American Journal of Primatology 77:200–210 (2015)
RESEARCH ARTICLE Sleep in the Spider Monkey (Ateles geoffroyi): A Semi‐Restrictive, Non‐Invasive, Polysomnographic Study MANUEL ALEJANDRO CRUZ‐AGUILAR1,2*, FRUCTUOSO AYALA‐GUERRERO3, ANABEL JIMÉNEZ‐ANGUIANO4, ANA MARÍA SANTILLÁN‐DOHERTY5, FRANCISCO GARCÍA‐ORDUÑA6, AND JAVIER VELÁZQUEZ‐MOCTEZUMA4 1 Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 2 Laboratorio de Sueño, Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Distrito Federal, México 3 Laboratorio de Neurociencias, Facultad de Psicología, Universidad Nacional Autónoma de México, Distrito Federal, México 4 Área de Neurociencias, Departamento Biología de la Reproducción, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 5 Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Distrito Federal, México 6 Instituto de Neuroetologia, Universidad Veracruzana, Xalapa, Veracruz, México
The normal sleep patterns of the spider monkey (Ateles geoffroyi) have not been described yet. The objective of this study was to characterize the electrophysiological patterns, sleeping postures, and sleep‐ wake cycle in semi‐restricted spider monkeys. Continuous 24‐hr polysomnographic (PSG) recordings, involving simultaneous recording of non‐invasive electroencephalographic (EEG), electro‐oculographic (EOG), and electromyographic (EMG) activities, were carried out in captive monkeys living in outdoor rainforest enclosures. Electrode placement was done according to the human international 10–20 system. Specific behaviors displayed by monkeys during the sleep‐wake cycles were correlated with the PSG recordings. The nycthemeral distribution of the sleep‐wake cycle was also calculated. The results show that electrophysiological N‐REM sleep patterns in spider monkeys are similar to those observed in other primates, including human beings. Furthermore, a vertical semi‐fetal posture was observed during N‐REM and REM sleep phases. The amount of nocturnal sleep was significantly higher than that of the diurnal period, showing that the spider monkey is a diurnal primate. An outstanding finding was the absence of muscular atonia during the spider monkey’s REM sleep, which suggests that arboreal primates have developed a neuromuscular mechanism specialized for sleeping in a vertical posture. Am. J. Primatol. 77:200–210, 2015. © 2014 Wiley Periodicals, Inc. Key words:
new world monkey; PSG; muscular atonia; REM
INTRODUCTION Sleep seems to be one of the most complex integrated behaviors for which the adaptive advantage remains unknown [Maquet et al., 1997; Mignot, 2008]. Animal and human studies suggest that its functions include, among others, recovery at the cellular, network and endocrine system levels [Mignot, 2008], thermoregulation [Gilbert et al., 2004], adaptation to an ecological niche [Webb, 1974], restoration of tissular integrity [Adams, 1980], and neuronal plasticity [Krueger et al., 1995]. Nevertheless, sleep in non‐human primates is rarely subject to systematic investigation [Anderson, 1998; Fruth & McGrew, 1998]. Most of the previous studies on electrophysiological and behavioral characteristics of the sleep‐wake cycle have been performed in Old World monkeys [Daley et al., 2006; Hsieh et al., 2008]. Nevertheless, from the point of view of adaptive behavior and
© 2014 Wiley Periodicals, Inc.
biological complexity of primates, the sleep analysis of New World monkeys can provide significant insights into sleep evolution in primates. Non‐human primates with an arboreal style life and prehensile tails, for example, can help us understand the anthropoid transition from arboreal to ground sleep through the study of their body postures.
