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Neurophysiological recordings in freely moving monkeys
Methods 38 (2006) 202–209 www.elsevier.com/locate/ymeth
Neurophysiological recordings in freely moving monkeys Ning Lei Sun a,d, Yan Lin Lei a, Byoung-Hoon Kim b, Jae-Wook Ryou c, Yuan-Ye Ma a, Fraser A.W. Wilson a,¤ a
Laboratory of Primate Neuroscience, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan 650223, China b Department of Physiology, University of Wisconsin, USA c Department of Neurology, Cornell University, USA d Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR china Accepted 15 September 2005
Abstract Recordings of neuronal activity in freely moving rats are common in experiments where electrical signals are transmitted using cables. Such techniques are not common in monkeys because their prehensile abilities are thought to preclude such techniques. However, analysis of brain mechanisms underlying spatial navigation and cognition require the subject to walk. We have developed techniques for recordings in freely moving monkeys in two diVerent situations: a 5 £ 5 m testing laboratory and in a 50 m2 open Weld environment. Neuronal signals are sent to ampliWers and data acquisition systems using cables or telemetry. These techniques provide high quality recordings of single neurons during behaviors such as foraging, walking, and the performance of memory tasks and thus provide a unique opportunity to study primate behavior in a semi-natural situation. © 2006 Elsevier Inc. All rights reserved. Keywords: Allocentric space; Egocentric space; Hippocampus; Prefrontal cortex primate; Putamen; Rhesus
1. Introduction The vast majority of neurophysiological studies in behaving monkeys use techniques originated by Edward Evarts (e.g. [3]) and remain very similar to those pioneering studies. For example, standard methods include immobilization of the head and a hydraulically driven drive to move a microelectrode. These techniques are well suited to studying many forms of brain function, but certain classes of behaviors and cognitive abilities such as learning about the spatial organization of the environment require a diVerent approach. O’Keefe and Dostrovsky [14] pioneered studies in recording hippocampal activity in freely moving rats with their original description of place cells. Many subsequent studies have conWrmed the original observations:
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Corresponding author. E-mail address: [email protected] (F.A.W. Wilson).
1046-2023/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.09.018
place cells are neurons that Wre selectively when rats walk through a part of, but not all of a familiar environment. Such cells are candidates for providing information about a ‘cognitive map’ of the environment and strongly suggest that the rat hippocampus is involved in the analysis of space [15]. In contrast, the functions of the non-human primate hippocampus are still controversial—it is simply not clear what function the hippocampus contributes. Our approach to this controversy has been to undertake studies in monkeys that replicate the basic paradigm established by O’Keefe. Other investigators have also adapted the freely moving rat paradigms to monkeys [16,17]. Like rats, non-human primates are foragers and travel considerable distances in search of food. Their feeding behavior is opportunistic [21], and is characterized by seasonal variations in the many diVerent foods that they sample [6,7]. One might expect that primate survival is mediated by the development of brain structures that provide the visual and spatial abilities to enable foraging, and laboratory
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studies amply attest to the ability of monkeys to learn about new food sources [22]. It is striking then that typical laboratory research with monkeys rarely studies foraging behavior as a means to examine fundamental processes of learning and memory. Part of the explanation for this neglect is that proper facilities for studying spatial memory are technically diYcult to develop and to place under experimental control. However, it is clear that brain mechanisms in humans are specialized for mapping the environment. For example, Habib and Sirigu [5] describe the loss of familiarity for places following brain damage. Although progress has been made in using brain imaging techniques to analyse these mechanisms in people [12], recordings of neuronal activity could contribute to studying the neural bases of learning about the spatial arrangements of cues in the environment by developing techniques for measuring behavior and neuronal activity in freely moving monkeys engaged in walking through extended spatial environments. In this article we document two diVerent approaches to studying neuronal activity in freely moving monkeys walking in extensive spatial environments. In one paradigm we describe procedures for recordings using cables and a commutator made in a testing room the size of a typical research laboratory. In a second paradigm we describe techniques for telemetric recordings in monkeys walking within a 50 m2 open Weld environment. 2. Description of methods 2.1. General behavioral and safety issues Rhesus monkeys (Macaca mulatta) are preferred to other species of monkeys (but see [10]), in part because they are intelligent and eminently trainable, are very resilient, work well on a training schedule, and because their anatomy and physiology is well known and in many respects comparable to people. A drawback is that rhesus monkeys can be relatively aggressive, and can inXict severe injuries. Accordingly, great care must be taken to avoid injury during freely moving monkey experiments. We have found that monkeys are very cooperative when they have learned that they will not be harmed and their testing regimen is routine. Under these conditions, monkeys can be lead around on leashes (Fig. 1) without incident although unexpected events can cause monkeys to respond aggressively. We prefer to begin training in juvenile (2–3 kg) monkeys, though we have successfully trained an 8-year-old 12 kg rhesus that had previously lived in a semi-natural environment with conspeciWcs in a monkey corral at the Kunming Primate Research Centre (www.kiz.ac.cn/introduction/map2.htm). Once trained, monkeys can participate in experimental work for up to 8 years, can be surgically implanted for neurophysiological studies, and may be retired at the end of the experiments in good health and weighing 16 kg. At this point; the implants are surgically removed and a skin graft is used to replace any defect. Recording locations in the brain can be identiWed with MRI scans and X-radiographs, thus obviating the need for histological examination of the brain.
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Fig. 1. Preliminary training for freely moving monkeys in the garden at the Kunming Institute of Zoology. Monkeys wear a collar on the neck which is attached to a leash drawn through a plastic tube.
2.2. A laboratory environment for testing freely moving monkeys 2.2.1. The testing rooms and feeders The testing room measures approximately 5.5 £ 6 m; the Xoors and walls are gray-green. Four feeders [24] are placed midway along each wall; the distance between the lids of the feeders is approximately 2.1 m. Fig. 2A shows the dimensions, task contingencies and equipment in the testing room. In the centre of the room is a post (0.5 m high) with a rotating spindle at the top. Mounted on the spindle is a housing that contains a retractable dog leash (Flexi Classic 1–5), which rotates as the monkey walks around the room. This leash is suYciently long (»2 m) to allow the monkeys to comfortably sit and forage at the four feeders but not long enough to allow them to climb the feeders. The feeders are 0.5 m high, with a 5 £ 5 cm lid (covering a 2 £ 4 cm slot housing a piece of food) that can be raised, triggering a computer to deliver a reward [24]. The feeders were designed so that eYcient retrieval required the monkeys to sit before opening the feeder lid. Monkeys usually use one hand to raise the lid and the other to retrieve the food. This skill requires good visuo-motor coordination, which took several days to learn. Each feeder is able to deliver 50 rewards (e.g., 1/8th grape, apple morsel, 1/2 peanut, raisin) per session. Feeders have several notable features, a white/blue LED (to identify it as the current target), a loudspeaker (to indicate a behavioral error), and a lid (with a magnetic sensor, RadioShack 49–496) that required lifting to initiate food delivery. Actuation of the sensor sent a signal to a laboratory computer (Pentium 133, DOS operating system; running TEMPO software, ReXective Computing, Inc.) when the lid is opened. Fig. 1C shows one of the feeders with a curtain behind it (see also http://w3.arizona.edu/~primate/). The curtains running behind the feeders are used to reduce the asymmetry of the room so that it is eVectively a
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Fig. 2. The freely moving monkey testing laboratory. (A) Layout of the laboratory.The dashed lines indicates the shortest pathways between the feeders. The path sequences are shown in part b. C/P refers to the position of the camera and commutator mounted in the ceiling. (B). Actual traces of the monkey’s movements in the testing room, obtained by plotting location as a function of head position. Typically, monkeys take a curved path between feeders; they rarely walk to the center of the room. (C) The south feeder. All feeders are identical, providing a homogeneous testing environment for neurophysiological experiments. The curtains running behind the feeders also contribute to this homogeneity.
