single-cell recording from the brain of freely moving monkeys

Journal of Neuroscience Methods 106 (2001) 179–187

Single-cell recording from the brain of freely moving monkeys

Nandor Ludvig *, Juan M. Botero, Hai M. Tang, Baiju Gohil, John G. KralDepartments of Physiology and Pharmacology, Anesthesiology, and Surgery, State Uni�ersity of New York, Health Science Center at Brooklyn,

450 Clarkson A�enue, 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 primatechair. However, this arrangement makes long-term recordings difficult, causes stress that may confound the data, and prevents themanifestation of natural behaviors. Extending our previous neurophysiological studies in non-human primates (Ludvig et al. BrainRes. Protocols 2000;5:75–85), we have developed a method for recording the electrical activity of single hippocampal neurons infreely moving squirrel monkeys (Saimiri sciureus). The recording sessions lasted for up to 6 h, during which the monkeys movedfreely around on the walls and the floor of a large test chamber and collected food pellets. Stable action potential waveforms werereadily kept throughout the sessions. The following factors proved to be critical in this study: (a) selecting squirrel monkeys forthe experiments, (b) using a driveable bundle of microwires for the recordings, (c) using a special recording cable, (d) implantingthe microwires into the brain without causing neurological deficits, and (e) running the recording sessions in a special testchamber. The described method allows long-term extracellular recordings from the brain of non-human primates, without thestress of chairing, during a wide range of natural behaviors. Using this model, new insights can be obtained into the unique firingrepertoire of the neurons of the primate brain. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Squirrel monkey; Free behavior; Hippocampus; Single-cell recording

www.elsevier.com/locate/jneumeth

1. Introduction

Our concepts on the biological laws that govern thefiring of neurons of the human central nervous system(CNS) are based on: (1) an enormous amount of dataobtained in invertebrates, rodents and other sub-pri-mate species, (2) on significantly less information col-lected from non-human primates, such as monkeys, and(3) on a very limited number of studies in patients withneurological disorders. The data obtained in sub-pri-mate species provide invaluable guidance for under-standing the basic electrophysiology of human braincells. However, they cannot shed light on the cellularmechanisms that are unique to the human brain. Thestudies in neurological patients generate critically im-portant information. However, this information is oftenextracted from abnormally functioning neurons, duringa very restricted set of behaviors. In this situation,

mapping the firing properties of neurons of the monkeybrain is especially useful. Unlike CNS neuronal record-ings from humans, those from monkeys can be per-formed in normal brain tissue and in a wider set ofbehaviors. At the same time, the cellular electrophysio-logical experiments in monkeys can yield data that aremore relevant to the human brain than those obtainedin sub-primate species.

Traditionally, recording of single neurons from thebrain of monkeys has been performed in head-re-strained animals seated in a primate chair. This methodhas been used regardless of whether the recordings weremade from the thalamus (Wiesel and Hubel, 1966), thecerebellum (Evarts, 1968), the neocortex (Kojima andGoldman-Rakic, 1982), the hippocampus (Ono et al.,1993), the locus coeruleus (Aston-Jones et al., 1997) orother structures. However, with this technique long-term neuronal recordings are difficult to obtain. Fur-thermore, these conditions are stressful for themonkeys, which may confound the generated data.Finally, this experimental arrangement does not permitthe manifestation of many natural behaviors. To im-

* Corresponding author. Tel.: +1-718-2701796; fax: +1-718-2703103.

E-mail address: [email protected] (N. Ludvig).

0165-0270/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 0270 (01 )00348 -X

N. Lud�ig et al. / Journal of Neuroscience Methods 106 (2001) 179–187180

prove this methodology, several investigators recentlyrecorded hippocampal neurons in monkeys seated in amoveable chair or cab (Rolls, 1999; Ono and Nishijo,1999). However, to date no single-cell recording hasbeen conducted from the brain of monkeys while theanimals are moving freely in a three-dimensional space.

