tend to distort tissues. This would increase the chance of misplacing the electrode tips in deep brain regions and damage the tissue. On the contrary,
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Fig. 5 An ECoG rhythm-driving STN firing unit and its time-shift superimposed spike waveforms. a Single-unit activities of a STN firing unit (top channel) was driven by theta oscillation (middle channel) of ECoG activities (bottom channel) during AE. The time duration of the signals was 4 s. b Power density spectrum of 30 s ECoG activities. c Spike autocorrelogram of the STN unit activities with theta rhythmicity. d Cross-correlogram theta rhythmic relationship between STN spike train and the filtered ECoG. e Polar histogram of the STN unit spike phase distribution to simultaneous ECoG theta oscillation. Red arrow represents the mean locked phase at 168.3 degree. f Tonic
firing mode with spindle-like background activities (arrow in bottom channel) of the STN unit and slow oscillation in delta band with positive-going SPW (arrow in top channel) of coincident ECoG activities during SWS. g ISIH observed the burst firing pattern of the STN unit during AE, in which bimodal distribution of intervals (two peaks marked by arrows) reflecting the interburst and intraburst intervals. h Time-shift superimposed spike waveforms of the STN unit show high level of similarity across the consecutive recording periods. Scales in (a) and (f) 250 ms in time axis, 50 mv in amplitude axis (color figure online)
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Fig. 6 Multi-unit recordings of STN. a Multi-unit signal of two STN units captured by an electrode wire at the first week. Duration 4 s. Scales 200 ms in time axis, 50 mv in amplitude axis. b A new tonic firing mode emerged with spikes marked in the same electrode wire
by dots above the channel. Scales 250 ms in time axis, 50 mv in amplitude axis. c Time-shift superimposed spike waveforms of the multi-unit STN signal across consecutive recording periods. d Voltage versus width scatter plot between the first and the fourth week
bundled microwires electrodes work well subcortical regions, such as hippocampus [32–34], amygdala [35, 36] and hypothalamus [37]. Recently, researchers have developed this kind of bundled electrode for recording trigeminal ganglion units in rats [14], as well as similar electrode with movable drive such as ‘‘Reitboeck’’ electrode for thalamic recordings in rabbits [38]. But the nucleus structure is much smaller and more slender in the BG and more difficult to position the electrode tips in the BG with accuracy. The bundled microwire array presented here can achieve high levels of accuracy and stability in STN single-unit recordings across 8 weeks in behaving rats. We obtained stable peak-to-peak amplitudes and waveforms, and acceptable cell lost rate across the consecutive recording sections. We successfully monitored the simultaneous
ECoG oscillations. We also analyzed the changes in phase relationship of the coupled STN spiking activities, especially the rhythmic burst firing that locked to the M1 ECoG theta oscillation in active exploration. This would further prove the state-dependent synchronization between STN and motor cortex [22, 24]. The length of the protruding parts of microwires is longer than the bundled electrode used in deep brain regions reported by Tseng et al. [14]. Longer protruding end can reduce the bending angle of the microwire. This can help the microwire tips push away brain blood vessels more easily, which are quite often confronted by the tip during its advancement to the deep brain regions. Longer protruding end can also improve stability in long-term behaving recording, especially when the animals shake the
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Fig. 7 Long-term performance of the movable microwire array. Peak-to-peak amplitude of all stable units (a) and new units (b) followed longitudinally across recording periods. Each line indicates a unit recorded by the same wire. Unfilled circles indicate units survived shorter than 1 week around the same wires
heads violently and cause the loss of recording units [13]. The rigidity of the long microwire protruding end is increased by the Cyanoacrylate gel and polyethylene glycol solidified at the microwire end, and the polyethylene glycol
