Journal of Neuroscience Methods 106 (2001) 179 187 www.elsevier.

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Single-cell recording from the brain of freely moving monkeys

Nandor Ludvig *, Juan M. Botero, Hai M. Tang, Baiju Gohil, John G. Kral
Departments of Physiology and Pharmacology, Anesthesiology, and Surgery, State Uni6ersity of New York, Health Science Center at Brooklyn, 450 Clarkson A6enue, Brooklyn, NY 11203, USA Received 2 January 2001; received in revised form 19 February 2001; accepted 19 February 2001

Abstract Single-cell recording from the brain of non-human primates has traditionally been performed in monkeys seated in a primate chair. However, this arrangement makes long-term recordings difcult, causes stress that may confound the data, and prevents the manifestation of natural behaviors. Extending our previous neurophysiological studies in non-human primates (Ludvig et al. Brain Res. Protocols 2000;5:7585), we have developed a method for recording the electrical activity of single hippocampal neurons in freely moving squirrel monkeys (Saimiri sciureus). The recording sessions lasted for up to 6 h, during which the monkeys moved freely around on the walls and the oor of a large test chamber and collected food pellets. Stable action potential waveforms were readily kept throughout the sessions. The following factors proved to be critical in this study: (a) selecting squirrel monkeys for the experiments, (b) using a driveable bundle of microwires for the recordings, (c) using a special recording cable, (d) implanting the microwires into the brain without causing neurological decits, and (e) running the recording sessions in a special test chamber. The described method allows long-term extracellular recordings from the brain of non-human primates, without the stress of chairing, during a wide range of natural behaviors. Using this model, new insights can be obtained into the unique ring repertoire of the neurons of the primate brain. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Squirrel monkey; Free behavior; Hippocampus; Single-cell recording

1. Introduction Our concepts on the biological laws that govern the ring of neurons of the human central nervous system (CNS) are based on: (1) an enormous amount of data obtained in invertebrates, rodents and other sub-primate species, (2) on signicantly less information collected from non-human primates, such as monkeys, and (3) on a very limited number of studies in patients with neurological disorders. The data obtained in sub-primate species provide invaluable guidance for understanding the basic electrophysiology of human brain cells. However, they cannot shed light on the cellular mechanisms that are unique to the human brain. The studies in neurological patients generate critically important information. However, this information is often extracted from abnormally functioning neurons, during a very restricted set of behaviors. In this situation,
* Corresponding author. Tel.: + 1-718-2701796; fax: +1-7182703103. E-mail address: ludvin10@bmec.hscbklyn.edu (N. Ludvig).

mapping the ring properties of neurons of the monkey brain is especially useful. Unlike CNS neuronal recordings from humans, those from monkeys can be performed in normal brain tissue and in a wider set of behaviors. At the same time, the cellular electrophysiological experiments in monkeys can yield data that are more relevant to the human brain than those obtained in sub-primate species. Traditionally, recording of single neurons from the brain of monkeys has been performed in head-restrained animals seated in a primate chair. This method has been used regardless of whether the recordings were made from the thalamus (Wiesel and Hubel, 1966), the cerebellum (Evarts, 1968), the neocortex (Kojima and Goldman-Rakic, 1982), the hippocampus (Ono et al., 1993), the locus coeruleus (Aston-Jones et al., 1997) or other structures. However, with this technique longterm neuronal recordings are difcult to obtain. Furthermore, these conditions are stressful for the monkeys, which may confound the generated data. Finally, this experimental arrangement does not permit the manifestation of many natural behaviors. To im-

0165-0270/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 7 0 ( 0 1 ) 0 0 3 4 8 - X