square (4.9 £ 4.9 m). Curtains provide a largely homogeneous visual environment and thus the view of the testing room is largely the same where ever the monkey happens to be looking. This measure is designed to reduce the visual cues that inXuence neuronal activity. Fluorescent lights were concealed in soWts close to the ceiling (4.5 m high) along all four walls. A chamber (1 m wide £ 1 m long £ 0.7 m deep) is located in the center of the room’s ceiling. It contained a color video camera (American Dynamics ADC762 1/3⬙) directed vertically down to record the monkey’s behavior, and an Air Flyte 72 channel slip-ring commutator (PN 1001498/72) that is modiWed (DragonXy Research and Development, Inc.) by providing a servo-controlled torque motor. The commutator is used to carry neurophysiological signals to power ampliWers in an adjacent control room. Investigators monitored equipment and the monkey’s behavior from the control room. 2.2.2. Behavioral tasks Monkeys were trained on several visual- and memory guided tasks in the testing laboratory [8]. One useful paradigm is the freely moving monkey version of the delayed alternation task, the performance of which is dependent upon the dorsolateral prefrontal cortex [20] and fornix [13]. In this task, the monkeys alternated between three of the four feeders in a stereotyped sequence (Fig. 2). There were two versions of this task. In the North task, monkeys alternated in the following way, west-north-east-north-west, etc. In the South task, the monkey alternated east-south-westsouth-east, etc. Thus, there were eight possible paths between the feeders in the two versions of the task. These tasks required the monkey to travel between the feeders in a stereotyped sequence. Each path is taken approximately 10–20 times during an experiment (replication is essential for neurophysiological studies) and the monkeys achieved average speeds of about 1 m/s, with peak speeds of 3–5 m/s between the feeders. In a 2 choice version of the delayed alternation task, the monkey alternated between the north
and south, or between the east and west feeders. Recordings of the visual- and memory guided tasks were performed sequentially in the same testing room. 2.2.3. Plotting head position and direction in the testing room Our techniques involve the chronic (2–6 months) implantation of recording electrodes in the brain. Surgical and implant construction techniques are standard [3] but use titanium nuts embedded within the dental acrylic so that bolts can be used to hold a helmet on the implant. The helmets (Fig. 3) were made of thermoplastic splinting sheets (Smith & Nephew EzeForm), and are used to protect electrodes [23] and preampliWers, and to allow the placement of LED arrays used to monitor the location of the monkeys within the testing room. The LED arrays were mounted on the helmet so that the LEDs were displaced 4.5⬙ left (red) and right (blue) of the centre of the helmet. The locations of the LEDs were tracked by a computer (Cheetah, Neuralynx, NL) interfaced with a ceiling-mounted video camera at a temporal resolution of 60 frames/s with 640 £ 480 pixel resolution provided by the NTSC signal. The area within which the monkeys walk is represented as a 100 £ 100 element grid where 1 pixel D 7 cm2 (PrimaTracker, Kim et al., 2004a; laboratory website: http://w3.arizona.edu/~primate/). Neuronal Wring rate is obtained by dividing the number of spikes emitted by the amount of time spent in a particular location (pixel). The software is used to determine the centre of the monkey’s head (location), the direction in which the head is facing, and the speed and angular velocity of movements of the head. A second method for analysing head movements during the memory tasks is to use cameras that were mounted inside the helmets [9]. This form of data provides comparable information about the viewing perspective of the monkeys but without adequate software is harder to interpret than reconstruction of head movements using the LED arrays. A movie of the scene from the head camera can be seen at the laboratory website (http://w3.arizona.edu/~primate/).
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Fig. 3. Helmet worn by freely moving monkeys. A Neuralynx headstage is mounted inside and to the rear of the helmet (10 cm long £ 12 cm wide £ 12 cm high) and is connected to a set of electrodes using a cable set built in the laboratory. A lightweight cable connects the headstage with a commutator and ampliWers. The skirt at the helmet base prevents the monkey from damaging the electrodes which are chronically implanted in the brain. The two holes at the base of the helmet accept bolts that attach the helmet to the implant; the single hole at the top of the helmet allows a camera to image the head scene.
2.2.4. Data acquisition and behavioral control system The Cheetah system (Neuralynx Inc.) is used to acquire neuronal waveforms. This system was developed by Casey Stengel and KristoV Agiello based on the DOS-based BrainWave system, which was originally designed by Stengel. The original Cheetah system was implemented on a Sun UltraSPARC 1 computer running the Solaris 7 operating system. This platform was believed to provide the necessary processing power and the original speciWcations envisaged two animal tracking devices. However, the rapid development and increasing power of CPUs and a stable operating system (Windows NT, 2000) made it possible to port the Cheetah system to an Intel processor-based system. The Cheetah action potential discrimination concept was designed to analyse waveform data acquired using electrode bundles in the tetrode conWguration. Action potentials are recorded diVerentially, buVered with a NL headstage-54 ampliWer (Fig. 3), sent by cable to a commutator and then to power ampliWers (NL 8). For monkeys, we use 36 gauge insulated wires to make a multichannel cable. The ampliWcation system is controlled by Cheetah software which selects waveforms for storage based on voltage threshold crossing. Cheetah data Wles incorporate Xagged signals that represent critical task events (e.g., reward delivery) that are generated by Tempo software (ReXective Computing, Inc.) which is responsible for selecting behavioral events such as trial type. Cheetah
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data Wles also store information about computed location of the monkey’s head. Action potential waveforms are discriminated oZine with semi-automated and manual sorting software obtained commercially (Plexon Inc., OZine sorter) or freeware written by ourselves and other investigators (ElectrodeSorter, B-H. Kim; Mclust, D. Reddish) (available upon request). Graphical analysis of neuronal Wring used NeuroExplorer (Plexon, Inc.) and VideoDecoder (B-H. Kim). Cheetah data Wles are relatively large; a 30 min recording session typically require 5 Mb for a hippocampal pyramidal cell with low Wring rates (75–400 total spikes), or in excess of 100 Mb of disk space for a neuron with a high Wring rate (hippocampal interneurons). While disk space for data storage is trivial given the low cost and large capacity of DVD recorders, the time requires to analyse large Cheetah Wles using available software is a major limitation. For example, a 70 Mb Wle generated by a single tetrode could take 2 days to discriminate action potential waveforms using a Dell computer with a Pentium III CPU and 512 Mb of RAM, even when the computer is dedicated to this analysis job. 2.2.5. Sample neurophysiological data The methods described above have enabled us to carry out recordings in dorsolateral prefrontal cortex [18,19], putamen (Kim et al., in preparation), and hippocampus [11,8]. Monkey hippocampus, like that of the rat [15] contains neurons whose Wring rate is maximal when the monkey walks through a particular region of the testing laboratory [11]. Such neurons are described as having place Welds: these cells Wre selectively when the monkey walks through a part of the laboratory but are inactive in walking through other parts. In the monkey, the appearance of neuronal place Welds is not simply related to a location in the laboratory as deWned by the presence of environmental cues [8]. For example, neuronal activity (Fig. 4A) is very weak in the Delayed Alternation task but a place Weld is
Fig. 4. Plot of hippocampal Wring rate in two behavioral tasks. In the Delayed Alternation task (both north and south versions) there is sporadic Wring in the south-east quadrant. In contrast, a clear place Weld emerges in the north-west quadrant during performance of the 2 choice alternation task. Intensity of Wring rate is color-coded, each colored pixel represents neuronal activity.