Correspondence to: Manuel Alejandro Cruz Aguilar, Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 09340, E‐mail: [email protected] Received 3 April 2014; revised 5 July 2014; revision accepted 11 July 2014 DOI: 10.1002/ajp.22322 Published online 17 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
Sleep in Spider Monkey / 201
At present, there are approximately 20 species of New World monkeys, but there is information about the circadian rhythm of sleep only for the common marmoset [Crofts et al., 2001; Sri Kantha & Suzuki, 2006], the squirrel monkey [Adams & Barratt, 1974; Breton et al., 1986; Edgar et al., 1993; Erny et al., 1985; Klerman et al., 1999; Sri Kantha et al., 2009; Wexler & Moore‐Ede, 1984, 1985, 1986], the owl monkey [Sri Kantha et al., 2009; Sri Kantha & Suzuki, 2006], and the tamarins [Sri Kantha & Suzuki, 2006]. Results have showed that, as in other primates, New World primates have sleep cycles with stages alternating between rapid eye movement (REM) and non‐rapid eye movement (N‐REM) sleep throughout the night, with the exception of the owl monkey, a uniquely nocturnal primate. In concordance with other non‐human primates, the duration of each of the sleep stages in New World monkeys is relatively short and punctuated with frequent waking. Electrophysiological patterns of sleep in New World monkeys show that both N‐REM and REM sleep are similar to those of other primates, including human beings. [Adams & Barratt, 1974; Breton et al., 1986; Crofts et al., 2001; Edgar et al., 1993; Wexler & Moore‐Ede, 1985]. Spider monkeys (Ateles geoffroyi) belong to the genus Ateles of the family Atelidae and the subfamily Atelinae; a group of New World monkeys with prehensile tails. In the wild, they are arboreal and found mainly in the higher strata of the forest canopy, spending most of their time travelling more than 20 m above the ground and rarely descending to the forest floor [Kellogg & Goldman, 1944; Takahashi, 2008]. The body structure and morphological characteristics of spider monkeys are adaptations to their completely arboreal lifestyle. Our field observations suggest that spider monkeys sleep on branches, in a vertical‐semi fetal position. Nevertheless, the sleep‐wake cycle, sleep postures, and concomitant electrophysiological sleep patterns of REM and N‐ REM sleep have never been documented in spider monkeys. Using an actigraphic technique, Muñoz‐Delgado and colleagues [2004], characterized the circadian motor rhythm in these primates in captivity. They observed that the Ateles species is diurnal in this condition, and daily activity starts about an hour after sunrise and ends about two hours after sunset. Their activity shows a bimodal pattern with two peaks, one before noon and one in late afternoon. Authors found that climate factors such as temperature during night and day, cloudiness, and humidity influence the onset and end of their daily activity. The gold standard for sleep studies in humans and animals is the polysomnographic technique (PSG). This involves simultaneous recording of electroencephalographic (EEG), electro‐oculographic (EOG), and the electromyographic (EMG) activities. N‐REM sleep is divided into stages 1, 2, and 3 or Delta
sleep, representing a continuum of relative deepness. Each stage presents different behavioral and electrophysiological characteristics. On the other hand, REM sleep is defined by the presence of low‐ voltage, mixed‐frequency brain activity. Muscle atonia and bursts of rapid eye movements are also present [Rechtschaffen & Kales, 1968]. In humans, the PSG is accomplished through the use of external electrodes that measure electrical activity at the level of the scalp. The main advantage of this technique is that it allows non‐invasive studies to be conducted [Rechtschaffen & Kales, 1968]. It is used to study, at both basic and clinical levels, the nature and pathology of sleep, as well as its relationship with certain affections of the central nervous system [Chesson et al., 1997]. Characterizing the sleep‐wake cycle, sleep postures, and the concomitant electrophysiological sleep patterns of REM and N‐REM sleep in the spider monkey would represent an excellent opportunity to study the sleeping patterns of the species at the basic and clinical levels. For this reason, sleep characteristics in spider monkeys, using a non‐invasive, semi‐restrictive PSG method are described in this experimental study. Based on our field observations and the close phylogenetic relatedness between spider monkeys and other arboreal primates, we pose the following hypothesis: (i) PSG patterns in spider monkeys will be similar to those in other arboreal primate mammals; (ii) a vertical, semi‐fetal position will be observed during both REM and N‐REM sleep; and (iii) the sleep‐wake cycle will be nocturnal‐monophasic. METHODS Ethics Statement Experiments were carried out in exemplars of spider monkeys maintained at the environmental management unit (UMA) “Doña Hilda Ávila de O’farril” from the Patronato Pro‐Universidad Veracruzana A.C. This study was approved by the Mexican Office for the Environment and Natural Resources (SEMARNAT; authorization number: SGPARN.03.VS.3644/11). The research adhered to the American Society of Primatologists: Principles for the Ethical Treatment of Non‐Human Primates. The study was also approved by the Ethical and Research Committee of the Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz” (authorization number: NC123340.2), according to statements of the Office of Animal Care and Use of the National Institutes of Health (USA). Location The UMA is located in the Mexican rainforest in Catemaco, Veracruz. It is located at 18° 260 –18° 280 ‘north latitude and 95° 010 –95° 030 west longitude 02’, 320 m above sea level. The area consists of 220 ha of
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202 / Cruz‐Aguilar et al.