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prove this methodology, several investigators recently recorded hippocampal neurons in monkeys seated in a moveable chair or cab (Rolls, 1999; Ono and Nishijo, 1999). However, to date no single-cell recording has been conducted from the brain of monkeys while the animals are moving freely in a three-dimensional space. Microwire electrodes have been proved to yield stable and long-term extracellular recordings from the hippocampus of freely moving rats (Kubie, 1984; Pavlides and Winson, 1989; Ludvig et al., 1994, 1996; Kentros et al., 1998; Ludvig, 1999), as well as in restrained monkeys (Aston-Jones et al., 1997; Nicolelis et al., 1998). Recently, we adapted this recording technique to squirrel monkeys seated in a traditional primate chair (Ludvig et al., 2000). We found that the microwires allowed the monitoring of single-cell ring for many hours, even without the use of head-restraint. Encouraged by these results, we devoted this study to determine whether it is possible to record single-cell ring from the brain of monkeys which are released from the primate chair and move freely on the walls and oor of a large test chamber. We focused our experiments on hippocampal neurons, because free and voluntary movement is especially important for the activation of these cells (Foster et al., 1989; Ludvig, 1999; OKeefe, 1999).

provide a concise description only. The assembly (Fig. 1) contained a 12-pin dual row prole Mill-Max connector (Mill-Max, Oyster Bay, NY), to which three driving screws were glued. The microelectrode array itself comprised 11 Stablohm 675 H- Formvar-coated nichrome wires (California Fine Wire, Grover Beach, CA), 25 mm diameter each. At one end they were secured to the pins of the Mill-Max connector with GC Silver Print conductive paint (Newark Electronics, Newark, NJ), whereas at the other (recording) end they protruded 4 mm from the tube. A 3 mm wire segment proximal to the tube was covered with epoxy to provide stiffness to the electrode array. In addition to the nichrome wires, a larger grounding wire was connected with conductive paint to the 12th pin of the Mill-Max socket. Within this assembly, the tip of the microelectrode array was 44 mm below the surface of the MillMax connector. This arrangement allowed the placement of the electrode tip 19.5 mm below the brain surface, while the bottom of the driving screws was still 12 mm above the skull. The driving screws were calibrated to advance the whole assembly 5 mm downward. This was enough to move the electrodes through all layers of the hippocampus. (We note that in our

2. Methods

2.1. Animals
Four squirrel monkeys (Saimiri sciureus; Suborder Anthropoidea) were used in this study. They were taken from the monkey colony kept at the SUNY Health Science Center at Brooklyn in a facility approved by the American Association for Accreditation of Laboratory Animal Care. Monkey No. 1 was male, 780 g and 11 years old; monkey No. 2 was female, 500 g and 12 years old. These animals were used for pilot studies to determine the optimal surgical technique and observe their behavior. Monkey No. 3 was female, 750 g and 5 years old; monkey No. 4 was male, 850 g and 22 years old. The data presented in the Results section were obtained in these latter animals. This study was conducted according to the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996) and was approved by the Institutional Animal Care and Use Committee at SUNY Health Science Center at Brooklyn.

2.2. Microelectrode assembly

A detailed description of the design and construction of the microelectrode assembly is available in our previous report (Ludvig et al., 2000). Therefore, here we

Fig. 1. The driveable microelectrode assembly. The craniotomy seal, initially taped to the assembly platform, is pulled down during surgery and glued to the skull. The entire assembly is anchored to the skull with dental cement applied around the cuff of each driving screw. The microelectrode array is advanced into the recording site by rotating the driving screws. The two hooks serve to tightly secure the assembly to the recording cable.

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operation was performed until the animal was habituated to the new environment and spontaneously moved between food ports.