can slowly dissolve when dipping into cerebrospinal fluid. These all facilitate the microwire tips advancing smoothly during electrode implantation. The microwire tips can penetrate the dura easily. However, we still recommend an incision or resection to the relative dura to reduce the concavity or distortion of the brain tissue. Otherwise, the ubiety between the puncture point and the target nucleus will change. We adjusted the tip of the reference electrode 0.5–1.0 mm shorter than those of the recording electrodes. This helped us achieve high level of common-mode rejection ratio of signals and lowered movement artifacts [14]. The distance of 0.5–1.0 mm also located the reference electrode tip in the region of ZI. It is possible to use the reference electrode for ZI stimulation, followed by simultaneous recording to STN single-unit firing. ZI is recently emphasized as a new superior target in deep brain stimulation for advanced Parkinson’s disease [39, 40]. Researchers could use our method to study the influence of ZI stimulation on STN spike activities. By adjusting the electrode depth, we can easily obtain larger amount of well-isolated units with favorable SNR. We could also observe the units that might have been silent during implantation under anesthesia using movable electrode [29]. We recommend using denser agar gel and smaller distance between microdrive bottom and skull to prevent too much agar gel from flowing into the outer cylinder. Even if the influx of the gel reaches to the bottom
Fig. 8 Stability analysis of allover STN units. a Scatterplot of peak-to-peak amplitude versus SNR for all STN units. b Mean number of cells lost weekly since cell acquisition. Error bars indicate SEs. c Predicted percentage (dashed line) and actual percentage (solid line) of cells retained on each subsequent week since cell acquisition. Error bars indicate SEs. d Distribution of all waveform similarities for the same units and different units of STN on different recording sections
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of microdrive piston, the electrode tips can still reach the target nucleus; because the gel is elastic and absorbable, and the distance to advance the electrode tips is small (preplacing the wire tips 500 lm above the STN in our experiment). We can prevent the piston and electrode tips from rotating by bending the needle at 90° and securing it with dental cement. This step is necessary for accurate and stable recording. We used a much smaller diameter screw for ECoG recording, and kept the underneath dura to reduce the suppression and irritation to the recording cortex. Using the same connector for ECoG and deep brain microelectrode array recording may be useful for multi-region recording with lighter recording accessories. Simultaneous cortical neural ensemble recording can be achieved by replacing the ECoG electrode to the traditional microarray electrode. Researchers can use this type of recording technology to study the relationship of spiking activities between cortex and BG. In conclusion, we presented a compact movable microwire array manufactured with readily available materials. This technology can be used for chronic single-unit recording of the BG in the deep brain regions, with simultaneous monitoring to the coupling ECoG activities in freely behaving rats. Researchers can apply this technique to investigate electrophysiological changes in basal ganglia–cortical circuits of rats in different physical statements. Acknowledgments This work was funded by Science Projects (Nos. 81371397/H0912, 81372345/H1607, 81172416/H1618), National Natural Science Foundation of China, and key Clinic Specialty Discipline Construction Program of Fujian, P.R.C. Conflict of interest
None.
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ORIGINAL ARTICLE
A movable microelectrode array for chronic basal ganglia singleunit electrocorticogram co-recording in freely behaving rats Xiaobin Zheng • Jia Zeng • Ting Chen • Yuanxiang Lin • Lianghong Yu Ying Li • Zhangya Lin • Xiyue Wu • Fuyong Chen • Dezhi Kang • Shizhong Zhang
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Received: 8 December 2013 / Accepted: 20 February 2014 Ó Springer-Verlag Italia 2014
Abstract The basal ganglia–cortical circuits are important for information process to brain function. However, chronic recording of single-unit activities in the basal ganglia nucleus has not yet been well established. We present a movable bundled microwire array for chronic subthalamic nucleus (STN) single-unit electrocorticogram co-recording. The electrode assembly contains a screwadvanced microdrive and a microwire array. The array consists of a steel guide tube, five recording wires and one referenced wire which form the shape of a guiding hand, and one screw electrode for cortico-recording. The electrode can acquire stable cortex oscillation-driven STN
firing units in rats under different behaving conditions for 8 weeks. We achieved satisfying signal-to-noise ratio, portions of cells retaining viability, and spike waveform similarities across the recording sections. Using this method, we investigated neural correlations of the basal ganglia–cortical circuits in different behaving conditions. This method will become a powerful tool for multi-region recording to study normal statements or movement disorders.