Microwire electrodes have been proved to yield stableand long-term extracellular recordings from thehippocampus of freely moving rats (Kubie, 1984;Pavlides and Winson, 1989; Ludvig et al., 1994, 1996;Kentros et al., 1998; Ludvig, 1999), as well as inrestrained monkeys (Aston-Jones et al., 1997; Nicoleliset al., 1998). Recently, we adapted this recording tech-nique to squirrel monkeys seated in a traditional pri-mate chair (Ludvig et al., 2000). We found that themicrowires allowed the monitoring of single-cell firingfor many hours, even without the use of head-restraint.Encouraged by these results, we devoted this study todetermine whether it is possible to record single-cellfiring from the brain of monkeys which are releasedfrom the primate chair and move freely on the wallsand floor of a large test chamber. We focused ourexperiments on hippocampal neurons, because free andvoluntary movement is especially important for theactivation of these cells (Foster et al., 1989; Ludvig,1999; O’Keefe, 1999).

2. Methods

2.1. Animals

Four squirrel monkeys (Saimiri sciureus ; SuborderAnthropoidea) were used in this study. They were takenfrom the monkey colony kept at the SUNY HealthScience Center at Brooklyn in a facility approved bythe American Association for Accreditation of Labora-tory Animal Care. Monkey No. 1 was male, 780 g and11 years old; monkey No. 2 was female, 500 g and 12years old. These animals were used for pilot studies todetermine the optimal surgical technique and observetheir behavior. Monkey No. 3 was female, 750 g and 5years old; monkey No. 4 was male, 850 g and 22 yearsold. The data presented in the Results section wereobtained in these latter animals. This study was con-ducted according to the Guide for the Care and Use ofLaboratory Animals (National Academy Press, Wash-ington, DC, 1996) and was approved by the Institu-tional Animal Care and Use Committee at SUNYHealth Science Center at Brooklyn.

2.2. Microelectrode assembly

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

provide a concise description only. The assembly (Fig.1) contained a 12-pin dual row profile Mill-Max con-nector (Mill-Max, Oyster Bay, NY), to which threedriving screws were glued. The microelectrode arrayitself comprised 11 Stablohm 675 H- Formvar-coatednichrome wires (California Fine Wire, Grover Beach,CA), 25 �m diameter each. At one end they weresecured to the pins of the Mill-Max connector with GCSilver Print conductive paint (Newark Electronics,Newark, NJ), whereas at the other (recording) end theyprotruded 4 mm from the tube. A 3 mm wire segmentproximal to the tube was covered with epoxy to providestiffness to the electrode array. In addition to thenichrome wires, a larger grounding wire was connectedwith conductive paint to the 12th pin of the Mill-Maxsocket. Within this assembly, the tip of the microelec-trode array was 44 mm below the surface of the Mill-Max connector. This arrangement allowed theplacement of the electrode tip 19.5 mm below the brainsurface, while the bottom of the driving screws was still1–2 mm above the skull. The driving screws werecalibrated to advance the whole assembly 5 mm down-ward. This was enough to move the electrodes throughall layers of the hippocampus. (We note that in our

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

N. Lud�ig et al. / Journal of Neuroscience Methods 106 (2001) 179–187 181

Fig. 2. The special test chamber. 1: Bars to allow the monkey to climbon the walls. 2: Smooth wall to prevent the monkey from movingabove the bars. 3: Food port. 4: Water bottle. 5: Straight and rigidrecording cable, invisible for the monkey in any position. 6: Protec-tive tube to prevent the monkey from grabbing the cable. 7: Pulleyassembly rotating together with the commutator. 8: Commutator. 9:Tube to prevent the cable’s counterweight from swinging. 10: Hori-zontal rod to prevent cable-twisting. 11: Camera, 280 cm above thefloor. 12: Curtain to regulate the monkey’s view to the laboratory.Each of these elements proved to be necessary for running thesingle-cell recording studies in freely moving squirrel monkeys forlong periods.

operation was performed until the animal was habitu-ated to the new environment and spontaneously movedbetween food ports.