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evident in the north-west quadrant when the monkey alternates between the north and south boxes in the 2 choice alternation task (Fig. 4B). The selective appearance of the place Welds occurs even though the monkey takes the same paths and directions in both tasks. Thus, when the monkey performs a diVerent task, new place Welds can appear and old Welds can disappear; such changes are termed remapping. Remapping occurs even though the environmental cues are constant. Our methods permit us to record from single neurons for long periods of time [4] and thus it is unlikely that the remapping of the place Weld in two sequentially performed tasks is an artifact due to changes in the recording isolation of a neuron. 2.3. Telemetric recordings of freely moving monkeys in a 50 m2 environment To improve the ability to record in a spatially extended environment that approximates that of monkeys in the wild, we realised that it would be necessary to adopt telemetered recordings. It is unlikely that investigators interested in primate social interactions would be able to obtain useful data when cables are attached to the helmets of monkeys as such cables would be quickly destroyed. However, cable-based recording systems are useful in the testing environment described above in which recordings take place from a single monkey as they can provide unparalleled data in terms of quality and quantity. One problem with telemetered recordings is that the ability to acquire data on multiple electrode channels simultaneously has proved to be technically challenging. For example, Neuralynx Inc. has worked on a miniature 32 channel telemetry system for some time, but currently more development is required before a reliable product is available commercially. However, for investigators interested in acquiring data on a limited (e.g., 2) number of independent channels, building a radio-telemetric system for freely moving monkeys is readily attainable [9]. We refer the reader to this paper for the design concepts and schematic diagrams of our ampliWer circuits. The development of our recording system faced several challenges, for example, how to eliminate electrical artifacts generated by the rapid limb and body movements of freely moving monkeys and how to provide information about the viewing perspective and eye position of the monkey. Our system is able to send neuronal signals from two electrodes as well as video signals from two cameras mounted on the head and used for monitoring head direction and eye position. For simplicity, we chose to focus our eVorts on designing the ampliWcation system for the neuronal signals but used a commercially available audio–visual transmitter for the actual telemetry; the neuronal signals were sent on the audio channels and the video channels were used for head and eye cameras. Monkeys are surprisingly strong for their size and we took advantage of this by mounting several power supplies for the equipment, three ampliWer stages, and the telemetry system in a jacket worn by the monkey [9].
The elimination of electrical artifacts in the recording system required a preampliWer with common mode rejection; two power supplies for the ampliWers; and a ground wire from the second ampliWer to the skull. The neuronal recordings employed diVerential recordings with a low impedance reference electrode located approximately 1 mm from the microelectrodes (0.5–1 Mohm). The bipolar recording conWguration allowed a common mode of input, reducing the interference generated by large movement artifacts which appeared on the recording and reference electrodes. Three ampliWcation stages were used with the preampliWer and the two main ampliWers having isolated power supplies. The reason for separating the ampliWers and power supplies is to avoid feedback-driven oscillations between successive ampliWcation stages as the output of a second or third stage ampliWer could aVect the power supply to the Wrst stage ampliWer. In addition, the power inputs of all operational ampliWers were bypassed with capacitors to reduce oscillations; this is particularly important for the front-end preampliWer. A unity-gain diVerential preampliWer received inputs from the recording and reference electrodes and is used to match the impedances of the electrical signals from the microelectrodes with the main ampliWers. Two further ampliWcation stages were used, powered by 12 V batteries. The output of the second ampliWer (gain D 50) is band-pass Wltered (900–6000 Hz) and fed to a third ampliWer (gain D 100) for a total gain of 5000. The separation of the two main ampliWers is to avoid the feedback ripple problem discussed above. Two video cameras were mounted on a helmet worn by the monkey. A wireless video camera (JLT-1.2G, Shen Zhen Company) is mounted above the eyes on the midline and is directed forward in order to record the “head scene,” i.e., the view straight ahead of the monkey. A second camera (203CA, Shen Zhen Company) is used to record eye movement and is mounted on a arm and directed toward the pupil. Both cameras measured 26 £ 26 £ 16 mm. The two video signals were sent to the audio–video transmitter in the jacket. Signals from electrodes were passed to the audio channel of an audio–video transmitter, in parallel with the camera signals which were sent on the video channels. These signals were sent telemetrically to the data storage devices located 50 m distant. The “data storage station” included two video receivers, a quad video processor, a videotape camcorder (held by the experimenter) and dc power supplies. The station is packaged inside a suitcase (26 £ 16 £ 20 cm high), weighed about 2.0 kg and is carried by the experimenter. One audio–video receiver is dedicated to the signals of neuronal activity and the video signals of the eye movements. Fig. 5 Another receiver (JLT-1.2 G, Shen Zhen, China, 11 £ 5 £ 2.2 cm) is used to receive the video signals from the head scene camera. Video images from all three cameras were then fed into a four channel black-white quad processor (Goldbeam, Qb-Bnc, 20 £ 22 £ 4.5 cm high); the composite picture of the three cameras as well as neuronal activity is
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Fig. 5. AmpliWers mounted on printed circuit boards.