evergreen high, mid and low forest, olmeca recta, acahual, palo mulato, and grassland as secondary vegetation. The local weather is hot and humid with rainfall throughout the year. It has an average annual temperature of 23.4°C with a maximum of 31.9°C and a minimum of 17.1°C. [Ibarra‐Manríquez et al., 1997]. The present study was performed in summer (June) of 2012.
movement during the recovery period from anesthesia, as their movements were clumsy and they tried to climb to the top. This procedure avoided any injury to the monkey. Once recovered, the height of the sleep PSG cage was adjusted allowing a greater range of motion. Inside the cage, trunks were placed transversely, where the animals could rest and sleep in their natural posture during PSG recordings.
Animals
PSG Recording Procedures
Six young individuals (mean weight of 7 kg.) from a colony composed of nine male spider monkeys housed in outdoor rain forest enclosures, participated in this study. Individuals of this colony had previously been housed in a large cage named “main closure”. The PSG recordings were done in this cage. The measurements of the cage are 78.74 in high 472.44 in long 236.22 in wide (Fig. 1) with a mesh roof and dirt floor. The main enclosure is sub‐divided into 12 individual cages (home cages) each measuring 78.74 in high 78.74 in long 97.11 in wide, each of which lodged one monkey. These enclosures allowed monkeys to be exposed throughout the nycthemeral cycle to natural environmental factors, such as temperature, humidity, rain, and natural light that modulate the circadian regulation of sleep [Gilbert et al., 2004; Sri Kantha & Suzuki, 2006]. Likewise, animals were exposed to natural environmental sounds, and could have vocal and visual interaction between themselves, as well as perceive animals living into the rain forest. Animals were given ad libitum access to food and water throughout the entire study.
Anesthesia To extract monkeys from their home cage for electrode application and placement into the PSG cage, we motivated the animal with some fruits to reach through cage mesh, upon which it was firmly held and anesthetic was applied in the forearm muscle. The animals were sedated with Zoletil 50 (2.4 mg/kg IM) for approximately 45 min in order to place the electrodes. In some cases, it was necessary to maintain the sedation for a longer period of time, administering an additional dose of 1.2 mg/kg. Monkey Restraint After removing the animal from its home cage, it was placed upright on the legs of one of the
PSG Cage PSG recordings were done in a “PSG cage” (Fig. 2), measuring 78.74 in high 78.74 long 39.37 in wide. This cage was constructed inside the main enclosure, in one of the home cages. The cage had a movable horizontal grid to adjust its height. This feature allowed the restriction of the monkeys’
Fig. 1. Main enclosure.
Am. J. Primatol.
Fig. 2. PSG cage. This cage was constructed in the interior of the home cage. In this condition, the monkeys have visual and vocal contact with the rest of the colony of spider monkeys during the study.
Sleep in Spider Monkey / 203
researchers with one hand grasping the back of the monkey and the other supporting the head (Fig. 3a). Electrodes were then placed under Zoletil anesthesia. Electrode Placement Electrode placement was done according to the human International 10–20 System [Jasper, 1958], an internationally recognized method to describe the location of scalp electrodes. The system is based on the relationship between the location of an electrode and the underlying area of cerebral cortex. The numbers “10” and “20” refer to the fact that the distances between adjacent electrodes are either 10% or 20% of the total front‐back or right‐left distance of the skull. Each site has a letter to identify the lobe and a number to identify the hemisphere location. Even numbers refer to electrode positions on the right hemisphere; odd numbers refer to electrode positions on the left hemisphere. We placed the electrodes in the O1‐O2, C4‐GND derivations of the human 10– 20 system. We cleaned each site with 70% alcohol solution, and electrodes were attached with collodion to the scalp (Fig. 3b and c). Electro‐oculogram (EOG) was registered from the outer canthi of both eyes and submental electromyographic (EMG) activity was acquired from electrodes attached to the mentalis muscle.