X. Zheng Y. Lin L. Yu Z. Lin X. Wu F. Chen D. Kang (&) Department of Neurosurgery, Key Clinic Specialty Discipline of Fujian, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China e-mail: [email protected]
Introduction
J. Zeng Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, USA T. Chen Department of Radiotherapy, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China Y. Li Intensive Care Unit, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China S. Zhang (&) Department of Neurosurgery, Institute of Neurosurgery, National Key Clinic Department, Key Laboratory on Brain Function Repair and Regeneration of Guangdong, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China e-mail: [email protected]
Keywords Microwire bundle Basal ganglia Single unit Electrocorticogram
The basal ganglia (BG) are interconnected to the cortex and thalamus [1, 2]. The basal ganglia–thalamo-cortical circuits play a key role in processing information within the global brain, such as motor control, motor learning [3], as well as cognition and memory processing [4]. Abnormal pattern switch of synchronous oscillation and discharge are underlying mechanisms of movement disorders and mental disorders [5, 6]. However, techniques for electrophysiological co-recording in BG and cortex are not well established. In the past two decades, most functional studies in the interactions between the BG and cortex have mainly been focused on immobile animals. Researchers combined electrocorticogram (ECoG) with extracellular recordings of single-unit spikes using glass micropipettes, during acute recording in animals under anesthesia [7–9]. Others recorded single-unit spikes in non-anesthetized animals with their head restrained by a U-shaped piece under sleep condition [10, 11]. Using either technique, researchers need to keep animals immobile and limit the number of
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recording channels. Neural ensemble recording, a revolutionizing technique, achieves simultaneous multi-channel recording in groups of well-isolated single units in behaving and freely moving animals. But it is so far mainly used in animal cortex [12, 13]. It is rarely applied in deep brain region, especially the BG nuclei which are smaller and more irregular, such as subthalamic nucleus (STN) and external segment of globus pallidus (GPe). We designed and developed a movable microelectrode array for simultaneous recording between the BG singleunit spiking activities and cortex oscillations. Using this method, we detected the phase relationship between STN single-unit activities and M1 cortex synchronized oscillations in rats under different behaving conditions.
Fig. 1 Construction of the movable microwire array and the implantation procedure
Materials and methods
Microelectrode construction
Drive design
Most of the operations below were performed under a microscope.
The structural elements of the microdrive include: a 2-cmlong cylinder cut from an insulin syringe (3/10, 31ga, Becton Drive, USA), a 1.5-cm core bar with piston from the same syringe, a stainless steel screw (8 mm long, 1.75 mm in diameter, 0.25 mm pitch) with matching knurled nut (3.0 mm in external diameter) as the propeller screw, two micro-screws (1.4 mm in diameter, 3 mm long) as the locking screw, and a 28-ga hypodermic needle from an 1-ml syringe as a keyway needle to the syringe piston (Fig. 1). The microdrive was installed as follows: 1.
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The cylinder was threaded by the two micro-screws after symmetrically perforating it using a flaming needle. The knurled nut was rotated to the cap end of the propeller screw. Then the screw’s tail end was vertically injection molded into the core bar at its mesial part, and reinforced by Cyanoacrylate gel (Chemmer Enterprise Co., Ltd. Taipei, Taiwan). The core bar and propeller screw were pushed into the cylinder, then the knurled nut was molded to the inner wall of the cylinder. The 28-ga hypodermic needle was bent at 90° about 7 mm away from the tip. After lubricated with mineral oil, the needle tip was vertically penetrated throughout the syringe piston at outer edge point. The needle tip was rotated slightly to bring the mineral oil into the needle trajectory for lubrication. The cap of the insulin syringe was cut at about 7 mm to the closed end to cover the propeller screw as its protective cap.
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A 10- to 12-mm-long 29-ga hypodermic stainless steel tube (330.2 lm outer diameter, 165.1 lm inner diameter, A-M Systems, Carlsborg, USA) was used as the guide tube. A 4-cm full-annealed stainless steel (PFA insulated, 76.2 lm bare diameter, 139.7 lm coated diameter, A-M Systems, Carlsborg, USA) was first stripped 1 cm for insulation. The stainless steel was tightly wound on the proximal end of the tube and fixed with silver paint (Electrolube, H K Wentworth.ltd, UK), serving as the ground wire. Five 5-cm platiniridium wires (PFA insulated, 25.4 lm bare diameter, 38.01 lm coated diameter, A-M Systems, Carlsborg, USA) was used as the recording electrodes. Each was slotted nearly 10 mm throughout the guide tube. One 4-cm half annealed stainless steel (PFA insulated, 50.8 lm bare diameter, 114.3 lm coated diameter, A-M Systems, Carlsborg, USA) was insulation stripped by a brief burn at its end. The bare section was trimmed to 0.5 mm using a microscissor. The stainless steel was slotted though the guide tube, with the bare tip protruding out 2.0–2.5 mm, serving as the reference electrode. The platiniridium wires were adjusted to form a fanlike shape with about 250 lm intervals at about 3 mm away from the tube end using a fine forceps. Cyanoacrylate gel was siphoned into the guide tube through a plastic capillary. A thin layer of gel was left out of the tube but kept free from the bare tip of the reference wire (Fig. 2a). This helped to keep the microwire arrangement and increase the tip rigidity.
