Dual-channel telemetry system for recording vocalization-correlated neuronal activity in freely moving squirrel monkeys

Journal of Neuroscience Methods 76 ( 1997) 7 I 7

Dual-channel telemetry system for recording vocalization-correlated neuronal activity in freely moving squirrel monkeys P. Grohrock, U. Hiiusler, U. Jiirgens * Germtrr~

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Centrr,

Giittirqyn.

Kellnerwe~~

3. II -17 077

Received S November 1996: received in revised form

17 March

Giirtwyerr.

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accepted 17

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March

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Abstract A miniature telemetric system is described which allows simultaneous measurements of neural activity and vocalization in freely moving monkeys within their social group. Single and multi-unit activities were detected with medium impedance electrodes that were fixed to self-made microdrives allowing accurate vertical positioning over a range of 8 mm. Vocalizations were registered by means of a piezo-ceramic device sensing the vocalization-induced skull vibrations. This allowed identification of the vocalizing animal in a larger group and eliminated environmental noise. Neuronal activity and vocalization were transmitted via separate channels of a FM transmitter using different carrier frequencies. The signals were decoded in two conventional FM receivers equipped with an automatic frequency control. The signals were stored for off-line analysis on a HiFi-videotape recorder. ‘is, IS97 Else\ ier Science B.V. Kt~?~~c~ortl.~: Bone-vibration Vocalization

sensor; Dual-channel telemetry; Miniature electrode drive: Single-unit recording: Squirrel monkey:

1. Introduction One problem in motor neurophysioiogy is to make the experimental subject repeat the behaviour under study often enough to allow the experimenter a comprehensive data acquisition. The usual approach to this problem is to train the animals in an operant conditionmg task with the motor pattern to be investigated as the operant. While some motor patterns are conditionable quite readily, others are less. Vocalization belongs to the latter category. In the rhesus monkey, Yamaguchi and Myers ( 1972), were unable to bring vocalization under stimulus control. Aitken and Wilson (1979) succeeded in vocal operant conditioning, but only if instead of food reward a shock-avoidance procedure (Sidman avoidance schedule) was used for reinforcemerit, Sutton et al. (1973), finally, were able to train

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macaques a vocal operant conditioning task with food as a reinforcer. They were unable. however. to control the call type. In order to circumvent these problems, some authors have used electrical brain stimulation as a means to produce vocalization repetitively (Sch uller, 1979: Miiller-Preuf3. 1988; Suga and Yajima, 1988; Kirzinger and Jiirgens, 1991). This procedure has the drawback. however. that the stimulus artefacts interfere with single-unit recording. Improvement is offered by chemical brain stimulation (Kirzinger and Jiirgens. 1990). This technique, however, suffers from the limited number of injections capable of eliciting vocalization from one and the samesite. Due to its artificial induction of bocalization, it also is unsuitable (similarly to electrical elicitation of vocalization) for detecting vocalizationcorrelated activity ‘upstream’ of the stimulation site. We therefore decided to develop a telemetric system allowing the recording of single and multi-unit activity during spontaneous vocal interactions in freely movirlg animals within their social group.

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2. Methods

Potentiometer case

The complete telemetric system consists of the following components: a headstage containing a low-noise preamplifier for the neuronal signal, the transmitting device, one to three microelectrode drives and a piezoceramic sensor monitoring the vocalization-induced skull vibrations. On the receiving side, there is an antenna placed within the cage and being connected by a coax-cable with two FM receivers outside of the animal room. From there, the signal is sent to a HiFi videotape recorder for storage. Within the animal room, there is in addition a videocamera with microphone and a radio frequency modulator for transformation of the video and acoustical signal into a radio signal that can be fed together with the telemetric information into the coax-cable. 2.1. Headstage

The basic unit of the headstage is the carrier platform (Fig. 1) with dimensions 20 x 15 x 5 mm. It contains 80 stainless steel tubes with an outer diameter of 0.8 mm and an inner diameter of 0.5 mm. The tubes are arranged in eight parallel rows of ten tubes connected with each other by solder and being embedded in dental acrylic (Paladur). The tubes serve to guide the microelectrode positioning devices. Additionally, the platform contains one screw rod of 30 mm length, that serves for mounting the transmitter and plastic protection cap. To mount the carrier platform on the skull, the animal was narcotized and fixed in a stereotaxic apparatus. With a midline incision of the scalp, the skull was exposed. Six special anchor screws, M2 x 10 mm flat headed, were mounted in such a way, that the heads of the screws became located between bone and dura. To achieve this, small T-shaped holes were drilled into

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Fig. 1. Carrier platform with guiding tube array and screw rod.