sent to a videotape camcorder (Sharp VL-E660U; or Panasonic NV-MX300), where it is stored on 8 mm cassettes together with the simultaneously recorded neuronal responses. A rechargeable battery (12 V, 7 Ah) is used to power all devices in the station except for the camcorder. 2.4. Synchronisation of behavioral events with telemetered signals One important problem that arises with telemetered data is the analysis and replication of the experimental conditions. Telemetry allows high quality recordings to be made when monkeys move within a large spatially extensive environment, as shown in Fig. 6. However, diYculties arise in analyzing the data in a way that provides information about desirable experimental variables. Typical neurophysiological experiments measure neuronal Wring rate with respect to a triggering event such as the onset of a visual stimulus on a video monitor, or the arrival of the monkey at a particular feeder in a freely moving monkey experiment [19]. In the open Weld experiment with a 50 m2 testing environment [9] the variability of the visual environment is a potential problem as the monkey can look at many diVerent objects from many viewing perspectives. This problem is compounded because it may be diYcult to obtain repetitions of the stimulus events that activate individual neurons. Nevertheless, the potential advantages of
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recording from freely moving monkeys in spatially extensive environments is very great—in principle, ethologists could examine neuronal activity from a monkey living in a social group, or neuropsychologists could record in freely moving monkeys during the initiation and maintenance of walking in order to study neuronal activity in the basal ganglia which would helpful for understanding their role in movement and thus yield insights into the problems of Parkinson’s disease [1]. The problem of synchronising behavioral events with telemetered signals is solvable in many ways. For example, a simple solution requires the investigator to design a structured series of events in which the monkey’s behavior can be coded and a method (e.g., synchronised clocks in computers) for linking telemetered neuronal activity to these coded events. Behavioral studies of animals commonly use computers programmed to encode speciWc events (e.g., vocalization, groom, etc.) via a keyboard in order to determine the frequency of behaviors of interest to the research (e.g. [2]). Entering such events into the data stream containing time-stamps of neuronal spikes is straightforward. We have used this technique to encode the onset and cessation of walking during recordings from the putamen and hippocampus (Kim et al., in preparation) and found that it produces reliable results for slowly occurring events such as the initiation of walking from a sitting position. The recordings obtained by the telemetry signal (three monkeys; [9]) from hippocampus and parietal cortex were satisfactory, comparable to those obtained by commercially available cabled systems we used in the laboratory (four monkeys; [19,8], and in preparation). As far as we are aware, we did not experience loss of signals at certain locations within the recording area though this needs further research. The bandwidth of the telemetry system resulted in low pass Wltering of action potentials but apart from some reduction in amplitude, the recording quality is acceptable (Fig. 6). 3. Concluding remarks There are many obstacles to obtaining high quality recordings from single neurons in freely moving monkeys. For example, failure to appreciate the frustration of an unattended monkey can lead to the destruction of an
Fig. 6. Telemetered recordings of hippocampal neurons in freely moving monkeys walking in the garden of the Kunming Institute of Zoology. Waveform analysis software is provided by Nan-Hui Chen.
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expensive equipment. However, a little common sense can usually eliminate these problems. Moreover, the equipment necessary for the experiments are very similar to that required in chair-housed monkeys. It is possible to buy virtually all the equipment from a commercial vendor, but in practice many items are easily constructed in a competent neurophysiology laboratory. Accordingly, we imagine that there are substantial gains to be made into understanding brain function in freely moving monkeys. Acknowledgments Support for the development of the methods described in this paper was provided by NIH Grants MH58415 and NS-36255; Chinese Academy of Sciences Grant KSCX2SW, National Basic Research Program of China Grant 2005CB522803; Chinese National Science Foundation Grants 30470553 and 30530270, the Yunnan Science Foundation.; and Whitehall Foundation Grant A89-04; National Science Foundation Grants of China (NSFC) 30530270 973 program project 2005CB522803 ChineseFinish International Collaborative Project-neuro (NSFC). Appendix A Air Flyte Electronics 56 New Hook Road Bayonne, NJ 07002, USA +1 201 436 2230 American Dynamics 6795 Flanders Drive San Diego, CA 92121, USA +1 800 507 6268 http://www.americandynamics.net/ DragonXy Research & Development PO Box 507, Ridgeley, West Virginia 26753-0507, USA +1 304 738 3609 http://www.dragonXyinc.com/ Goldbeam Electronics, Inc. 1741 W. Rosecrans Avenue, Gardena, CA 90249, USA +1 800 777 4760 HYGENIC Perm Line Repair Resin Akron, OH Neuralynx, Inc 2434 North Pantano Road Tucson, AZ 85715, USA +1 520 722 8144 http://www.neuralynx.com/ Panasonic Corporation 1 Panasonic Way Secaucus, NJ 07094, USA
+1 201 348 7000 http://www.panasonic.com Primate Products PO Box 620415 Woodside, CA 94062,USA +1 650 529 0419 http://www.primateproducts.com/ ReXective Computing, Inc. Sheldon HoVman 917 Alanson Dr St. Louis, MO 63132, USA +1 314 993 6132 voice [email protected] Sharp Electronics Corporation [USA] 1300 Naperville Drive, Romeoville, IL 60446, USA +1 800 237 4277 http://www.sharpusa.com/ SHENZHEN OMENT ELECTRONICS CO., LTD. 5B Xingyunge, Zhongfu Building Fumin Road, Futian Shenzhen 518048, China +86 0755 83866791 models JLT-1.2G (wireless) and 203CA Smith & Nephew Wound Management PO Box 81 101 Hessle Road HULL HU3 2BN, UK Tel: +44 (0)1482 225181 http://www.smith-nephew.com/ References [1] J.-P. Azulay, S. Mesure, B. Amblard, O. Blin, I. Sangla, J. Pouget, Brain 122 (1999) 111–120. [2] S.A. Castner, P.S. Goldman-Rakic, Neuropsychopharmacology 20 (1999) 10–28. [3] E. Evarts, J. Neurophysiol. 29 (1966) 1011–1027. [4] P.A. Greenberg, F.A.W. Wilson, J. Neurophysiol. 92 (2004) 1042– 1055. [5] M. Habib, A. Sirigu, Cortex 23 (1987) 73–85. [6] D.A. Hill, Am. J. Primatol. 43 (1997) 305–322. [7] K. Hill, C. Boesch, J. Goodall, A. Pusey, J. Williams, R. Wrangham, J. Hum. Evol. 40 (2001) 437–450. [8] B.-H. Kim, S.-L. Lim, J.-W. Ryou, F.A.W. Wilson, Soc. Neurosci. Absts. 30 (2004) 1007.2. [9] Y. Lei, N. Sun, F.A.W. Wilson, X. Wang, N. Chen, J. Yang, Y. Peng, J. Wang, S. Tian, M. Wang, Y. Miao, W. Xhu, H. Qi, Y.Y. Ma, J. Neurosci. Methods 135 (2004) 35–41. [10] N. Ludvig, J.M. Botero, H.M. Tang, B. Gohil, J.G. Kral, J. Neurosci. Methods 106 (2001) 179–187. [11] Y.-Y. Ma, J.-W. Ryou, B.-H. Kim, F.A.W. Wilson, Spatially directed movement and neuronal activity in freely moving monkeys, in: S. Mori, D.G. Stuart, M. Wiesendanger (Eds.), Brain Mechanisms for the Integration of Posture and Movement, Progress in Brain Research, vol. 143, Elsevier, Amsterdam, 2003, pp. 505–512.