Neurol Sci Fig. 2 a The outward arrangement of microwire tips. b The inward arrangement of microwire tips. c The movable electrode consists of one microdrive with keyway needle (unfilled arrow), one microwire electrode array, and one 10-pin electrode connector with one 76.2-lm-diameter ECoG recording wire (solid arrow). d The intraoperative brain penetration with tiny dimples. It pushes away blood vessel without hemorrhage. e The side view of the electrode assembly implanted in the rat’s brain with a protective cap. f Right side view of the electrode assembly implanted in the rat’s brain
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The recording microwire tips were linearly trimmed at 35–45 horizontal degree from right to left (for left STN implantation) using a microscissors with 3.0–3.5 mm left out of the guide tube. So that the recording tips are 0.5–1.0 mm longer than the reference wire tip, and the arrangement of electrode tips formed a guiding-hand shape (Fig. 2a, b). The free ends of all the microwires were 5 mm insulation stripped and wound tightly to a 10-pin subminiature connector (DRP-10-vv-ESR, Omnetics Connector, Minneapolis, MN, USA). A 5-cm fullannealed stainless steel with the same diameter as the ground wire was 5 mm insulation stripped at both ends for connecting to the connector and the ECoG recording screw (Fig. 2c). Silver paste was applied to all the connection points. The pins and the base of the connector were sealed by thin layer epoxy glue (Chemmer Enterprise Co., Ltd.
Taipei, Taiwan). This stabilized the microwire connections after the silver paste solidified. The tip impedance of the recording electrodes was 0.5–1.0 MX at 1 kHz (TH2893 impedance instrument, Tonghui Electronics, China).
Assembly of microelectrode system and preparation before implantation The microwires in guide tube were properly bent at the proximal end, and vertically anchored to the mesial part of the microdrive piston bottom with epoxy glue. After epoxy glue solidified, the microdrive piston was pulled about 5 mm back into the outer cylinder by counter-rotating the propeller screw. Before implanted, the microelectrode was first sterilized with ethylene epoxide. The end of the electrode was
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secured with a tiny spot of melted aseptic polyethylene glycol, which further reinforced the microwire tips (Fig. 2a). The gross weight of the microelectrode system was 13.8 g, including 6.6 g in the microdrive. Implant surgery Six Wistar rats (weight 280–300 g) were anesthetized with i.p. injection of 90 mg/kg ketamine and i.m. 5–9 mg/kg xylazine, with a supplementary dose (25 mg/kg, im) of ketamine if necessary. Each rat’s head was fixed to a stereotaxic frame with -3.3 mm incisor bar. After skull exposure, four stainless steel screws were secured into the skull, including one electrical screw (0.8 mm diameter, 1.6 mm long, Plasticsone, USA) at the dura above the M1 cortex (AP: ?2.00 mm, ML: ?2.80 mm, Paxinos and Watson 2005) as the ECoG recording electrode. One 3-mmdiameter hole was drilled just above the STN coordinate (AP: -3.70 mm, ML: ?2.20 mm, 7.90–8.10 mm below cortex, Paxinos and Watson 2005), and the dura was resected carefully using a hooked needle tip. Before implantation, the microdrive was held by a micromanipulator (MP-225, Sutter Instrument); the connector was held by a helping hand (A-M Systems, Carlsborg, USA). The ECoG microwire was wound onto the ECoG recording screw and fixed by silver paste. The row of electrode tips was adjusted to be parallel to the coronal section of the brain. The lowering speed was first set at 300 lm/min or manually controlled, following the dissolution of the polyethylene glycol. This facilitated the tips to penetrate the brain and push the blood vessels, without bending the microwire or generating big brain dimples (Fig. 2d). After the guide tube’s distal end touched the brain surface, the lowering speed
Fig. 3 a The illustration from the rat brain atlas [modified from Paxinos and Watson (2005)] with the tip trajectory from VPM, ZI to STN. b A 2 s duration representation of tracings of neuronal activities in the VPM, ZI and STN under ketamine anesthesia. c One track of