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Fig. 2. Microdrive with electrode triplets.

the bone and the screws were inserted with the heads first. Screws were fixed with nuts and dental cement. The positions of the screws were in the frontal, the upper temporal and in the upper occipital area. The platform then was brought into the desired position with the aid of a stereotaxic manipulator and the space between platform and screws was filled with dental acrylic. After that, the skin was aligned to the edges of the acrylic cement and sewed up at the front and rear end. 2.2. Microdrives

For positioning of the microelectrodes a modified spindle potentiometer was used (Fig. 2). In order to reduce the size of the microdrive, the potentiometer was milled down to a cross section of 5 x 3 mm (height 20 mm), so that only the spindle drive remained. The original slide was removed and replaced by a drop of acrylic cement embracing the worm. To prevent adhesion between acrylic and worm, the latter was moistened with silicone oil. The microelectrodes were glued to the slide with another drop of acrylic. Each drive was provided with three electrodes with a tip-to-tip distance of 0.5 mm. We used quartz-insulated platinumtungsten microelectrodes (Thomas Recording, Marburg/Germany) with a pencil-shaped tip of 20 pm and a shaft diameter of 80 pm; the impedance at 1 kHz was about 1 MOhm. The microelectrodes were running within a cannula that was glued to the case of the spindle potentiometer. The cannula had an outer diameter of 0.47 mm and an inner diameter of 0.23 mm; it was filled with low viscosity silicone oil to improve electrode movement and prevent entrance of bacteria. For implantation of the microelectrodes, a hole of 0.5 mm was drilled through the bone using the platform tube for guidance. Then the electrode assembly was directed through the platform tube and bone opening into the brain. During insertion, the microelectrodes

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were pulled back into the 0.47 mm cannula to prevent damage of the electrode tips. After insertion, the microdrive was fixed to the carrier platform with a drop of acrylic cement and the electrodes were advanced into the brain by turning the worm of the spindle potentiometer with a screw driver. One turn corresponded to 0.32 mm. The electrodes could be moved over a total distance of ,Ymm.

The transmitter consisted of two independent channels. The RF stages of both channels were single stage HF oscillators with a frequency in the range between 120 and 150 MHz. Frequency modulation was achieved by capacitive diodes (Fig. 3). Large oscillator coils with approximately 15 mm diameter were used; they also served as antennae to radiate RF energy. One of the RF stages was directly modulated by a piezo-ceramic vibration sensor (Fig. 4). Vibration sensors were made of small piezo-ceramic plates measuring 4 x 8 x 0.3 mm (PXE Philips) that were soldered at one end onto the transmitter’s baseplate. The other end contained an electric contact with a connection wire but

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otherwise was free to vibrate. The vocalization-induced vibrations generated voltages large enough to modulate the RF stage directly. Sensitivity of the vibration sensor was line-tuned to avoid over-modulation of the RF stage during the most intense calls. !-‘me-tuning was carried out by Larying the amount of solder at the connection-wire contact. The second channel was used for the transmission of the neuronal activity picked up by one of the implanted electrodes mounted on the headstage. Amplification of the neuronal signal was achieved by a MOSFET operational amplifier which combines low power consumption with low noise. The microcircuit TLC25M2 (Texas Instruments) gave 2 /IV input noise at 7 kHz bandwidth. This is much less than the thermal noise produced even by low-impedance recording electrodes. With an electrode of I MOhm impedance and 7 kHz bandwidth, one will get U, = ti/4 kT E 1 MOhm*7 kHz = 10 ,i~Vt<~~ with 4 kT = 1.6 x 10”‘” at tissue temperature. Voltage amplification was set to ca. 50, so that input amplitudes of 20 ,uV were able to modulate the RF stage. AC coupling between electrode, amplifier and RF stage was used to suppress polarization of the electrode and DC offset of the amplifier. In addition, a filtering network in the feedback stage (Fig. 3; C2. R2. R?) served to set the low frequency cut-off to about 0.3 kHz. The 7 kHz high-frequency cut-off was automatically determined by the slew rate, if the medium bias version of the TLC 25 series microcircuit was used. In this way, extra circuitry could be avoided. The remaining operational amplifier of the TLC 25MZ circuit was used to generate a virtual ground. The circuit was completely built with surfacemounted technology (SMT) components and assembled on ;I single 20 x 2X mm printed circuit hoard. Transmit-