2.4. Microelectrode implantation

All surgical tools and the stereotaxic apparatus wereautoclaved. After the injection of atropine (0.05 mg/kg,i.m.) and bicillin (100 000 units/kg, i.m.), anesthesia wasinduced with a mixture of ketamine (11 mg/kg, i.m.)and xylazine (0.5 mg/kg, i.m.). The animal wasweighed, the head and extremities were shaved, and thenon-invasive ECG, blood pressure, SpO2 and ECGmonitors were placed. A second injection of ketaminealone (11 mg/kg, i.m.) was given during these proce-dures. By a facemask the animal was preoxygenatedwith 100% O2, and the anesthesia was deepened with2.5% halothane for 5 min. Laryngoscopy was per-formed with 0.1 ml of 2% lidocaine sprayed onto thebase of the vocal folds before endotracheal intubation.Anesthesia was maintained with 1.2–2% Isoflurane inoxygen using a Narkomed Compact system. The tailvein was cannulated for fluid (lactated Ringer) adminis-tration. The tail of monkey No. 3 was injured prior tothis study, therefore in this animal we used the femoralvein for cannulation. A Propaq 106 system served tomonitor blood pressure, ECG, SpO2, respiratory rate,end tidal CO2, as well as rectal temperature. The animalwas kept normothermic with a warming blanket andwas placed in the stereotaxic frame. After positioningthe head, the skull was exposed, and a 3 mm diametercraniotomy was made with a dental drill. The coordi-nates of the center of the hole on the skull were: 0.2mm anterior to the line between the ear bars, and 9 mmright to the midline, according to the atlas of Gergenand MacLean (1962). Three anchoring screws wereplaced in the bone around the craniotomy. Betadinewas extensively applied to the operated area. Next, thedura mater and the pia mater were excised from thebrain surface. This was accompanied with minor bleed-ing in two animals. In these cases, we prevented theperidural diffusion of blood by draining it out of thecraniotomy. The microelectrode assembly was sterilizedwith alcohol and betadine, and its electrode unit wasintroduced into the brain at an 8° angle through thecenter of the hole (Fig. 3). The tip of the unit waspositioned to be 19.5 mm below the brain surface. Thegrounding wire of the assembly was connected to oneof the screws with conductive paint. The paint wasallowed to dry for 45 min. The assembly was secured tothe skull and the screws with dental cement. A plasticring with sterile bone wax and Panalog ointment on itssurface sealed the craniotomy. Next, the base of aprotective cap was positioned around the assembly, andsecured to the skull with interior and exterior screwsand dental cement, as described (Ludvig et al., 2000).

design the microelectrode assembly also incorporates amicrodialysis probe-guide. In fact, after completing thepresent recording studies, we performed simultaneoussingle-cell recording and microdialysis in the monkeys.The results of these experiments have been compiled fora separate report.)

2.3. Preoperati�e protocol

Three weeks before microelectrode implantation, themonkeys were taken from their social group and placedin individual cages (60 cm L×80 cm H×60 cm W).However, the cages were close to each other so that themonkeys could interact. Purina monkey chow and wa-ter was available for the animals ad libitum. In addi-tion, banana was given to them three times per week. Inthe last preoperative week, the monkeys were trained (1h/day) to move around in the test chamber (Fig. 2) andcollect food pellets (fruit loops) from 16 food ports.These food ports were 3 cm diameter and 8 cm longplastic tubes secured to the walls of the chamber. No


The removable cover of the protective cap was thenattached to the base. The skin was approximated with aone-layer interrupted horizontal mattress closure usinga 3-0 nylon suture. Anesthesia was reversed, and theanimal was extubated before being returned to thehome cage. Monkey No. 2 died 16 h after surgery.Because of her low body weight of 500 g we suspectthat she may have been chronically ill. This is consis-tent with the experience of Gergen and MacLean (1962)with very low weight squirrel monkeys. The rest of themonkeys (n=3) vigorously climbed on the walls oftheir home cage and consumed food and water nor-mally within 3 h after operation.

2.5. Postoperati�e care and neurologic examinations

On the first and second postoperative days analgesicwas given to the monkeys twice per day (buprenex, 0.01mg/kg, i.m.). The neurological status of the animalswere monitored and documented twice a day for a fullweek. The neurological monitoring included the exami-nation of physical appearance, overall behavior, eatingand drinking habits, motor and sensory functions andcoordination. Food and water was available for theanimals ad libitum. In the third postoperative week, the

animals were placed in the test chamber and theirbehavior was observed. After 3 weeks of recovery thedecision to proceed with recording was made if theanimals collected food pellets in the same way as beforeoperation.