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was slowed down to 150 lm/min. The craniotomy was filled with gelfoam granule and then covered with tiny spot melted agar gel to avoid the leakage of the cerebrospinal fluid. Continuous extracellular recording was performed in the ventral posteromedial thalamic nucleus (VPM) and zona incerta (ZI) (Fig. 3a) along the microwire tip trajectory. The VPM neurons should show discrete spikes with irregular inter spike interval (ISI) and occasional short burst activity. ZI neurons had typical high amplitude spikes with longer regular ISI and lower background noise (Fig. 3b). Finally, more melted agar gel was applied to cover the skull hole and microdrive bottom for isolation. This avoided the influx of the dental cement into the microdrive bottom during electrode fixation. The electrode connector was placed at 45° to the microdrive. The whole assembly was fixed to the skull with dental cement (Fig. 2e, f). Rats were i.m. injected with 30 mg/kg lincomycin and 0.30 mg/kg buprenorphine for 3 days. Postoperative advancement and histology The electrodes were advanced 125 lm/day by rotating the propeller screw clockwise after the rats had recovered for 3 days. When the electrode tips were advanced into the STN, extracellular signal would show high cell density and background noise with occasional longer burst than VPM neurons (Fig. 3b). Before advancing the electrode tips, the locking screws were loosened. They were fastened again after the advancement. At the end of the recording period, the recording sites were electrolytic lesion marked. The brain coronal frozen sections were Nissl stained as Tseng et al. [14] described. Electrode tip tracts and lesion sites (Fig. 3c) were validated under a microscope (eclipse 80i, Nikon, Japan).
electrode tips (arrow) and electrolytic lesion (asterisk) within STN boundary (dashed line) in brain coronal section with Nissl staining. Scale bar 200 lm
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Data acquisition and analysis STN single units and ECoG activities were monitored during behaving conditions. Signals were headstage pre-amplified and transmitted via a light-connecting cable to two differential AC-coupled amplifiers (Model 1700, A-M Systems, Carlsborg, USA). They were analog/digital converted by the Powerlab data acquisition system (Model ML870, ADInstruments, USA), and recorded by the labChart software (professional version 7.3). The STN spike trains were filtered between 300 and 3,000 Hz and sampled at 20 kHz rate. The ECoG was filtered between 0.1 and 300 Hz, and sampled at 1 kHz rate. For the unit stability analysis, STN neuronal activities were recorded weekly for 8 weeks. STN spikes were template-matching sorted. ISI was another factor for verifying unit identities when spike amplitude changed largely during long term following recordings. Units were considered to be alive only when both coincident ISI and similar waveforms still remained, rather than being lost. Unit size was quantified by the peakto-peak amplitude of the averaged waveforms collected from the 10-min period in the conscious condition [13, 14]. Signal-to-noise ratio (SNR) was calculated for unit quantification as well [15]. STN units stabilities were evaluated by the similarity among spike waveforms, which was defined as the linear correlation (r) values between timeshifted average waveforms (r = 1 indicates identical spike waveforms) [13]. ECoG rhythms were detected using methods described in Zhang et al. paper [16]. The relationship between ECoG rhythms and STN unit activities was analyzed using two methods. The first method used cross-correlogram to show the oscillation of correlated activities [7, 17]. The second method used Hilbert transform to decompose the filtered ECoG to into instantaneous amplitude and phase components. The peak of the up state in ECoG was defined at 180° [18, 19] (Fig. 4). STN spike phase was determined by the angle of the analytic ECoG signal at its location. Finally, phase distribution and mean phase of STN spikes were showed by polar histogram. Rayleigh test was performed for circular uniformity (P \ 0.001 indicates significant phase locking between ECoG and STN spike trains) [16, 18, 20].
Results All the six rats survived longer than 9 weeks after implantation surgery. We successfully obtained STN single-unit recordings and ECoG. 90 % (27/30) microelectrode tips tract went through the STN boundary. We acquired 46 STN units. 67.4 % (31/46) STN units survived more than 1 week and were defined as the stable units. Nine STN new units appeared during the recordings.