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Neurophysiological recordings in freely moving monkeys Ning Lei Sun a,d, Yan Lin Lei a, Byoung-Hoon Kim b, Jae-Wook Ryou c, Yuan-Ye Ma a, Fraser A.W. Wilson a,¤ a
Laboratory of Primate Neuroscience, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan 650223, China b Department of Physiology, University of Wisconsin, USA c Department of Neurology, Cornell University, USA d Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR china Accepted 15 September 2005
Abstract Recordings of neuronal activity in freely moving rats are common in experiments where electrical signals are transmitted using cables. Such techniques are not common in monkeys because their prehensile abilities are thought to preclude such techniques. However, analysis of brain mechanisms underlying spatial navigation and cognition require the subject to walk. We have developed techniques for recordings in freely moving monkeys in two diVerent situations: a 5 £ 5 m testing laboratory and in a 50 m2 open Weld environment. Neuronal signals are sent to ampliWers and data acquisition systems using cables or telemetry. These techniques provide high quality recordings of single neurons during behaviors such as foraging, walking, and the performance of memory tasks and thus provide a unique opportunity to study primate behavior in a semi-natural situation. © 2006 Elsevier Inc. All rights reserved. Keywords: Allocentric space; Egocentric space; Hippocampus; Prefrontal cortex primate; Putamen; Rhesus
1. Introduction The vast majority of neurophysiological studies in behaving monkeys use techniques originated by Edward Evarts (e.g. [3]) and remain very similar to those pioneering studies. For example, standard methods include immobilization of the head and a hydraulically driven drive to move a microelectrode. These techniques are well suited to studying many forms of brain function, but certain classes of behaviors and cognitive abilities such as learning about the spatial organization of the environment require a diVerent approach. O’Keefe and Dostrovsky [14] pioneered studies in recording hippocampal activity in freely moving rats with their original description of place cells. Many subsequent studies have conWrmed the original observations:
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Corresponding author. E-mail address: [email protected] (F.A.W. Wilson).
1046-2023/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2005.09.018
place cells are neurons that Wre selectively when rats walk through a part of, but not all of a familiar environment. Such cells are candidates for providing information about a ‘cognitive map’ of the environment and strongly suggest that the rat hippocampus is involved in the analysis of space [15]. In contrast, the functions of the non-human primate hippocampus are still controversial—it is simply not clear what function the hippocampus contributes. Our approach to this controversy has been to undertake studies in monkeys that replicate the basic paradigm established by O’Keefe. Other investigators have also adapted the freely moving rat paradigms to monkeys [16,17]. Like rats, non-human primates are foragers and travel considerable distances in search of food. Their feeding behavior is opportunistic [21], and is characterized by seasonal variations in the many diVerent foods that they sample [6,7]. One might expect that primate survival is mediated by the development of brain structures that provide the visual and spatial abilities to enable foraging, and laboratory
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studies amply attest to the ability of monkeys to learn about new food sources [22]. It is striking then that typical laboratory research with monkeys rarely studies foraging behavior as a means to examine fundamental processes of learning and memory. Part of the explanation for this neglect is that proper facilities for studying spatial memory are technically diYcult to develop and to place under experimental control. However, it is clear that brain mechanisms in humans are specialized for mapping the environment. For example, Habib and Sirigu [5] describe the loss of familiarity for places following brain damage. Although progress has been made in using brain imaging techniques to analyse these mechanisms in people [12], recordings of neuronal activity could contribute to studying the neural bases of learning about the spatial arrangements of cues in the environment by developing techniques for measuring behavior and neuronal activity in freely moving monkeys engaged in walking through extended spatial environments. In this article we document two diVerent approaches to studying neuronal activity in freely moving monkeys walking in extensive spatial environments. In one paradigm we describe procedures for recordings using cables and a commutator made in a testing room the size of a typical research laboratory. In a second paradigm we describe techniques for telemetric recordings in monkeys walking within a 50 m2 open Weld environment. 2. Description of methods 2.1. General behavioral and safety issues Rhesus monkeys (Macaca mulatta) are preferred to other species of monkeys (but see [10]), in part because they are intelligent and eminently trainable, are very resilient, work well on a training schedule, and because their anatomy and physiology is well known and in many respects comparable to people. A drawback is that rhesus monkeys can be relatively aggressive, and can inXict severe injuries. Accordingly, great care must be taken to avoid injury during freely moving monkey experiments. We have found that monkeys are very cooperative when they have learned that they will not be harmed and their testing regimen is routine. Under these conditions, monkeys can be lead around on leashes (Fig. 1) without incident although unexpected events can cause monkeys to respond aggressively. We prefer to begin training in juvenile (2–3 kg) monkeys, though we have successfully trained an 8-year-old 12 kg rhesus that had previously lived in a semi-natural environment with conspeciWcs in a monkey corral at the Kunming Primate Research Centre (www.kiz.ac.cn/introduction/map2.htm). Once trained, monkeys can participate in experimental work for up to 8 years, can be surgically implanted for neurophysiological studies, and may be retired at the end of the experiments in good health and weighing 16 kg. At this point; the implants are surgically removed and a skin graft is used to replace any defect. Recording locations in the brain can be identiWed with MRI scans and X-radiographs, thus obviating the need for histological examination of the brain.
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Fig. 1. Preliminary training for freely moving monkeys in the garden at the Kunming Institute of Zoology. Monkeys wear a collar on the neck which is attached to a leash drawn through a plastic tube.
2.2. A laboratory environment for testing freely moving monkeys 2.2.1. The testing rooms and feeders The testing room measures approximately 5.5 £ 6 m; the Xoors and walls are gray-green. Four feeders [24] are placed midway along each wall; the distance between the lids of the feeders is approximately 2.1 m. Fig. 2A shows the dimensions, task contingencies and equipment in the testing room. In the centre of the room is a post (0.5 m high) with a rotating spindle at the top. Mounted on the spindle is a housing that contains a retractable dog leash (Flexi Classic 1–5), which rotates as the monkey walks around the room. This leash is suYciently long (»2 m) to allow the monkeys to comfortably sit and forage at the four feeders but not long enough to allow them to climb the feeders. The feeders are 0.5 m high, with a 5 £ 5 cm lid (covering a 2 £ 4 cm slot housing a piece of food) that can be raised, triggering a computer to deliver a reward [24]. The feeders were designed so that eYcient retrieval required the monkeys to sit before opening the feeder lid. Monkeys usually use one hand to raise the lid and the other to retrieve the food. This skill requires good visuo-motor coordination, which took several days to learn. Each feeder is able to deliver 50 rewards (e.g., 1/8th grape, apple morsel, 1/2 peanut, raisin) per session. Feeders have several notable features, a white/blue LED (to identify it as the current target), a loudspeaker (to indicate a behavioral error), and a lid (with a magnetic sensor, RadioShack 49–496) that required lifting to initiate food delivery. Actuation of the sensor sent a signal to a laboratory computer (Pentium 133, DOS operating system; running TEMPO software, ReXective Computing, Inc.) when the lid is opened. Fig. 1C shows one of the feeders with a curtain behind it (see also http://w3.arizona.edu/~primate/). The curtains running behind the feeders are used to reduce the asymmetry of the room so that it is eVectively a
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Fig. 2. The freely moving monkey testing laboratory. (A) Layout of the laboratory.The dashed lines indicates the shortest pathways between the feeders. The path sequences are shown in part b. C/P refers to the position of the camera and commutator mounted in the ceiling. (B). Actual traces of the monkey’s movements in the testing room, obtained by plotting location as a function of head position. Typically, monkeys take a curved path between feeders; they rarely walk to the center of the room. (C) The south feeder. All feeders are identical, providing a homogeneous testing environment for neurophysiological experiments. The curtains running behind the feeders also contribute to this homogeneity.