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Cap

Spindle electrode Battery

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Chair fixation platform Dental acrylic ?Skin /

Transmitter 4 Platform ,A4

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Fig. 5. Lateral view of the complete setup of the transmitter headstage system.

ting coils and RF stages were mounted on one side, while the other side received the preamplifier together with the 3 V lithium battery, a battery switch, the vibration sensor and the electrode connector sockets. Transmitting coils and electronic components were sealed with epoxy resin. The total weight of the transmitter plus headstage was 13 g. A schematic view of the complete system is shown in Fig. 5. Fig. 6 shows the system implanted on a squirrel monkey with protection cap removed. 2.4. Receiving system

The RF signal from both transmitters was picked up by an antenna inside the shielded animal cage and was amplified by a RF preamplifier selective for the range

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of 115- 150 MHz (SSB-Electronic, Iserlohn, Germany). Interference with powerful commercial radio stations could be avoided in this way. Two commercially available FM receivers (Yaesu FRG-9600) were used for demodulation. An automatic frequency control system (AFC) was added to these receivers to deal with frequency shifts appearing when the transmitter comes close to metallic material. The signals of both channels were stored on a HiFi VHS videotape recorder (Sony SLV-825) together with the vocalizations picked up by a room microphone. The animal’s behaviour was monitored by a video camera with a wide-angle lens and stored on the same videotape recorder. 2.5. Recording procedure

Electrode drives were fixed to the headstage above regions in which vocalization-correlated neuronal activity was expected, as described above. Then the transmitter was fixed and one of the electrodes connected to the transmitter input. Once or twice a day, the animal was placed in a monkey chair, with the headstage fixed to a holder. The battery was checked and replaced if necessary. With the telemetric system running, the microelectrodes were advanced by the aid of the spindle drive until a stable single or multiple-unit recording was obtained. The animal was then released from the chair and brought back to its home cage where vocal interactions with conspecifics started immediately. The group cage had a size of ca. 3 x 3 x 3 m. Recording was carried out until a sufficient number of different call types had occurred. This usually took l-2 h per recording site.

3. Results

Fig. 6. Squirrel monkey with implanted transmitter system, protection cap removed.

The recordings made with our telemetric system were nearly identical to conventional recordings made with the same electrodes. Single-unit as well as multiple-unit recordings were obtained (Fig. 7 and Fig. 8). The recordings were surprisingly stable and free of most types of artefacts, electrical contamination with 50 Hz cycles or interference with nearby radio stations. During the animal’s vocalization, the recordings remained stable and no electrical or mechanically induced crosstalk was observed between the two transmitter channels leading to false correlations between vocalization and neuronal activity (Fig. 7). Movement artefacts were not obtained during the normal activity of the animal, even if the animal leaped from perch to perch. The only artefact we met was caused by rapid changes of the carrier frequency in combination with a low amplitude of the carrier. In this case, the automatic frequency-control circuit lost the carrier and locked onto the second RF channel. By momentarily switching

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f ‘ig. 7 fhrer telemetric recording episodes (b c, d) of’ single-unit and vocal ,Ictlvity (~1 Show5 d time-expanded piece of’ episode (b). In each eptsode. the upper tract shows the neuronal activity, the lower trace displays vocal activity. The recording site was in the dorsofatera1 periaqueductaf grey 01‘ the midbrain. This site showed no vocalization-correlated neuronal activity. Episodes (a) and (d) demonstrate the akncc of’ vocalization-induced movement and microphonic arte/‘.LClS.

off the automatic frequency control, it was possible to regain the original channel. This did not take more than I :, and could be easily done by hand while monitoring the recording. The frequency of such an incident MYIS quite low and did not present a major problem The implanted microelectrode arrays could be used for exploration over a period of several months without a decrease in recording quality.