Fig. 4 The steps for determining phase relationship between STN spiking activities and theta-ECoG oscillation. a, b The raw ECoG. We applied a band-pass filter to (a) at 4–12 Hz and got (b). c The instantaneous phase of filtered trace after Hilbert transformation. The 180° gray dashed line corresponds to the peak of the ECoG oscillation. d Shows the phases of STN spikes (red numbers in c) which are determined by the instantaneous phase of filtered ECoG trace (red dashed lines) (color figure online)
ECoG rhythm-driving STN single units Previous studies showed the relationship between cortex synchronous oscillation and the BG activities under physiological state [21–24]. We also found some STN neurons driven by ECoG rhythms. Figure 5 shows spiking activities of a STN unit and the coincident ECoG oscillations during different physiological states. Under active exploration, the real-time ECoG showed theta rhythm (Fig. 5a). In power analysis, the principal frequency shows theta rhythm; the frequency of sub-peaks shows gamma band oscillations (Fig. 5b). The STN unit showed rhythmic burst activities that tended to occur at the ascending phase close to the peak of ECoG activities (Fig. 5a). The spike autocorrelogram of the STN unit consistently characterized a high level of theta rhythmicity (Fig. 5c). The STN unit is prone to burst at about 18 ms before the peak of the ECoG theta oscillation, according to the cross-correlogram (Fig. 5d). Phase relationship analysis showed that the STN spike trains phase locked the ECoG theta oscillation, with a mean phase at 168.3 degree (Rayleigh test, P \ 0.001) (Fig. 5e). The ISI histogram also demonstrated that the prominent
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peak indicated the intraburst interval; the sub-peak at about 0.15 s indicated the interburst interval. This meant about 7 Hz burst occurrence of this STN unit during active exploration, which coincided with the simultaneous ECoG theta oscillation (Fig. 5g). Interestingly, during slow wave sleep (SWS), the STN unit turned into a tonic firing mode when slow wave (*1 Hz) oscillated in the coincident ECoG. Spindle-like background activities emerged in short-term burst activities of the STN unit intermittently. We observed simultaneous positive-going sharp waves (SPW) in the corresponding ECoG (Fig. 5f). In the fourth week, the spike peak-to-peak amplitude decreased. However, the spike shapes overlapped well and showed a similarity with the waveforms in previous periods, compared with superimposed spike waveforms in 10 s of different recording periods (Fig. 5h). This indicated a robust living state of this STN unit. STN multi-unit recordings Most multi-unit recordings had relatively low peak-to-peak amplitude and SNR, indicating high-density neurodendrites or synapses around the microwires. Thus, new units probably arose in the recording of such microwires. An example of STN multi-unit recording is shown in Fig. 6. This microwire captured two stable units in the first week, which lasted for more than 2 weeks. In the third week, the spike waveforms in one of the units had a low level of similarity and lower amplitude. This unit disappeared in the fourth week. However, in the subsequent days, a new unit emerged in the recording of the same microwire with different spike waveforms (Fig. 6c) and a smaller pulse width (Fig. 6d) compared with the disappeared unit. Tonic firing pattern was also different from the long burst firing pattern of the survived unit (Fig. 6b).
from the recorded neurons would hinder long-term stable recording. This was also supported by the fact that SNR of survived units accumulated at 5–10 (Fig. 7c). The mean SNR of stable units was 7.848 ± 2.282, which was larger than new units (5.550 ± 1.138). It indicated that new units tended to emerge at the further region from the electrode tips. Both stable units and new units showed significant linear correlations between peak-to-peak amplitude and SNR (stable units, r = 0.8892, P \ 1e-4; new units, r = 0.5265, P = 0.0118) (Fig. 8a). This illustrated high quality and stability of recordings in STN. We calculated the weekly cell lost rate of all the acquired STN units. Units tended to be lost in the first week after we advanced electrodes, at a lost rate of 32.6 % (15/ 46). This rate fell to 20.9 % in the second week and remained at the same level until the fifth week when it reached 9.2 %. This was because quite a small amount of units survived longer than 5 weeks (Fig. 8b). We also predicted the proportion of cells that would be kept retained on each recording period, by cumulating the loss rates in the following weeks. This prediction (dashed line in Fig. 8c) suggested that nearly half (46.4 %) of the units on the initially acquisition week would survive more than 2 weeks. However, the actual proportion of retained cells was higher than the predicted, which was opposite to the result of Jackson et al. paper [13]. It is because new units arose intermittently and were counted (Fig. 8c). Similarity between spike shapes of the cells over consecutive recording sessions was characterized by the maximum linear correlation coefficient between averaged waveforms in Fig. 8d. 84.6 % of waveforms in the same STN units were similar (r [ 0.95) in different recording weeks. Only 29.5 % of waveforms were similar in different units. We concluded that the waveforms stayed stable when we tracked the activities of each individual cell using this electrode assembly.