2.6. Special test chamber

A 150 cm long, 150 cm wide and 280 cm highwooden chamber (Fig. 2) was built for this study. Thewalls of this chamber were composed of bars to aheight of 120 cm, while above this line the walls wereconstructed of smooth plywood. This design was madeto prevent the monkeys from moving or jumping abovea defined area. Four food ports were placed on eachwall, with a water bottle secured to one corner. Therecording cable (G-Tech, Cortland Manor, NY) wasconnected to a commutator (Crist Instruments, Hager-stown, MD) placed on the ceiling of the chamber. A 3mm diameter and 135 cm long plastic rod was tapedalong the lower portion of the cable to make it stiff andinvisible to the monkey at any position. A 3.5 cmdiameter and 15 cm long plastic tube was attached tothe bottom of the cable (Fig. 4A and B) to prevent themonkey from grabbing and damaging the cable. Theweight of the cable was counterbalanced by a 13 mmdiameter metal cylinder sliding in a 150 cm long plastictube. This prevented the counterweight from swinging.The monkey could not reach the counterweight tubebecause this latter was at a 80 cm horizontal distancefrom the recording cable. A lightweight horizontal rod,made of stainless steel, was designed to move thecounterweight tube if the cable twisted. A camera wasplaced on the ceiling to follow the monkey’s behavioron a monitor. Finally, a curtain was used to open orclose the monkey’s view to the laboratory.

2.7. Single-cell recording

The animal was lightly sedated in its cage with 15mg/kg ketamine, i.m., and transported to the test cham-ber. The animal was restrained in a chair for 15–20 minduring which the cover of the protective cap was re-moved. Ethanol and betadine were applied to the inte-rior of the base using sterile rubber gloves. Therecording cable was connected to the Mill-Max socket.Operational amplifiers were built into this cable toeliminate movement artifacts from the recording. Also,a stainless steel hook was cemented to the cable whichwas juxtaposed to the hook located on the microelec-trode assembly. The two hooks were tightly boundtogether with a wire, preventing displacement of thecable. The protective tube was attached to the base ofthe protective cap.

The electrode/probe unit was advanced 50 �m incre-ments by rotating the driving screws. The extracellular

Fig. 3. Sagittal radiograph showing the intracranial position of themicroelectrode array in monkey No. 4. Note that the electrode wasintroduced into the brain at an angle to avoid penetration throughthe basal ganglia and the motor cortex. This technique assuredhippocampal microelectrode implantation without causing neurologi-cal deficits. Also note the correct localization of the electrode tipbetween the density of the petrous part of the temporal bone and thatof the hypophyseal fossa. The diameter of the head of each skullscrew is 3.5 mm.


Fig. 4. Demonstration of single-cell recording in the hippocampus of a freely moving squirrel monkey (monkey No. 3). (A) shows the animaltaking a piece of food from the hand of the experimenter. This illustrates that in these conditions the monkey readily cooperates with theinvestigator. (Glove absent for photograph only.) (B) captures a moment when the animal is climbing on the wall of the test chamber to find fruitloops in a food port. Note the protective tube (weight=38 g) around the recording cable, which apparently does not disturb the animal’smovement 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 firingmodes of the neuron: bursting mode (asterisk) and single-spike mode. (A videotape of this experiment, with the simultaneously recordedbehavioral and cellular events, is available from the corresponding author.)

recordings were made between pairs of microwires. Thesignals were fed into differential AC amplifiers. Thecellular discharges were amplified 10 000 times andfiltered between 300 and 10 000 Hz. The electrical activ-ity of the neurons was displayed on oscilloscopes. Typ-ically, it took about 10–20 min to detect clear, highamplitude action potentials, such as those shown inFig. 4C. For about 20 additional minutes, the detected

cells were observed to determine the stability of therecording. Then the monkey was released from thechair into the test chamber.