Wires and Electrodes Protection To protect the electrode wire, we used the technique of tethered cable, similar to that used in electrophysiological studies in cats and rats. All wires were joined as a braid and isolated with plastic adhesive tape; this prevents the animal from biting or pulling them, damaging the PSG montage (Fig. 3d). To protect the electrodes, a cotton‐lycra cap was designed and placed on the monkey’s head (Fig. 3e and f). We used a standard measure based on the average extent of the head of a young‐adult spider monkey. The cap was adjusted at the time of placement by sewing with needle and thread through the back and basement, allowing the braid out through the top of the cap. Two caps were placed on each monkey to give a greater resistance to the PSG montage. It is necessary to sew the braid to the two caps. Scissors were used to make orifices for the eyes, mouth, and nose (Fig. 3g). Habituation Period After the placement of electrodes, the monkey was introduced into the sleeping cage (9:00 AM, approximately), and the habituation period began, which included the recovery time from anesthesia (approximately 1 hr). Animals were habituated to PSG montage discomfort and restriction of sleep cage
Fig. 3. Method and sleep postures: (a) anesthesia; (b, c) surface electrode placement; (d) wire protection; (e) electrode protection, (f, g) adjustment cap; (h) recovered active and eating monkey; (i) freedom of movement; (j) N‐REM posture; (k) REM posture; and (l) safe removal of electrodes.
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204 / Cruz‐Aguilar et al.
for 22 continuous hours, from the time they were put in the habituation cage until the next day. It was necessary to monitor, clean and assist the animal continuously to achieve complete habituation (Fig. 3h and i). PSG Recording The PSG recordings were performed following the 22 hr period of habituation. PSG paper recordings were obtained during 24 continuous hours under light/dark cycles from 07:00 AM to 07:00 AM of the next day. Recordings were carried out on a Grass Model 7‐D polygraph at a speed of 12 mm/s. A band‐ passing filter was used for an EEG of 1–25 Hz, EOG of 3–10 Hz, and EMG of 10–100 Hz. Electrode Removals Using the movable horizontal grid, the animal was immobilized and anesthetized with 1.2 mg/kg intramuscular Zoletil. The animal was then extracted from the monkey sleeping cage, caps were snipped off and the electrodes were detached by mean cotton soaked in acetone (Fig. 3l). The monkey was then was returned to their home cage. Data Analysis The effectiveness of the habituation period was evaluated, and pulling the tether and caps behavior across 22 hr was quantified; two‐tailed Student’s t‐tests were performed to compare the number of pulling the tether and caps events between the first 3 hr versus the last 3 hr. The different sleep stages were visually appraised in continuous epochs of 12 s
using the Rechtschaffen and Kales, [1968], qualitative description of PSG traces made concomitantly with natural sleep postures exhibited by animals. The temporal distribution of wake‐sleep stages was analyzed across a continuous 24 hr light–dark cycle. Sleep efficiency (EFFIC), total time of sleep (TTS), sleep latency (SLA), and percentage of total time occupied by each sleep stage (TT) were obtained. The average duration of N‐REM/REM sleep cycle (calculated as the cumulative amount of sleep time from the start of one REM sleep episode to the start of the next) and average duration of REM sleep were calculated. To differentiate between the light and dark periods over sleep stage parameters, two‐tailed Student’s t‐tests were performed. For both the effectiveness of the habituation period and the temporal distribution of wake‐sleep, a P‐value less than 0.05 was considered to be statistically significant. RESULTS Habituation Period Exemplars of spider monkeys showed an adequate habituation to condition recordings because attempted cap‐pulling behavior decreased significantly when compared to the first 3 hr (36.50 SD 12.69 attempt numbers) with the last 3 hr (3 SD 2 attempt numbers) of the habituation period (t ¼ 6.38, P ¼