Stability of STN single-unit recordings Discussion Average peak-to-peak amplitudes of STN stable and new units across different recording periods were shown in Fig. 7a and b. Stable units had larger peak-to-peak amplitudes than new units (152.4 ± 48.63 versus 115.5 ± 19.55 lV). Average peak-to-peak amplitudes did not correlate with the recording periods following the electrode implantation in both stable units (Pearson’s r = -0.13, two tailed, P = 0.11) and new units (r = -0.14, P = 0.5377). At the end of the recording, we still observed amplitudes at around 184.7 lV in stable units and up to 146.3 lV in new units. Most units that survived less than 1 week (Fig. 7a, unfilled circles) had peak-to-peak amplitude either larger than 250 lV or less than 100 lV. It indicated that positioning the wire tip too close to or too far
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So far, long-term neural ensemble recording has mainly been used in rodents or primates cortex using microarray electrodes [12, 15, 25–28]. Researchers have made great progress in seeking more well-isolated and stable single units of interest after implantation [29]. Recently, movable microelectrode with tether [26] or wireless neurochip [13, 30, 31] becomes one of the prevailing techniques in awake animals. However, this advanced neural ensemble recording technique above has not yet been applied in deep brain regions, especially in the BG. It may be because traditional microarray electrodes tend to distort tissues. This would increase the chance of misplacing the electrode tips in deep brain regions and damage the tissue. On the contrary,
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Fig. 5 An ECoG rhythm-driving STN firing unit and its time-shift superimposed spike waveforms. a Single-unit activities of a STN firing unit (top channel) was driven by theta oscillation (middle channel) of ECoG activities (bottom channel) during AE. The time duration of the signals was 4 s. b Power density spectrum of 30 s ECoG activities. c Spike autocorrelogram of the STN unit activities with theta rhythmicity. d Cross-correlogram theta rhythmic relationship between STN spike train and the filtered ECoG. e Polar histogram of the STN unit spike phase distribution to simultaneous ECoG theta oscillation. Red arrow represents the mean locked phase at 168.3 degree. f Tonic
firing mode with spindle-like background activities (arrow in bottom channel) of the STN unit and slow oscillation in delta band with positive-going SPW (arrow in top channel) of coincident ECoG activities during SWS. g ISIH observed the burst firing pattern of the STN unit during AE, in which bimodal distribution of intervals (two peaks marked by arrows) reflecting the interburst and intraburst intervals. h Time-shift superimposed spike waveforms of the STN unit show high level of similarity across the consecutive recording periods. Scales in (a) and (f) 250 ms in time axis, 50 mv in amplitude axis (color figure online)
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Fig. 6 Multi-unit recordings of STN. a Multi-unit signal of two STN units captured by an electrode wire at the first week. Duration 4 s. Scales 200 ms in time axis, 50 mv in amplitude axis. b A new tonic firing mode emerged with spikes marked in the same electrode wire
by dots above the channel. Scales 250 ms in time axis, 50 mv in amplitude axis. c Time-shift superimposed spike waveforms of the multi-unit STN signal across consecutive recording periods. d Voltage versus width scatter plot between the first and the fourth week
bundled microwires electrodes work well subcortical regions, such as hippocampus [32–34], amygdala [35, 36] and hypothalamus [37]. Recently, researchers have developed this kind of bundled electrode for recording trigeminal ganglion units in rats [14], as well as similar electrode with movable drive such as ‘‘Reitboeck’’ electrode for thalamic recordings in rabbits [38]. But the nucleus structure is much smaller and more slender in the BG and more difficult to position the electrode tips in the BG with accuracy. The bundled microwire array presented here can achieve high levels of accuracy and stability in STN single-unit recordings across 8 weeks in behaving rats. We obtained stable peak-to-peak amplitudes and waveforms, and acceptable cell lost rate across the consecutive recording sections. We successfully monitored the simultaneous