2.4. Microelectrode implantation

All surgical tools and the stereotaxic apparatus were autoclaved. After the injection of atropine (0.05 mg/kg, i.m.) and bicillin (100 000 units/kg, i.m.), anesthesia was induced with a mixture of ketamine (11 mg/kg, i.m.) and xylazine (0.5 mg/kg, i.m.). The animal was weighed, the head and extremities were shaved, and the non-invasive ECG, blood pressure, SpO2 and ECG monitors were placed. A second injection of ketamine alone (11 mg/kg, i.m.) was given during these procedures. By a facemask the animal was preoxygenated with 100% O2, and the anesthesia was deepened with 2.5% halothane for 5 min. Laryngoscopy was performed with 0.1 ml of 2% lidocaine sprayed onto the base of the vocal folds before endotracheal intubation. Anesthesia was maintained with 1.22% Isourane in oxygen using a Narkomed Compact system. The tail vein was cannulated for uid (lactated Ringer) administration. The tail of monkey No. 3 was injured prior to this study, therefore in this animal we used the femoral vein for cannulation. A Propaq 106 system served to monitor blood pressure, ECG, SpO2, respiratory rate, end tidal CO2, as well as rectal temperature. The animal was kept normothermic with a warming blanket and was placed in the stereotaxic frame. After positioning the head, the skull was exposed, and a 3 mm diameter craniotomy was made with a dental drill. The coordinates of the center of the hole on the skull were: 0.2 mm anterior to the line between the ear bars, and 9 mm right to the midline, according to the atlas of Gergen and MacLean (1962). Three anchoring screws were placed in the bone around the craniotomy. Betadine was extensively applied to the operated area. Next, the dura mater and the pia mater were excised from the brain surface. This was accompanied with minor bleeding in two animals. In these cases, we prevented the peridural diffusion of blood by draining it out of the craniotomy. The microelectrode assembly was sterilized with alcohol and betadine, and its electrode unit was introduced into the brain at an 8 angle through the center of the hole (Fig. 3). The tip of the unit was positioned to be 19.5 mm below the brain surface. The grounding wire of the assembly was connected to one of the screws with conductive paint. The paint was allowed to dry for 45 min. The assembly was secured to the skull and the screws with dental cement. A plastic ring with sterile bone wax and Panalog ointment on its surface sealed the craniotomy. Next, the base of a protective cap was positioned around the assembly, and secured to the skull with interior and exterior screws and dental cement, as described (Ludvig et al., 2000).

Fig. 2. The special test chamber. 1: Bars to allow the monkey to climb on the walls. 2: Smooth wall to prevent the monkey from moving above the bars. 3: Food port. 4: Water bottle. 5: Straight and rigid recording cable, invisible for the monkey in any position. 6: Protective tube to prevent the monkey from grabbing the cable. 7: Pulley assembly rotating together with the commutator. 8: Commutator. 9: Tube to prevent the cables counterweight from swinging. 10: Horizontal rod to prevent cable-twisting. 11: Camera, 280 cm above the oor. 12: Curtain to regulate the monkeys view to the laboratory. Each of these elements proved to be necessary for running the single-cell recording studies in freely moving squirrel monkeys for long periods.

design the microelectrode assembly also incorporates a microdialysis probe-guide. In fact, after completing the present recording studies, we performed simultaneous single-cell recording and microdialysis in the monkeys. The results of these experiments have been compiled for a separate report.)

2.3. Preoperati6e protocol

Three weeks before microelectrode implantation, the monkeys were taken from their social group and placed in individual cages (60 cm L80 cm H 60 cm W). However, the cages were close to each other so that the monkeys could interact. Purina monkey chow and water was available for the animals ad libitum. In addition, banana was given to them three times per week. In the last preoperative week, the monkeys were trained (1 h/day) to move around in the test chamber (Fig. 2) and collect food pellets (fruit loops) from 16 food ports. These food ports were 3 cm diameter and 8 cm long plastic tubes secured to the walls of the chamber. No

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The removable cover of the protective cap was then attached to the base. The skin was approximated with a one-layer interrupted horizontal mattress closure using a 3-0 nylon suture. Anesthesia was reversed, and the animal was extubated before being returned to the home cage. Monkey No. 2 died 16 h after surgery. Because of her low body weight of 500 g we suspect that she may have been chronically ill. This is consistent with the experience of Gergen and MacLean (1962) with very low weight squirrel monkeys. The rest of the monkeys (n= 3) vigorously climbed on the walls of their home cage and consumed food and water normally within 3 h after operation.

animals were placed in the test chamber and their behavior was observed. After 3 weeks of recovery the decision to proceed with recording was made if the animals collected food pellets in the same way as before operation.