RESEARCH ARTICLE Sleep in the Spider Monkey (Ateles geoffroyi): A Semi‐Restrictive, Non‐Invasive, Polysomnographic Study MANUEL ALEJANDRO CRUZ‐AGUILAR1,2*, FRUCTUOSO AYALA‐GUERRERO3, ANABEL JIMÉNEZ‐ANGUIANO4, ANA MARÍA SANTILLÁN‐DOHERTY5, FRANCISCO GARCÍA‐ORDUÑA6, AND JAVIER VELÁZQUEZ‐MOCTEZUMA4 1 Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 2 Laboratorio de Sueño, Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Distrito Federal, México 3 Laboratorio de Neurociencias, Facultad de Psicología, Universidad Nacional Autónoma de México, Distrito Federal, México 4 Área de Neurociencias, Departamento Biología de la Reproducción, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 5 Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Distrito Federal, México 6 Instituto de Neuroetologia, Universidad Veracruzana, Xalapa, Veracruz, México
The normal sleep patterns of the spider monkey (Ateles geoffroyi) have not been described yet. The objective of this study was to characterize the electrophysiological patterns, sleeping postures, and sleep‐ wake cycle in semi‐restricted spider monkeys. Continuous 24‐hr polysomnographic (PSG) recordings, involving simultaneous recording of non‐invasive electroencephalographic (EEG), electro‐oculographic (EOG), and electromyographic (EMG) activities, were carried out in captive monkeys living in outdoor rainforest enclosures. Electrode placement was done according to the human international 10–20 system. Specific behaviors displayed by monkeys during the sleep‐wake cycles were correlated with the PSG recordings. The nycthemeral distribution of the sleep‐wake cycle was also calculated. The results show that electrophysiological N‐REM sleep patterns in spider monkeys are similar to those observed in other primates, including human beings. Furthermore, a vertical semi‐fetal posture was observed during N‐REM and REM sleep phases. The amount of nocturnal sleep was significantly higher than that of the diurnal period, showing that the spider monkey is a diurnal primate. An outstanding finding was the absence of muscular atonia during the spider monkey’s REM sleep, which suggests that arboreal primates have developed a neuromuscular mechanism specialized for sleeping in a vertical posture. Am. J. Primatol. 77:200–210, 2015. © 2014 Wiley Periodicals, Inc. Key words:
new world monkey; PSG; muscular atonia; REM
INTRODUCTION Sleep seems to be one of the most complex integrated behaviors for which the adaptive advantage remains unknown [Maquet et al., 1997; Mignot, 2008]. Animal and human studies suggest that its functions include, among others, recovery at the cellular, network and endocrine system levels [Mignot, 2008], thermoregulation [Gilbert et al., 2004], adaptation to an ecological niche [Webb, 1974], restoration of tissular integrity [Adams, 1980], and neuronal plasticity [Krueger et al., 1995]. Nevertheless, sleep in non‐human primates is rarely subject to systematic investigation [Anderson, 1998; Fruth & McGrew, 1998]. Most of the previous studies on electrophysiological and behavioral characteristics of the sleep‐wake cycle have been performed in Old World monkeys [Daley et al., 2006; Hsieh et al., 2008]. Nevertheless, from the point of view of adaptive behavior and
© 2014 Wiley Periodicals, Inc.
biological complexity of primates, the sleep analysis of New World monkeys can provide significant insights into sleep evolution in primates. Non‐human primates with an arboreal style life and prehensile tails, for example, can help us understand the anthropoid transition from arboreal to ground sleep through the study of their body postures.
Correspondence to: Manuel Alejandro Cruz Aguilar, Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana‐Iztapalapa, Distrito Federal, México 09340, E‐mail: [email protected] Received 3 April 2014; revised 5 July 2014; revision accepted 11 July 2014 DOI: 10.1002/ajp.22322 Published online 17 September 2014 in Wiley Online Library (wileyonlinelibrary.com).