4. Discussion For being able to correlate vocal behaviour with neuronal activity. a recording system has to fulfill sev-

eral conditions. It has been ntentioncd Ctlrcady that monkeys cannot be trained to utter different call types voluntarily. The only way to obtain a high number of different call types if their artilicial eli&ation by elcctrical or chemical brain stimulation is tc~ bc avoided ih to collect the calls during spont:tncnus vocal conversation within the social group. Such con\ ersations, ho\~:ever. take place only in unrestrained ,~mim;zls. 7’his made it necessary to have a telemetric s;>stem. There are several commercially available telemetric systems f’ot the transmission ol’ electroencephalo+phic acti\,itl (e.g. BioMetric Systems. D-64 33 1 W::itrrstadt; Data Sciences international, St, Paul. Minn6.5otii: Technical and Scientific Equipment, D-61 348 Bad Homburg). Due to their restricted bandwidth. I(u p~>wer and, in some cases. IOU input impedance. the:, lucre not suitable for our purposes. In order 10 avoiil the problems associated with the ctevelopment 06‘ a I! JL~-weight. lowsize. high-input impedance, wide-bandwidth. powerful preamplifier-transmitter system, \amr :r:lthorh ctudying neuronal activity in freely moving animais have rccurred to cable recording using ;i commutator swivel suspended from the ceiling of’ the citgc IO prevent twisting and breaking of the cable during locomotion (Fontani. 1981; Kubie. 1984: Bordi ct ,:I. / 993; Szabo and Marczynski. 1993). This method i$ suitable in single animals restricted in their locomotion to the floor of the cage, for instance, rals. tt is nc 1~ applicable in monkeys interacting with their group ;md roving ovx the cage in all three dimensicxx. T‘rue telemetric~ singlcunit recording has been reported by Eichcnbanm et al, ( 1977) for the rat ;tnd rabbit and h> Pinkwart and Borchers ( 1387) for the toad. Both systems were limilcd to one channel. howc’r’er. A special feature of the transmitter ‘;j stem dtascribecl in the present study is the combination \xi” ;I channel t’or neural activity with a chamiel for- bitne vibration\. Telemetric transmissron of \,ocalizatiol; -induced bone Librations wcrc reported already by Naurus and SYabolcs ( 1972). The system was used to id1:nrif.y individually vocalizing animals in ii larger gr(bup of xluirrtl monkeys. As the report does not give any &tail< about the transmitter circuit and the M;I) the latter cvas COUpled to the piezo-ceramic sensor. wc ,:ouId not hut approach the problem anew. The debice presented in this stud? yields v,ocal signals of high c>u,tlity. Due to the lack of echoes. bound interferences ajld background noise. the quality cjf the sensor-transmitlzd ~~~a1 signal was always superior to the ~~ocal si,rna! pickcii up b\ the room microplion~: Another novei feature of‘ our system 1s :he microdrikc used. Microdrives suitable for single-unit recording in free& moving animals have been described by several authors (e.g. .4insworth and O’Kcet‘i:. 1’!77: Sinnamon and Woodward. j977: Deadwyler et al I C179: Kubic. 1984: Dian;i et ;[I. 19Y7: Boisson;,tic i:t ;!’ . 1991’ Banks

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Fig. 8. Telemetrically recorded vocalization-correlated neuronal activity from the ventrolateral periaqueductal grey. Trace A shows neuronal activity, trace B displays the vocalization-induced bone vibration, trace C is a spectrographic representation of the signal displayed in B.

et al., 1993). However, due to the special conditions of our experiments, none of these microdrives was optimal. The first requirement our microdrive had to fulfill was a size small enough to accommodate the microdrive together with the complete transmitter system on a platform of about 20 x 15 mm. Our microdrive has a cross section of 3 x 5 mm and thus, is the smallest microdrive reported until now in the literature. The second condition was low weight. Headstage plus transmitter system plus vibration sensor plus microdrive should not exceed 15 g. Our microdrive has a weight of 0.6 g and thus is the lightest microdrive we know of. In order to save animals, the third condition was to be able to run numerous electrode tracks in the same animal. With our device, we are able to place electrode tracks at intervals of 0.8 mm medio-laterally as well as antero-posteriorly over an area of 6.4 x 8 mm. A fourth condition, finally, was that the construction of the microdrive should be simple. Our microdrive, in fact, does not need machining of complicated pieces but consists of a slightly modified conventional spindle potentiometer. The electrodes we use are triplets of mediumimpedance quartz-insulated platinum-tungsten electrodes with pencil-shaped tips. First tests with high-impedance electrodes bearing sharply pointed tips were unsuccessful as cells were lost when the animal moved. The reason that we preferred stiff electrodes over microwire bundles, despite their lower recording stability, was that exact positioning of the electrodes and histological verification of individual electrode tracks is much less problematical in the first. A limiting factor of the system is that we are not able at the moment to change the position of an electrode telemetrically. For each change, the animal has to be