2.6. Special test chamber

A 150 cm long, 150 cm wide and 280 cm high wooden chamber (Fig. 2) was built for this study. The walls of this chamber were composed of bars to a height of 120 cm, while above this line the walls were constructed of smooth plywood. This design was made to prevent the monkeys from moving or jumping above a dened area. Four food ports were placed on each wall, with a water bottle secured to one corner. The recording cable (G-Tech, Cortland Manor, NY) was connected to a commutator (Crist Instruments, Hagerstown, MD) placed on the ceiling of the chamber. A 3 mm diameter and 135 cm long plastic rod was taped along the lower portion of the cable to make it stiff and invisible to the monkey at any position. A 3.5 cm diameter and 15 cm long plastic tube was attached to the bottom of the cable (Fig. 4A and B) to prevent the monkey from grabbing and damaging the cable. The weight of the cable was counterbalanced by a 13 mm diameter metal cylinder sliding in a 150 cm long plastic tube. This prevented the counterweight from swinging. The monkey could not reach the counterweight tube because this latter was at a 80 cm horizontal distance from the recording cable. A lightweight horizontal rod, made of stainless steel, was designed to move the counterweight tube if the cable twisted. A camera was placed on the ceiling to follow the monkeys behavior on a monitor. Finally, a curtain was used to open or close the monkeys view to the laboratory.

2.5. Postoperati6e care and neurologic examinations

On the rst and second postoperative days analgesic was given to the monkeys twice per day (buprenex, 0.01 mg/kg, i.m.). The neurological status of the animals were monitored and documented twice a day for a full week. The neurological monitoring included the examination of physical appearance, overall behavior, eating and drinking habits, motor and sensory functions and coordination. Food and water was available for the animals ad libitum. In the third postoperative week, the

2.7. Single-cell recording

The animal was lightly sedated in its cage with 15 mg/kg ketamine, i.m., and transported to the test chamber. The animal was restrained in a chair for 15 20 min during which the cover of the protective cap was removed. Ethanol and betadine were applied to the interior of the base using sterile rubber gloves. The recording cable was connected to the Mill-Max socket. Operational ampliers were built into this cable to eliminate movement artifacts from the recording. Also, a stainless steel hook was cemented to the cable which was juxtaposed to the hook located on the microelectrode assembly. The two hooks were tightly bound together with a wire, preventing displacement of the cable. The protective tube was attached to the base of the protective cap. The electrode/probe unit was advanced 50 mm increments by rotating the driving screws. The extracellular

Fig. 3. Sagittal radiograph showing the intracranial position of the microelectrode array in monkey No. 4. Note that the electrode was introduced into the brain at an angle to avoid penetration through the basal ganglia and the motor cortex. This technique assured hippocampal microelectrode implantation without causing neurological decits. Also note the correct localization of the electrode tip between the density of the petrous part of the temporal bone and that of the hypophyseal fossa. The diameter of the head of each skull screw is 3.5 mm.

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Fig. 4. Demonstration of single-cell recording in the hippocampus of a freely moving squirrel monkey (monkey No. 3). (A) shows the animal taking a piece of food from the hand of the experimenter. This illustrates that in these conditions the monkey readily cooperates with the investigator. (Glove absent for photograph only.) (B) captures a moment when the animal is climbing on the wall of the test chamber to nd fruit loops in a food port. Note the protective tube (weight = 38 g) around the recording cable, which apparently does not disturb the animals movement in three dimensions. (C) shows the action potentials of a single complex-spike cell, as it was recorded during the experimental session. Note that the amplitude of these discharges (negativity up) is at least four-times higher than that of the background electrical noise. (D) demonstrates that the action potential waveforms could be kept safely for many hours in the freely moving monkey. (E) illustrates the two ring modes of the neuron: bursting mode (asterisk) and single-spike mode. (A videotape of this experiment, with the simultaneously recorded behavioral and cellular events, is available from the corresponding author.)

recordings were made between pairs of microwires. The signals were fed into differential AC ampliers. The cellular discharges were amplied 10 000 times and ltered between 300 and 10 000 Hz. The electrical activity of the neurons was displayed on oscilloscopes. Typically, it took about 1020 min to detect clear, high amplitude action potentials, such as those shown in Fig. 4C. For about 20 additional minutes, the detected

cells were observed to determine the stability of the recording. Then the monkey was released from the chair into the test chamber. Data collection started 4 h after the release of the animal. This assured that the monkey completely recovered from the effects of ketamine sedation. At the dose used, the behavioral and electrophysiological effects of ketamine completely disappear within 12 h (Popilskis

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cleaned again with betadine. The cover of the cap was reattached, sealed with bone wax, and the animal was transported back to the home cage.