ECoG oscillations. We also analyzed the changes in phase relationship of the coupled STN spiking activities, especially the rhythmic burst firing that locked to the M1 ECoG theta oscillation in active exploration. This would further prove the state-dependent synchronization between STN and motor cortex [22, 24]. The length of the protruding parts of microwires is longer than the bundled electrode used in deep brain regions reported by Tseng et al. [14]. Longer protruding end can reduce the bending angle of the microwire. This can help the microwire tips push away brain blood vessels more easily, which are quite often confronted by the tip during its advancement to the deep brain regions. Longer protruding end can also improve stability in long-term behaving recording, especially when the animals shake the
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Fig. 7 Long-term performance of the movable microwire array. Peak-to-peak amplitude of all stable units (a) and new units (b) followed longitudinally across recording periods. Each line indicates a unit recorded by the same wire. Unfilled circles indicate units survived shorter than 1 week around the same wires
heads violently and cause the loss of recording units [13]. The rigidity of the long microwire protruding end is increased by the Cyanoacrylate gel and polyethylene glycol solidified at the microwire end, and the polyethylene glycol
can slowly dissolve when dipping into cerebrospinal fluid. These all facilitate the microwire tips advancing smoothly during electrode implantation. The microwire tips can penetrate the dura easily. However, we still recommend an incision or resection to the relative dura to reduce the concavity or distortion of the brain tissue. Otherwise, the ubiety between the puncture point and the target nucleus will change. We adjusted the tip of the reference electrode 0.5–1.0 mm shorter than those of the recording electrodes. This helped us achieve high level of common-mode rejection ratio of signals and lowered movement artifacts [14]. The distance of 0.5–1.0 mm also located the reference electrode tip in the region of ZI. It is possible to use the reference electrode for ZI stimulation, followed by simultaneous recording to STN single-unit firing. ZI is recently emphasized as a new superior target in deep brain stimulation for advanced Parkinson’s disease [39, 40]. Researchers could use our method to study the influence of ZI stimulation on STN spike activities. By adjusting the electrode depth, we can easily obtain larger amount of well-isolated units with favorable SNR. We could also observe the units that might have been silent during implantation under anesthesia using movable electrode [29]. We recommend using denser agar gel and smaller distance between microdrive bottom and skull to prevent too much agar gel from flowing into the outer cylinder. Even if the influx of the gel reaches to the bottom
Fig. 8 Stability analysis of allover STN units. a Scatterplot of peak-to-peak amplitude versus SNR for all STN units. b Mean number of cells lost weekly since cell acquisition. Error bars indicate SEs. c Predicted percentage (dashed line) and actual percentage (solid line) of cells retained on each subsequent week since cell acquisition. Error bars indicate SEs. d Distribution of all waveform similarities for the same units and different units of STN on different recording sections
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of microdrive piston, the electrode tips can still reach the target nucleus; because the gel is elastic and absorbable, and the distance to advance the electrode tips is small (preplacing the wire tips 500 lm above the STN in our experiment). We can prevent the piston and electrode tips from rotating by bending the needle at 90° and securing it with dental cement. This step is necessary for accurate and stable recording. We used a much smaller diameter screw for ECoG recording, and kept the underneath dura to reduce the suppression and irritation to the recording cortex. Using the same connector for ECoG and deep brain microelectrode array recording may be useful for multi-region recording with lighter recording accessories. Simultaneous cortical neural ensemble recording can be achieved by replacing the ECoG electrode to the traditional microarray electrode. Researchers can use this type of recording technology to study the relationship of spiking activities between cortex and BG. In conclusion, we presented a compact movable microwire array manufactured with readily available materials. This technology can be used for chronic single-unit recording of the BG in the deep brain regions, with simultaneous monitoring to the coupling ECoG activities in freely behaving rats. Researchers can apply this technique to investigate electrophysiological changes in basal ganglia–cortical circuits of rats in different physical statements. Acknowledgments This work was funded by Science Projects (Nos. 81371397/H0912, 81372345/H1607, 81172416/H1618), National Natural Science Foundation of China, and key Clinic Specialty Discipline Construction Program of Fujian, P.R.C. Conflict of interest
None.
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