square (4.9 £ 4.9 m). Curtains provide a largely homogeneous visual environment and thus the view of the testing room is largely the same where ever the monkey happens to be looking. This measure is designed to reduce the visual cues that inXuence neuronal activity. Fluorescent lights were concealed in soWts close to the ceiling (4.5 m high) along all four walls. A chamber (1 m wide £ 1 m long £ 0.7 m deep) is located in the center of the room’s ceiling. It contained a color video camera (American Dynamics ADC762 1/3⬙) directed vertically down to record the monkey’s behavior, and an Air Flyte 72 channel slip-ring commutator (PN 1001498/72) that is modiWed (DragonXy Research and Development, Inc.) by providing a servo-controlled torque motor. The commutator is used to carry neurophysiological signals to power ampliWers in an adjacent control room. Investigators monitored equipment and the monkey’s behavior from the control room. 2.2.2. Behavioral tasks Monkeys were trained on several visual- and memory guided tasks in the testing laboratory [8]. One useful paradigm is the freely moving monkey version of the delayed alternation task, the performance of which is dependent upon the dorsolateral prefrontal cortex [20] and fornix [13]. In this task, the monkeys alternated between three of the four feeders in a stereotyped sequence (Fig. 2). There were two versions of this task. In the North task, monkeys alternated in the following way, west-north-east-north-west, etc. In the South task, the monkey alternated east-south-westsouth-east, etc. Thus, there were eight possible paths between the feeders in the two versions of the task. These tasks required the monkey to travel between the feeders in a stereotyped sequence. Each path is taken approximately 10–20 times during an experiment (replication is essential for neurophysiological studies) and the monkeys achieved average speeds of about 1 m/s, with peak speeds of 3–5 m/s between the feeders. In a 2 choice version of the delayed alternation task, the monkey alternated between the north
and south, or between the east and west feeders. Recordings of the visual- and memory guided tasks were performed sequentially in the same testing room. 2.2.3. Plotting head position and direction in the testing room Our techniques involve the chronic (2–6 months) implantation of recording electrodes in the brain. Surgical and implant construction techniques are standard [3] but use titanium nuts embedded within the dental acrylic so that bolts can be used to hold a helmet on the implant. The helmets (Fig. 3) were made of thermoplastic splinting sheets (Smith & Nephew EzeForm), and are used to protect electrodes [23] and preampliWers, and to allow the placement of LED arrays used to monitor the location of the monkeys within the testing room. The LED arrays were mounted on the helmet so that the LEDs were displaced 4.5⬙ left (red) and right (blue) of the centre of the helmet. The locations of the LEDs were tracked by a computer (Cheetah, Neuralynx, NL) interfaced with a ceiling-mounted video camera at a temporal resolution of 60 frames/s with 640 £ 480 pixel resolution provided by the NTSC signal. The area within which the monkeys walk is represented as a 100 £ 100 element grid where 1 pixel D 7 cm2 (PrimaTracker, Kim et al., 2004a; laboratory website: http://w3.arizona.edu/~primate/). Neuronal Wring rate is obtained by dividing the number of spikes emitted by the amount of time spent in a particular location (pixel). The software is used to determine the centre of the monkey’s head (location), the direction in which the head is facing, and the speed and angular velocity of movements of the head. A second method for analysing head movements during the memory tasks is to use cameras that were mounted inside the helmets [9]. This form of data provides comparable information about the viewing perspective of the monkeys but without adequate software is harder to interpret than reconstruction of head movements using the LED arrays. A movie of the scene from the head camera can be seen at the laboratory website (http://w3.arizona.edu/~primate/).
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Fig. 3. Helmet worn by freely moving monkeys. A Neuralynx headstage is mounted inside and to the rear of the helmet (10 cm long £ 12 cm wide £ 12 cm high) and is connected to a set of electrodes using a cable set built in the laboratory. A lightweight cable connects the headstage with a commutator and ampliWers. The skirt at the helmet base prevents the monkey from damaging the electrodes which are chronically implanted in the brain. The two holes at the base of the helmet accept bolts that attach the helmet to the implant; the single hole at the top of the helmet allows a camera to image the head scene.
2.2.4. Data acquisition and behavioral control system The Cheetah system (Neuralynx Inc.) is used to acquire neuronal waveforms. This system was developed by Casey Stengel and KristoV Agiello based on the DOS-based BrainWave system, which was originally designed by Stengel. The original Cheetah system was implemented on a Sun UltraSPARC 1 computer running the Solaris 7 operating system. This platform was believed to provide the necessary processing power and the original speciWcations envisaged two animal tracking devices. However, the rapid development and increasing power of CPUs and a stable operating system (Windows NT, 2000) made it possible to port the Cheetah system to an Intel processor-based system. The Cheetah action potential discrimination concept was designed to analyse waveform data acquired using electrode bundles in the tetrode conWguration. Action potentials are recorded diVerentially, buVered with a NL headstage-54 ampliWer (Fig. 3), sent by cable to a commutator and then to power ampliWers (NL 8). For monkeys, we use 36 gauge insulated wires to make a multichannel cable. The ampliWcation system is controlled by Cheetah software which selects waveforms for storage based on voltage threshold crossing. Cheetah data Wles incorporate Xagged signals that represent critical task events (e.g., reward delivery) that are generated by Tempo software (ReXective Computing, Inc.) which is responsible for selecting behavioral events such as trial type. Cheetah
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data Wles also store information about computed location of the monkey’s head. Action potential waveforms are discriminated oZine with semi-automated and manual sorting software obtained commercially (Plexon Inc., OZine sorter) or freeware written by ourselves and other investigators (ElectrodeSorter, B-H. Kim; Mclust, D. Reddish) (available upon request). Graphical analysis of neuronal Wring used NeuroExplorer (Plexon, Inc.) and VideoDecoder (B-H. Kim). Cheetah data Wles are relatively large; a 30 min recording session typically require 5 Mb for a hippocampal pyramidal cell with low Wring rates (75–400 total spikes), or in excess of 100 Mb of disk space for a neuron with a high Wring rate (hippocampal interneurons). While disk space for data storage is trivial given the low cost and large capacity of DVD recorders, the time requires to analyse large Cheetah Wles using available software is a major limitation. For example, a 70 Mb Wle generated by a single tetrode could take 2 days to discriminate action potential waveforms using a Dell computer with a Pentium III CPU and 512 Mb of RAM, even when the computer is dedicated to this analysis job. 2.2.5. Sample neurophysiological data The methods described above have enabled us to carry out recordings in dorsolateral prefrontal cortex [18,19], putamen (Kim et al., in preparation), and hippocampus [11,8]. Monkey hippocampus, like that of the rat [15] contains neurons whose Wring rate is maximal when the monkey walks through a particular region of the testing laboratory [11]. Such neurons are described as having place Welds: these cells Wre selectively when the monkey walks through a part of the laboratory but are inactive in walking through other parts. In the monkey, the appearance of neuronal place Welds is not simply related to a location in the laboratory as deWned by the presence of environmental cues [8]. For example, neuronal activity (Fig. 4A) is very weak in the Delayed Alternation task but a place Weld is
Fig. 4. Plot of hippocampal Wring rate in two behavioral tasks. In the Delayed Alternation task (both north and south versions) there is sporadic Wring in the south-east quadrant. In contrast, a clear place Weld emerges in the north-west quadrant during performance of the 2 choice alternation task. Intensity of Wring rate is color-coded, each colored pixel represents neuronal activity.