2.8. Data analysis

The extracellular recording data were analyzed offline with the CP Analysis software of DataWave Technologies in the same fashion as in our previous rat and monkey studies (Ludvig, 1999; Ludvig et al., 2000). Briey, the raw data were subjected to cluster cutting to discriminate the action potentials of each detected single neuron. The action potential waveforms were overlaid and examined. Such overlaid action potential waveforms are shown in Fig. 4D. In addition, the discriminated spikes were played back to analyze characteristic recording segments (Fig. 4E). Finally, ring rate histograms (Fig. 5) were generated. For this report, single-cell ring data were collected from the third and fourth monkeys, in six recording sessions. A total of 10 well-discriminated neurons were examined.

2.9. X-ray and histological studies

X-ray studies were conducted within 3 weeks after microelectrode implantation, with the use of a Siemens Mobilett machine. The animals were anesthetized with 20 mg/kg ketamine, i.m., for these procedures. Both frontal and sagittal (Fig. 3) images were generated. The histological studies were conducted at the end of the experiments, 34 months after operation. These studies were performed as described (Ludvig et al., 2000), except that this time sagittal brain sections were prepared. Representative areas of the sections containing the microelectrode track were documented with the use of a SONY DKC500 digital imaging system. A printout of one of these digital images with the hippocampal electrode track is shown in Fig. 6.

Fig. 5. The ring frequency of two different hippocampal cell types in freely moving monkeys. (A) ring rate histogram showing the electrical activity of a slow-ring complex- spike cell, as recorded from monkey No. 3. (B) ring rate histogram showing the electrical activity of a fast-ring cell, presumably an interneuron, from the hippocampus of monkey No. 4. Both recordings were started 10 min after the release of the animals into the test chamber. Lights were on; view to the laboratory was open. X-axis: recording time (60 min); Y-axis: ring rate (maximum =30 spikes/s). Flag C indicates the moment when the animal climbed to the wall for the rst time. Note the steady increase of the ring rate of both neurons, due to recovery from ketamine sedation. Also note the characteristic ring of the cell in (A): 3 6 Hz bursts alternate with very low frequency/silent periods.

3. Results and Kohn, 1997; Ludvig, unpublished observations). This is illustrated in Fig. 4A and B. The extracellular signals were collected on a hard disk of a computer with the Discovery data acquisition software of DataWave Technologies (Longmont, CO). Eating, drinking and resting periods, as well as vocalizations (twitters; Newman, 1985) were marked in the DataWave les with manually entered ags. The durations of the recording sessions usually ranged between 2 and 6 h. After data collection, one of the experimenters entered into the test chamber and gently restrained the animal. Under very light ketamine sedation (10 mg/kg, i.m.), the recording cable and the protective tube were disconnected, and the interior of the protective cap was

3.1. Neurological examinations

The motor functions were normal. Neither paresis, paralysis or rigidity was observed in the upper and lower limbs. Tail movements were normal. Neither intention nor rest tremor was displayed by the animals. Hypokinesia, akinesia, bradykinesia or dystonia were not observed. The animals responded to acoustic stimuli in both directions. No abnormal head or eye movements were detected. Indeed, the animals moved in their home cage, climbed and hung on the walls in a manner that was indistinguishable from normal. The sensory functions were also normal: the animals reacted appropriately well to cutaneous stimuli. No

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Fig. 6. Computer-captured micrograph of a sagittal, Nissl-stained section indicating the localization of the microelectrode track (arrow) in monkey No. 4. Calibration bar as indicated. Note the correct localization of the electrode tip in the CA1 region of the hippocampus (Hip). The dentate gyrus is located medial to this sagittal plane and is therefore not seen. Recordings were made while the microelectrode array was gradually advanced 3 mm from the original implantation site (19.5 mm below brain surface) to the end-point indicated in the micrograph (arrow). GL: corpus geniculatum laterale.

hypesthesia or anesthesia was found in the upper or lower limbs. Upon presentation of food from various horizontal angles, the monkeys reached for food equally to the left and right: hemineglect was not observed. Ataxia, convulsions, gait or stance abnormalities did not develop. Overall, the animals looked healthy, with no signs of infection, bleeding or abrasions on the skin.