Sleep in Spider Monkey / 201
At present, there are approximately 20 species of New World monkeys, but there is information about the circadian rhythm of sleep only for the common marmoset [Crofts et al., 2001; Sri Kantha & Suzuki, 2006], the squirrel monkey [Adams & Barratt, 1974; Breton et al., 1986; Edgar et al., 1993; Erny et al., 1985; Klerman et al., 1999; Sri Kantha et al., 2009; Wexler & Moore‐Ede, 1984, 1985, 1986], the owl monkey [Sri Kantha et al., 2009; Sri Kantha & Suzuki, 2006], and the tamarins [Sri Kantha & Suzuki, 2006]. Results have showed that, as in other primates, New World primates have sleep cycles with stages alternating between rapid eye movement (REM) and non‐rapid eye movement (N‐REM) sleep throughout the night, with the exception of the owl monkey, a uniquely nocturnal primate. In concordance with other non‐human primates, the duration of each of the sleep stages in New World monkeys is relatively short and punctuated with frequent waking. Electrophysiological patterns of sleep in New World monkeys show that both N‐REM and REM sleep are similar to those of other primates, including human beings. [Adams & Barratt, 1974; Breton et al., 1986; Crofts et al., 2001; Edgar et al., 1993; Wexler & Moore‐Ede, 1985]. Spider monkeys (Ateles geoffroyi) belong to the genus Ateles of the family Atelidae and the subfamily Atelinae; a group of New World monkeys with prehensile tails. In the wild, they are arboreal and found mainly in the higher strata of the forest canopy, spending most of their time travelling more than 20 m above the ground and rarely descending to the forest floor [Kellogg & Goldman, 1944; Takahashi, 2008]. The body structure and morphological characteristics of spider monkeys are adaptations to their completely arboreal lifestyle. Our field observations suggest that spider monkeys sleep on branches, in a vertical‐semi fetal position. Nevertheless, the sleep‐wake cycle, sleep postures, and concomitant electrophysiological sleep patterns of REM and N‐ REM sleep have never been documented in spider monkeys. Using an actigraphic technique, Muñoz‐Delgado and colleagues [2004], characterized the circadian motor rhythm in these primates in captivity. They observed that the Ateles species is diurnal in this condition, and daily activity starts about an hour after sunrise and ends about two hours after sunset. Their activity shows a bimodal pattern with two peaks, one before noon and one in late afternoon. Authors found that climate factors such as temperature during night and day, cloudiness, and humidity influence the onset and end of their daily activity. The gold standard for sleep studies in humans and animals is the polysomnographic technique (PSG). This involves simultaneous recording of electroencephalographic (EEG), electro‐oculographic (EOG), and the electromyographic (EMG) activities. N‐REM sleep is divided into stages 1, 2, and 3 or Delta
sleep, representing a continuum of relative deepness. Each stage presents different behavioral and electrophysiological characteristics. On the other hand, REM sleep is defined by the presence of low‐ voltage, mixed‐frequency brain activity. Muscle atonia and bursts of rapid eye movements are also present [Rechtschaffen & Kales, 1968]. In humans, the PSG is accomplished through the use of external electrodes that measure electrical activity at the level of the scalp. The main advantage of this technique is that it allows non‐invasive studies to be conducted [Rechtschaffen & Kales, 1968]. It is used to study, at both basic and clinical levels, the nature and pathology of sleep, as well as its relationship with certain affections of the central nervous system [Chesson et al., 1997]. Characterizing the sleep‐wake cycle, sleep postures, and the concomitant electrophysiological sleep patterns of REM and N‐REM sleep in the spider monkey would represent an excellent opportunity to study the sleeping patterns of the species at the basic and clinical levels. For this reason, sleep characteristics in spider monkeys, using a non‐invasive, semi‐restrictive PSG method are described in this experimental study. Based on our field observations and the close phylogenetic relatedness between spider monkeys and other arboreal primates, we pose the following hypothesis: (i) PSG patterns in spider monkeys will be similar to those in other arboreal primate mammals; (ii) a vertical, semi‐fetal position will be observed during both REM and N‐REM sleep; and (iii) the sleep‐wake cycle will be nocturnal‐monophasic. METHODS Ethics Statement Experiments were carried out in exemplars of spider monkeys maintained at the environmental management unit (UMA) “Doña Hilda Ávila de O’farril” from the Patronato Pro‐Universidad Veracruzana A.C. This study was approved by the Mexican Office for the Environment and Natural Resources (SEMARNAT; authorization number: SGPARN.03.VS.3644/11). The research adhered to the American Society of Primatologists: Principles for the Ethical Treatment of Non‐Human Primates. The study was also approved by the Ethical and Research Committee of the Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz” (authorization number: NC123340.2), according to statements of the Office of Animal Care and Use of the National Institutes of Health (USA). Location The UMA is located in the Mexican rainforest in Catemaco, Veracruz. It is located at 18° 260 –18° 280 ‘north latitude and 95° 010 –95° 030 west longitude 02’, 320 m above sea level. The area consists of 220 ha of
Am. J. Primatol.