Data collection started 4 h after the release of theanimal. This assured that the monkey completely recov-ered from the effects of ketamine sedation. At the doseused, the behavioral and electrophysiological effects ofketamine completely disappear within 1–2 h (Popilskis


Fig. 5. The firing frequency of two different hippocampal cell types infreely moving monkeys. (A) firing rate histogram showing the electri-cal activity of a slow-firing complex- spike cell, as recorded frommonkey No. 3. (B) firing rate histogram showing the electrical activityof a fast-firing cell, presumably an interneuron, from the hippocam-pus of monkey No. 4. Both recordings were started 10 min after therelease of the animals into the test chamber. Lights were on; view tothe laboratory was open. X-axis: recording time (60 min); Y-axis:firing rate (maximum=30 spikes/s). Flag C indicates the momentwhen the animal climbed to the wall for the first time. Note thesteady increase of the firing rate of both neurons, due to recoveryfrom ketamine sedation. Also note the characteristic firing of the cellin (A): 3–6 Hz bursts alternate with very low frequency/silent peri-ods.

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

2.8. Data analysis

The extracellular recording data were analyzed off-line with the CP Analysis software of DataWave Tech-nologies in the same fashion as in our previous rat andmonkey studies (Ludvig, 1999; Ludvig et al., 2000).Briefly, the raw data were subjected to cluster cutting todiscriminate the action potentials of each detected sin-gle neuron. The action potential waveforms were over-laid and examined. Such overlaid action potentialwaveforms are shown in Fig. 4D. In addition, thediscriminated spikes were played back to analyze char-acteristic recording segments (Fig. 4E). Finally, firingrate histograms (Fig. 5) were generated. For this report,single-cell firing data were collected from the third andfourth monkeys, in six recording sessions. A total of 10well-discriminated neurons were examined.

2.9. X-ray and histological studies

X-ray studies were conducted within 3 weeks aftermicroelectrode implantation, with the use of a SiemensMobilett machine. The animals were anesthetized with20 mg/kg ketamine, i.m., for these procedures. Bothfrontal and sagittal (Fig. 3) images were generated. Thehistological studies were conducted at the end of theexperiments, 3–4 months after operation. These studieswere performed as described (Ludvig et al., 2000),except that this time sagittal brain sections were pre-pared. Representative areas of the sections containingthe microelectrode track were documented with the useof a SONY DKC500 digital imaging system. A printoutof one of these digital images with the hippocampalelectrode track is shown in Fig. 6.

3. Results

3.1. Neurological examinations

The motor functions were normal. Neither paresis,paralysis or rigidity was observed in the upper andlower limbs. Tail movements were normal. Neitherintention nor rest tremor was displayed by the animals.Hypokinesia, akinesia, bradykinesia or dystonia werenot observed. The animals responded to acoustic stim-uli in both directions. No abnormal head or eye move-ments were detected. Indeed, the animals moved intheir home cage, climbed and hung on the walls in amanner that was indistinguishable from normal.

The sensory functions were also normal: the animalsreacted appropriately well to cutaneous stimuli. No

and Kohn, 1997; Ludvig, unpublished observations).This is illustrated in Fig. 4A and B. The extracellularsignals were collected on a hard disk of a computerwith the Discovery data acquisition software of Dat-aWave Technologies (Longmont, CO). Eating, drinkingand resting periods, as well as vocalizations (‘twitters’;Newman, 1985) were marked in the DataWave fileswith manually entered flags. The durations of therecording sessions usually ranged between 2 and 6 h.

After data collection, one of the experimenters en-tered into the test chamber and gently restrained theanimal. Under very light ketamine sedation (10 mg/kg,i.m.), the recording cable and the protective tube weredisconnected, and the interior of the protective cap was


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

to climb on the bars of the chamber. In the next hour,their behavior was completely normalized. This may beillustrated in photographs Fig. 4A and B which weretaken 3 hours after ketamine injection.

Our major concerns were that while in the test cham-ber and connected to the recording apparatus the mon-keys may grab the recording cable, jump to the ceiling,or be unable to move normally with the cable-protec-tive tube on their head. However, none of these behav-iors occurred, and the monkeys’ movement pattern wasindistinguishable from that before the operation. Theeating and drinking habits of the animals were alsoundisturbed by the ongoing recordings. Aggressive be-havior was not observed. In fact, the animals readilycooperated with the experimenter (Fig. 4A).