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evident in the north-west quadrant when the monkey alternates between the north and south boxes in the 2 choice alternation task (Fig. 4B). The selective appearance of the place Welds occurs even though the monkey takes the same paths and directions in both tasks. Thus, when the monkey performs a diVerent task, new place Welds can appear and old Welds can disappear; such changes are termed remapping. Remapping occurs even though the environmental cues are constant. Our methods permit us to record from single neurons for long periods of time [4] and thus it is unlikely that the remapping of the place Weld in two sequentially performed tasks is an artifact due to changes in the recording isolation of a neuron. 2.3. Telemetric recordings of freely moving monkeys in a 50 m2 environment To improve the ability to record in a spatially extended environment that approximates that of monkeys in the wild, we realised that it would be necessary to adopt telemetered recordings. It is unlikely that investigators interested in primate social interactions would be able to obtain useful data when cables are attached to the helmets of monkeys as such cables would be quickly destroyed. However, cable-based recording systems are useful in the testing environment described above in which recordings take place from a single monkey as they can provide unparalleled data in terms of quality and quantity. One problem with telemetered recordings is that the ability to acquire data on multiple electrode channels simultaneously has proved to be technically challenging. For example, Neuralynx Inc. has worked on a miniature 32 channel telemetry system for some time, but currently more development is required before a reliable product is available commercially. However, for investigators interested in acquiring data on a limited (e.g., 2) number of independent channels, building a radio-telemetric system for freely moving monkeys is readily attainable [9]. We refer the reader to this paper for the design concepts and schematic diagrams of our ampliWer circuits. The development of our recording system faced several challenges, for example, how to eliminate electrical artifacts generated by the rapid limb and body movements of freely moving monkeys and how to provide information about the viewing perspective and eye position of the monkey. Our system is able to send neuronal signals from two electrodes as well as video signals from two cameras mounted on the head and used for monitoring head direction and eye position. For simplicity, we chose to focus our eVorts on designing the ampliWcation system for the neuronal signals but used a commercially available audio–visual transmitter for the actual telemetry; the neuronal signals were sent on the audio channels and the video channels were used for head and eye cameras. Monkeys are surprisingly strong for their size and we took advantage of this by mounting several power supplies for the equipment, three ampliWer stages, and the telemetry system in a jacket worn by the monkey [9].
The elimination of electrical artifacts in the recording system required a preampliWer with common mode rejection; two power supplies for the ampliWers; and a ground wire from the second ampliWer to the skull. The neuronal recordings employed diVerential recordings with a low impedance reference electrode located approximately 1 mm from the microelectrodes (0.5–1 Mohm). The bipolar recording conWguration allowed a common mode of input, reducing the interference generated by large movement artifacts which appeared on the recording and reference electrodes. Three ampliWcation stages were used with the preampliWer and the two main ampliWers having isolated power supplies. The reason for separating the ampliWers and power supplies is to avoid feedback-driven oscillations between successive ampliWcation stages as the output of a second or third stage ampliWer could aVect the power supply to the Wrst stage ampliWer. In addition, the power inputs of all operational ampliWers were bypassed with capacitors to reduce oscillations; this is particularly important for the front-end preampliWer. A unity-gain diVerential preampliWer received inputs from the recording and reference electrodes and is used to match the impedances of the electrical signals from the microelectrodes with the main ampliWers. Two further ampliWcation stages were used, powered by 12 V batteries. The output of the second ampliWer (gain D 50) is band-pass Wltered (900–6000 Hz) and fed to a third ampliWer (gain D 100) for a total gain of 5000. The separation of the two main ampliWers is to avoid the feedback ripple problem discussed above. Two video cameras were mounted on a helmet worn by the monkey. A wireless video camera (JLT-1.2G, Shen Zhen Company) is mounted above the eyes on the midline and is directed forward in order to record the “head scene,” i.e., the view straight ahead of the monkey. A second camera (203CA, Shen Zhen Company) is used to record eye movement and is mounted on a arm and directed toward the pupil. Both cameras measured 26 £ 26 £ 16 mm. The two video signals were sent to the audio–video transmitter in the jacket. Signals from electrodes were passed to the audio channel of an audio–video transmitter, in parallel with the camera signals which were sent on the video channels. These signals were sent telemetrically to the data storage devices located 50 m distant. The “data storage station” included two video receivers, a quad video processor, a videotape camcorder (held by the experimenter) and dc power supplies. The station is packaged inside a suitcase (26 £ 16 £ 20 cm high), weighed about 2.0 kg and is carried by the experimenter. One audio–video receiver is dedicated to the signals of neuronal activity and the video signals of the eye movements. Fig. 5 Another receiver (JLT-1.2 G, Shen Zhen, China, 11 £ 5 £ 2.2 cm) is used to receive the video signals from the head scene camera. Video images from all three cameras were then fed into a four channel black-white quad processor (Goldbeam, Qb-Bnc, 20 £ 22 £ 4.5 cm high); the composite picture of the three cameras as well as neuronal activity is
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Fig. 5. AmpliWers mounted on printed circuit boards.