3.2. Beha6ioral obser6ations

During behavioral training, the animals learned within a few hours to move around in the test chamber, remove fruit loops from the food ports and drink from the water bottle. They mostly stayed on the bars and moved around, but occasionally climbed down and explored the oor of the chamber. For short periods, they rested on the oor, but long sleeping periods were not observed. From their vocalizations (twitters) and overall behavior (e.g. their careful exploration of the laboratory setting) we concluded that the animals were comfortable in the test chamber. In fact, this chamber was larger than their individual cage and exposed the monkeys to an enriched, novel environment. After releasing the monkeys from the primate chair to the test chamber, there was a 20 40 min period characterized by ataxic movements and disoriented behavior. This was the consequence of the ketamine sedation. During this period, however, the ring of the cells continued and could be detected without difculty (Fig. 5). Within 1 h after drug injection, the monkeys started

to climb on the bars of the chamber. In the next hour, their behavior was completely normalized. This may be illustrated in photographs Fig. 4A and B which were taken 3 hours after ketamine injection. Our major concerns were that while in the test chamber and connected to the recording apparatus the monkeys may grab the recording cable, jump to the ceiling, or be unable to move normally with the cable-protective tube on their head. However, none of these behaviors occurred, and the monkeys movement pattern was indistinguishable from that before the operation. The eating and drinking habits of the animals were also undisturbed by the ongoing recordings. Aggressive behavior was not observed. In fact, the animals readily cooperated with the experimenter (Fig. 4A). At the end of the recording sessions, the monkeys were gently restrained allowing them to be disconnected from the recording setup. This took less than a minute using light sedation with ketamine. In between the recording sessions, no changes were observed in the animals behavior. In the second postoperative month the food consumption of monkey No. 3 decreased and within 2 weeks the animal died. Autopsy and histological studies showed no evidence of infection, abscess, extensive necrosis or intracerebral hemorrhage in the vicinity of the electrode. However, an examination of the ve-year Animal Care and Use Record of this monkey revealed that her tail was chewed off a few months after her birth. This trauma may explain her vulnerability. We have not seen any such behavioral abnormality in any of our previously implanted squirrel monkeys (Ludvig et al., 2000). Indeed, in the present study, monkeys Nos. 1 and 4 displayed perfectly normal behavior well over 4 months, until they were sacriced for the histological studies.

3.3. Single-cell recordings

Clear action potentials with amplitude at least threetimes higher than the 5070 mV background noise (Fig. 4C) were recorded after 610, one-quarter turns on the driving screws. These action potentials were remarkably stable: we found no difculties in safely recording the cells for many hours (Fig. 4D). Slow-ring complexspike cells (n=7), similar to those that dominate the recordings in the rat hippocampus, were detected. However, complex spikes were less prevalent than in rats, even when the monkeys rested on the oor. For the slow-ring cells, the most characteristic ring pattern was the alternation of single spikes and occasional bursts (Fig. 4E). The average ring rate of these cells varied between 0.1 and 1.8 Hz, with the rate of their bursts ranging from 2.5 to 25.8 Hz. A thorough analysis of the behavioral and spatial correlates of these bursts was not the objective of this methodological study. In addition to the slow-ring cells, fast-ring

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neurons (n= 3) were also detected. The average ring rate of these cells varied between 9.2 and 35.0 Hz. These neurons red rather evenly throughout the recording sessions, without displaying bursts. Fig. 5 demonstrates the different ring pattern of a slow ring and a fast-ring cell. Perhaps the most important observation was that after releasing the monkeys into the test chamber, the cells were not lost, that is, their action potential waveforms could be clearly detected for long periods despite the movement of the animals in the three-dimensional space of the chamber. Furthermore, although the initial ketamine injection suppressed the ring of the neurons (Fig. 5), the ring pattern of the cells returned to normal within 1 h. Therefore, after this period, the ring of the cells in a variety of natural behaviors (drinking, eating, exploration, etc.) could readily be studied. Finally, the quality of recordings in the monkey recording setup (Fig. 2) was similar to what can be achieved in any well-tested apparatus for rats (Ludvig et al., 1996; Ludvig, 1999).

moving animals. Furthermore, during daytime squirrel monkeys have a natural tendency to climb around in their cage for many hours. This is also advantageous for studies in freely moving subjects. In addition, the cerebrovascular pulsation in these animals is quite moderate. This greatly contributes to the successful conduct of long-term extracellular recordings.