At the end of the recording sessions, the monkeyswere gently restrained allowing them to be disconnectedfrom the recording setup. This took less than a minuteusing light sedation with ketamine. In between therecording sessions, no changes were observed in theanimals’ behavior. In the second postoperative monththe food consumption of monkey No. 3 decreased andwithin 2 weeks the animal died. Autopsy and histologi-cal studies showed no evidence of infection, abscess,extensive necrosis or intracerebral hemorrhage in thevicinity of the electrode. However, an examination ofthe five-year Animal Care and Use Record of thismonkey revealed that her tail was chewed off a fewmonths after her birth. This trauma may explain hervulnerability. We have not seen any such behavioralabnormality in any of our previously implanted squirrelmonkeys (Ludvig et al., 2000). Indeed, in the presentstudy, monkeys Nos. 1 and 4 displayed perfectly nor-mal behavior well over 4 months, until they weresacrificed for the histological studies.

3.3. Single-cell recordings

Clear action potentials with amplitude at least three-times higher than the 50–70 �V background noise (Fig.4C) were recorded after 6–10, one-quarter turns on thedriving screws. These action potentials were remarkablystable: we found no difficulties in safely recording thecells for many hours (Fig. 4D). Slow-firing complex-spike cells (n=7), similar to those that dominate therecordings in the rat hippocampus, were detected. How-ever, complex spikes were less prevalent than in rats,even when the monkeys rested on the floor. For theslow-firing cells, the most characteristic firing patternwas the alternation of single spikes and occasionalbursts (Fig. 4E). The average firing rate of these cellsvaried between 0.1 and 1.8 Hz, with the rate of theirbursts ranging from 2.5 to 25.8 Hz. A thorough analy-sis of the behavioral and spatial correlates of thesebursts was not the objective of this methodologicalstudy. In addition to the slow-firing cells, fast-firing

hypesthesia or anesthesia was found in the upper orlower limbs. Upon presentation of food from varioushorizontal angles, the monkeys reached for foodequally to the left and right: hemineglect was notobserved.

Ataxia, convulsions, gait or stance abnormalities didnot develop. Overall, the animals looked healthy, withno signs of infection, bleeding or abrasions on the skin.

3.2. Beha�ioral obser�ations

During behavioral training, the animals learnedwithin a few hours to move around in the test chamber,remove fruit loops from the food ports and drink fromthe water bottle. They mostly stayed on the bars andmoved around, but occasionally climbed down andexplored the floor of the chamber. For short periods,they rested on the floor, but long sleeping periods werenot observed. From their vocalizations (‘twitters’) andoverall behavior (e.g. their careful exploration of thelaboratory setting) we concluded that the animals werecomfortable in the test chamber. In fact, this chamberwas larger than their individual cage and exposed themonkeys to an enriched, novel environment.

After releasing the monkeys from the primate chairto the test chamber, there was a 20–40 min periodcharacterized by ataxic movements and disoriented be-havior. This was the consequence of the ketamine seda-tion. During this period, however, the firing of the cellscontinued and could be detected without difficulty (Fig.5). Within 1 h after drug injection, the monkeys started


neurons (n=3) were also detected. The average firingrate of these cells varied between 9.2 and 35.0 Hz.These neurons fired rather evenly throughout therecording sessions, without displaying bursts. Fig. 5demonstrates the different firing pattern of a slow firingand a fast-firing cell.

Perhaps the most important observation was thatafter releasing the monkeys into the test chamber, thecells were not ‘lost’, that is, their action potentialwaveforms could be clearly detected for long periodsdespite the movement of the animals in the three-di-mensional space of the chamber. Furthermore, al-though the initial ketamine injection suppressed thefiring of the neurons (Fig. 5), the firing pattern of thecells returned to normal within 1 h. Therefore, after thisperiod, the firing of the cells in a variety of naturalbehaviors (drinking, eating, exploration, etc.) couldreadily be studied. Finally, the quality of recordings inthe monkey recording setup (Fig. 2) was similar to whatcan be achieved in any well-tested apparatus for rats(Ludvig et al., 1996; Ludvig, 1999).