sent to a videotape camcorder (Sharp VL-E660U; or Panasonic NV-MX300), where it is stored on 8 mm cassettes together with the simultaneously recorded neuronal responses. A rechargeable battery (12 V, 7 Ah) is used to power all devices in the station except for the camcorder. 2.4. Synchronisation of behavioral events with telemetered signals One important problem that arises with telemetered data is the analysis and replication of the experimental conditions. Telemetry allows high quality recordings to be made when monkeys move within a large spatially extensive environment, as shown in Fig. 6. However, diYculties arise in analyzing the data in a way that provides information about desirable experimental variables. Typical neurophysiological experiments measure neuronal Wring rate with respect to a triggering event such as the onset of a visual stimulus on a video monitor, or the arrival of the monkey at a particular feeder in a freely moving monkey experiment [19]. In the open Weld experiment with a 50 m2 testing environment [9] the variability of the visual environment is a potential problem as the monkey can look at many diVerent objects from many viewing perspectives. This problem is compounded because it may be diYcult to obtain repetitions of the stimulus events that activate individual neurons. Nevertheless, the potential advantages of
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recording from freely moving monkeys in spatially extensive environments is very great—in principle, ethologists could examine neuronal activity from a monkey living in a social group, or neuropsychologists could record in freely moving monkeys during the initiation and maintenance of walking in order to study neuronal activity in the basal ganglia which would helpful for understanding their role in movement and thus yield insights into the problems of Parkinson’s disease [1]. The problem of synchronising behavioral events with telemetered signals is solvable in many ways. For example, a simple solution requires the investigator to design a structured series of events in which the monkey’s behavior can be coded and a method (e.g., synchronised clocks in computers) for linking telemetered neuronal activity to these coded events. Behavioral studies of animals commonly use computers programmed to encode speciWc events (e.g., vocalization, groom, etc.) via a keyboard in order to determine the frequency of behaviors of interest to the research (e.g. [2]). Entering such events into the data stream containing time-stamps of neuronal spikes is straightforward. We have used this technique to encode the onset and cessation of walking during recordings from the putamen and hippocampus (Kim et al., in preparation) and found that it produces reliable results for slowly occurring events such as the initiation of walking from a sitting position. The recordings obtained by the telemetry signal (three monkeys; [9]) from hippocampus and parietal cortex were satisfactory, comparable to those obtained by commercially available cabled systems we used in the laboratory (four monkeys; [19,8], and in preparation). As far as we are aware, we did not experience loss of signals at certain locations within the recording area though this needs further research. The bandwidth of the telemetry system resulted in low pass Wltering of action potentials but apart from some reduction in amplitude, the recording quality is acceptable (Fig. 6). 3. Concluding remarks There are many obstacles to obtaining high quality recordings from single neurons in freely moving monkeys. For example, failure to appreciate the frustration of an unattended monkey can lead to the destruction of an
Fig. 6. Telemetered recordings of hippocampal neurons in freely moving monkeys walking in the garden of the Kunming Institute of Zoology. Waveform analysis software is provided by Nan-Hui Chen.
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expensive equipment. However, a little common sense can usually eliminate these problems. Moreover, the equipment necessary for the experiments are very similar to that required in chair-housed monkeys. It is possible to buy virtually all the equipment from a commercial vendor, but in practice many items are easily constructed in a competent neurophysiology laboratory. Accordingly, we imagine that there are substantial gains to be made into understanding brain function in freely moving monkeys. Acknowledgments Support for the development of the methods described in this paper was provided by NIH Grants MH58415 and NS-36255; Chinese Academy of Sciences Grant KSCX2SW, National Basic Research Program of China Grant 2005CB522803; Chinese National Science Foundation Grants 30470553 and 30530270, the Yunnan Science Foundation.; and Whitehall Foundation Grant A89-04; National Science Foundation Grants of China (NSFC) 30530270 973 program project 2005CB522803 ChineseFinish International Collaborative Project-neuro (NSFC). Appendix A Air Flyte Electronics 56 New Hook Road Bayonne, NJ 07002, USA +1 201 436 2230 American Dynamics 6795 Flanders Drive San Diego, CA 92121, USA +1 800 507 6268 http://www.americandynamics.net/ DragonXy Research & Development PO Box 507, Ridgeley, West Virginia 26753-0507, USA +1 304 738 3609 http://www.dragonXyinc.com/ Goldbeam Electronics, Inc. 1741 W. Rosecrans Avenue, Gardena, CA 90249, USA +1 800 777 4760 HYGENIC Perm Line Repair Resin Akron, OH Neuralynx, Inc 2434 North Pantano Road Tucson, AZ 85715, USA +1 520 722 8144 http://www.neuralynx.com/ Panasonic Corporation 1 Panasonic Way Secaucus, NJ 07094, USA
+1 201 348 7000 http://www.panasonic.com Primate Products PO Box 620415 Woodside, CA 94062,USA +1 650 529 0419 http://www.primateproducts.com/ ReXective Computing, Inc. Sheldon HoVman 917 Alanson Dr St. Louis, MO 63132, USA +1 314 993 6132 voice [email protected] Sharp Electronics Corporation [USA] 1300 Naperville Drive, Romeoville, IL 60446, USA +1 800 237 4277 http://www.sharpusa.com/ SHENZHEN OMENT ELECTRONICS CO., LTD. 5B Xingyunge, Zhongfu Building Fumin Road, Futian Shenzhen 518048, China +86 0755 83866791 models JLT-1.2G (wireless) and 203CA Smith & Nephew Wound Management PO Box 81 101 Hessle Road HULL HU3 2BN, UK Tel: +44 (0)1482 225181 http://www.smith-nephew.com/ References [1] J.-P. Azulay, S. Mesure, B. Amblard, O. Blin, I. Sangla, J. Pouget, Brain 122 (1999) 111–120. [2] S.A. Castner, P.S. Goldman-Rakic, Neuropsychopharmacology 20 (1999) 10–28. [3] E. Evarts, J. Neurophysiol. 29 (1966) 1011–1027. [4] P.A. Greenberg, F.A.W. Wilson, J. Neurophysiol. 92 (2004) 1042– 1055. [5] M. Habib, A. Sirigu, Cortex 23 (1987) 73–85. [6] D.A. Hill, Am. J. Primatol. 43 (1997) 305–322. [7] K. Hill, C. Boesch, J. Goodall, A. Pusey, J. Williams, R. Wrangham, J. Hum. Evol. 40 (2001) 437–450. [8] B.-H. Kim, S.-L. Lim, J.-W. Ryou, F.A.W. Wilson, Soc. Neurosci. Absts. 30 (2004) 1007.2. [9] Y. Lei, N. Sun, F.A.W. Wilson, X. Wang, N. Chen, J. Yang, Y. Peng, J. Wang, S. Tian, M. Wang, Y. Miao, W. Xhu, H. Qi, Y.Y. Ma, J. Neurosci. Methods 135 (2004) 35–41. [10] N. Ludvig, J.M. Botero, H.M. Tang, B. Gohil, J.G. Kral, J. Neurosci. Methods 106 (2001) 179–187. [11] Y.-Y. Ma, J.-W. Ryou, B.-H. Kim, F.A.W. Wilson, Spatially directed movement and neuronal activity in freely moving monkeys, in: S. Mori, D.G. Stuart, M. Wiesendanger (Eds.), Brain Mechanisms for the Integration of Posture and Movement, Progress in Brain Research, vol. 143, Elsevier, Amsterdam, 2003, pp. 505–512.
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