4.2. De6eloping a proper electrode-implantation technique

Since even a slight motor decit affects the experiment in a freely moving monkey, it is critical to implant the microelectrodes without damaging the motor cortex or the basal ganglia. To avoid these structures, we introduced the microelectrode array into the hippocampus at an angle. We also reduced the risk of peridural hemorrhage. Tightly sealing the craniotomy and rmly anchoring the microelectrode/protective cap assembly to the skull diminished the potential for intracranial infection.

4.3. Using microwire electrodes for recording

4. Discussion This study demonstrated for the rst time that it is possible to record the ring of single neurons in the brain of monkeys while the animals are moving freely in a three-dimensional space. The described method has several advantages over the traditional techniques employing chaired monkeys. First, since the animals are comfortable in the test chamber, long-term neuronal monitoring can be readily performed. This is important for studying the plastic ring pattern changes that occur over time in primate CNS neurons. Second, since the animals are not restrained, the confounding neural and endocrine effects of immobilization-related stress are eliminated. For examining the cellular mechanisms of cognition and emotion in primates, this is especially valuable. Third, since the animals are allowed to move freely in a relatively large three-dimensional space, the cellular electrophysiological correlates of a wide range of natural behaviors, including exploration and social interactions, can be studied. In fact, for clarifying the neural mechanisms underlying spatial memory/cognitive map formation in the primate temporal lobe memory system (Squire and Zola, 1996; Fried et al., 1997), the present method offers a very useful model. The following factors proved to be critical in our experiments. The advantages of these electrodes for electrophysiological studies in monkeys were recognized in our previous study (Ludvig et al., 2000). The present experiments conrmed this observation. We are now convinced that a driveable array of microwires, protected by a removable cap, is an ideal device for long-term neuronal recording from the brain of freely moving monkeys.

4.4. Designing a special recording cable

It was necessary to protect a segment of the recording cable, just above the microelectrode assembly. Also, we found that the cable should be rigid and straight to be kept out of the sight of the monkey. Furthermore, it was important to place the recording cables counterweight into a tube to prevent its swinging when the animal moved quickly over a large distance. Finally, it was necessary to position a horizontal rod between the recording cable and the counterweight tube to prevent cable twisting and keep the tube out of the monkeys reach.

4.5. Constructing a special test chamber

It was essential to have a high, smooth wall above the monkeys climbing space. In this way the monkeys do not attempt to jump toward the ceiling which might damage the commutator and the camera. Other components of the chamber design, such as the food ports and the curtain, provided an enriched environment for the monkeys. This was important to keep the animals awake and moving for long periods.

4.1. Selecting squirrel monkeys for the studies

In contrast to macaques and many other primate species, squirrel monkeys are easy to handle. This is important for any electrophysiological study in freely

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Ultimately, wireless radiotelemetric single-cell recording (Nieder, 2000) might replace our described method. At present, however, the recording cable allows the collection of low-noise signals from a much larger neuron population than the use of radiotelemetry. Furthermore, a recording cable can be complemented with additional tubes and wires to perform voltammetry, microdialysis, intracerebral drug administration and many other procedures simultaneously with the electrophysiological recording. Certainly, one day these procedures will also be replaced by portable, remotely controlled devices. Yet, for the foreseeable future, the technique presented in this report offers a viable approach to examine neuronal ring in the brain of freely moving non-human primates. As such, the method has the potential to signicantly contribute to the determination of the cellular electrophysiological mechanisms that are unique to the cognitive, emotional and communicational systems of the primate brain.

Acknowledgements We are grateful to Dr Steven E. Fox for his encouragements and to Dr Ivan Bodis-Wollner for his guidance for the neurological examinations. The technical assistance of Nancy Loporcaro, Mark Shkop and Johnny Richardson are greatly appreciated. This project was supported by NIH Grant MH56800 and a Research investment Initiative Grant from SUNY HSCB to Nandor Ludvig.

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