4. Discussion

This study demonstrated for the first time that it ispossible to record the firing of single neurons in thebrain of monkeys while the animals are moving freelyin a three-dimensional space. The described method hasseveral advantages over the traditional techniques em-ploying chaired monkeys. First, since the animals arecomfortable in the test chamber, long-term neuronalmonitoring can be readily performed. This is importantfor studying the plastic firing pattern changes thatoccur over time in primate CNS neurons. Second, sincethe animals are not restrained, the confounding neuraland endocrine effects of immobilization-related stressare eliminated. For examining the cellular mechanismsof cognition and emotion in primates, this is especiallyvaluable. Third, since the animals are allowed to movefreely in a relatively large three-dimensional space, thecellular electrophysiological correlates of a wide rangeof natural behaviors, including exploration and socialinteractions, can be studied. In fact, for clarifying theneural mechanisms underlying spatial memory/cogni-tive map formation in the primate temporal lobe mem-ory 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 ourexperiments.

4.1. Selecting squirrel monkeys for the studies

In contrast to macaques and many other primatespecies, squirrel monkeys are easy to handle. This isimportant for any electrophysiological study in freely

moving animals. Furthermore, during daytime squirrelmonkeys have a natural tendency to climb around intheir cage for many hours. This is also advantageousfor studies in freely moving subjects. In addition, thecerebrovascular pulsation in these animals is quite mod-erate. This greatly contributes to the successful conductof long-term extracellular recordings.

4.2. De�eloping a proper electrode-implantationtechnique

Since even a slight motor deficit affects the experi-ment in a freely moving monkey, it is critical to implantthe microelectrodes without damaging the motor cortexor the basal ganglia. To avoid these structures, weintroduced the microelectrode array into the hippocam-pus at an angle. We also reduced the risk of periduralhemorrhage. Tightly sealing the craniotomy and firmlyanchoring the microelectrode/protective cap assemblyto the skull diminished the potential for intracranialinfection.

4.3. Using microwire electrodes for recording

The advantages of these electrodes for electrophysio-logical studies in monkeys were recognized in our previ-ous study (Ludvig et al., 2000). The present experimentsconfirmed this observation. We are now convinced thata driveable array of microwires, protected by a remov-able cap, is an ideal device for long-term neuronalrecording from the brain of freely moving monkeys.

4.4. Designing a special recording cable

It was necessary to protect a segment of the record-ing cable, just above the microelectrode assembly. Also,we found that the cable should be rigid and straight tobe kept out of the sight of the monkey. Furthermore, itwas important to place the recording cable’s counter-weight into a tube to prevent its swinging when theanimal moved quickly over a large distance. Finally, itwas necessary to position a horizontal rod between therecording cable and the counterweight tube to preventcable twisting and keep the tube out of the monkey’sreach.

4.5. Constructing a special test chamber

It was essential to have a high, smooth wall abovethe monkeys’ climbing space. In this way the monkeysdo not attempt to jump toward the ceiling which mightdamage the commutator and the camera. Other compo-nents of the chamber design, such as the food ports andthe curtain, provided an enriched environment for themonkeys. This was important to keep the animalsawake and moving for long periods.


Ultimately, wireless radiotelemetric single-cell record-ing (Nieder, 2000) might replace our described method.At present, however, the recording cable allows thecollection of low-noise signals from a much largerneuron population than the use of radiotelemetry. Fur-thermore, a recording cable can be complemented withadditional tubes and wires to perform voltammetry,microdialysis, intracerebral drug administration andmany other procedures simultaneously with the electro-physiological recording. Certainly, one day these proce-dures will also be replaced by portable, remotelycontrolled devices. Yet, for the foreseeable future, thetechnique presented in this report offers a viable ap-proach to examine neuronal firing in the brain of freelymoving non-human primates. As such, the method hasthe potential to significantly contribute to the determi-nation of the cellular electrophysiological mechanismsthat are unique to the cognitive, emotional and commu-nicational systems of the primate brain.

Acknowledgements

We are grateful to Dr Steven E. Fox for his encour-agements and to Dr Ivan Bodis-Wollner for his guid-ance for the neurological examinations. The technicalassistance of Nancy Loporcaro, Mark Shkop andJohnny Richardson are greatly appreciated. This pro-ject was supported by NIH Grant MH56800 and aResearch investment Initiative Grant from SUNYHSCB to Nandor Ludvig.

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