caught, placed into the monkey chair and the electrode adjusted manually. In comparison with conventional single-unit recordings in which tracks of several mm length can be explored within one session, this is a very time-consuming procedure. At present, we are going to develop a telemetrically controlled microswitch allowing us to switch from one electrode to the other by remote control. This would reduce the amount of animal handling necessary and help to save a good deal of time. References Ainsworth A, O’Keefe J. A light weight microdrive for the simultaneous recording of several units in the awake, freely moving rat. J. Physiol. (London) 1977;269:8- 10. Aitken PG, Wilson WA Jr. Discriminative vocal conditioning in rhesus monkeys: evidence for volitional control? Brain Lang. 1979:8:227740. Banks D, Kuriakose M, Matthews B. A technique for recording the activity of brain stem neurones in awake, unrestrained cats using microwires and an implantable micromanipulator. J. Neurosci. Meth. 1993;46:83-8. Boissonade FM, Banks D, Matthews B. A technique for recording from brain-stem neurones in awake, unrestrained cats.. J. Neurosci. Meth. 1991;38:41-6. Bordi F, LeDoux J, Clugnet MC, Pavlides C. Single-unit activity in the lateral nucleus of the amygdala and overlying areas of the striatum in freely behaving rats: rates, discharge patterns, and responses to acoustic stimuli. Behav. Neurosci. 1993;107:757-69. Deadwyler SA, Biela J, Rose G, West M, Lynch G. A microdrive for use with glass or metal micro-electrodes in recording from freely moving rats. Electroenceph. Clin. Neurophysiol. 1979;47:752-4. Diana M, Garcia-Munoz M, Freed CR. Wire electrodes for chronic single unit recording of dopamine cells in substantia nigra pars compacta of awake rats. J. Neurosci. Meth. 1987;21:71-9. Eichenbaum H, Pettijohn D, Deluca AM, Chorover SL. Compact miniature microelectrode-telemetry system. Physiol. Behav. 1977;18:117558.

Fontam G. .A technique for long term recording from single neurons in unrestrained behaving animals. Physiol. Behav. 1981;26:331 -3. Kirzinger A. Jiirgens U. Chemical brain stimulation as a means to ctrsumvent electrical stimulation artefacts in single-unit recording studies of evoked vocalization. J. Neurosci. Meth. 1990:33:165 70 Kirzmger A. Jiirgens U. Vocalization-correlated single-unit activity in the brain stem of the squirrel monkey. Exp. Brain Res. I99 I :X4:545 60. Kubte J. A driveahle bundle of microwires for collecting single-unit dala from freely moving rats. Physiol. Behav. 1984;32: 115.. 8. Maurus M. Szabolcs J. Kleinstsender fiir die Ubertragung von Affenlauten. Naturwissenschaften 197X8:273 4. ‘vliiller-Preub P. Neural correlates of audio-vocal behavior: properties ol‘ anterior Iimbic cortex and related areas. In: Newman JD, edttor. The Physiological Control of Mammalian Vocalization. New, York: Plenum. 1988:24S 62. !‘inkwart C‘. Botchers HW. Miniature three-function transmitting

system for single neuron recording, wireless br,rin stnnulatton and marking. J. Neurosci. Meth. 1987;20:341 52 Schuller G. Vocalization influences auditory processing in collicular neurons of the CF-FM-bat, Rhinolophus ferrumequinum. J. Comp. Physiol. I979:132:39 46. Sinnamon HM. Woodward DJ. Microdrtve anti method for single unit recording in the active rat. Physiol. Beh;;v. 1977:19:451 .i. Suga N. Yajnna L Auditory-vocal integratton in the midbrain of the mustached bat: periaqueductal gray and retit ular formation, In: Newman JD. editor. The Physiological COOI rol t,f M,rmmalian Vocalization. New York: Plenum. 1988:87 lit’: Sutton D. Larson C. Taylor EM, Lindeman KC V