202 / Cruz‐Aguilar et al.
evergreen high, mid and low forest, olmeca recta, acahual, palo mulato, and grassland as secondary vegetation. The local weather is hot and humid with rainfall throughout the year. It has an average annual temperature of 23.4°C with a maximum of 31.9°C and a minimum of 17.1°C. [Ibarra‐Manríquez et al., 1997]. The present study was performed in summer (June) of 2012.
movement during the recovery period from anesthesia, as their movements were clumsy and they tried to climb to the top. This procedure avoided any injury to the monkey. Once recovered, the height of the sleep PSG cage was adjusted allowing a greater range of motion. Inside the cage, trunks were placed transversely, where the animals could rest and sleep in their natural posture during PSG recordings.
Animals
PSG Recording Procedures
Six young individuals (mean weight of 7 kg.) from a colony composed of nine male spider monkeys housed in outdoor rain forest enclosures, participated in this study. Individuals of this colony had previously been housed in a large cage named “main closure”. The PSG recordings were done in this cage. The measurements of the cage are 78.74 in high 472.44 in long 236.22 in wide (Fig. 1) with a mesh roof and dirt floor. The main enclosure is sub‐divided into 12 individual cages (home cages) each measuring 78.74 in high 78.74 in long 97.11 in wide, each of which lodged one monkey. These enclosures allowed monkeys to be exposed throughout the nycthemeral cycle to natural environmental factors, such as temperature, humidity, rain, and natural light that modulate the circadian regulation of sleep [Gilbert et al., 2004; Sri Kantha & Suzuki, 2006]. Likewise, animals were exposed to natural environmental sounds, and could have vocal and visual interaction between themselves, as well as perceive animals living into the rain forest. Animals were given ad libitum access to food and water throughout the entire study.
Anesthesia To extract monkeys from their home cage for electrode application and placement into the PSG cage, we motivated the animal with some fruits to reach through cage mesh, upon which it was firmly held and anesthetic was applied in the forearm muscle. The animals were sedated with Zoletil 50 (2.4 mg/kg IM) for approximately 45 min in order to place the electrodes. In some cases, it was necessary to maintain the sedation for a longer period of time, administering an additional dose of 1.2 mg/kg. Monkey Restraint After removing the animal from its home cage, it was placed upright on the legs of one of the
PSG Cage PSG recordings were done in a “PSG cage” (Fig. 2), measuring 78.74 in high 78.74 long 39.37 in wide. This cage was constructed inside the main enclosure, in one of the home cages. The cage had a movable horizontal grid to adjust its height. This feature allowed the restriction of the monkeys’
Fig. 1. Main enclosure.
Am. J. Primatol.
Fig. 2. PSG cage. This cage was constructed in the interior of the home cage. In this condition, the monkeys have visual and vocal contact with the rest of the colony of spider monkeys during the study.
Sleep in Spider Monkey / 203
researchers with one hand grasping the back of the monkey and the other supporting the head (Fig. 3a). Electrodes were then placed under Zoletil anesthesia. Electrode Placement Electrode placement was done according to the human International 10–20 System [Jasper, 1958], an internationally recognized method to describe the location of scalp electrodes. The system is based on the relationship between the location of an electrode and the underlying area of cerebral cortex. The numbers “10” and “20” refer to the fact that the distances between adjacent electrodes are either 10% or 20% of the total front‐back or right‐left distance of the skull. Each site has a letter to identify the lobe and a number to identify the hemisphere location. Even numbers refer to electrode positions on the right hemisphere; odd numbers refer to electrode positions on the left hemisphere. We placed the electrodes in the O1‐O2, C4‐GND derivations of the human 10– 20 system. We cleaned each site with 70% alcohol solution, and electrodes were attached with collodion to the scalp (Fig. 3b and c). Electro‐oculogram (EOG) was registered from the outer canthi of both eyes and submental electromyographic (EMG) activity was acquired from electrodes attached to the mentalis muscle.
Wires and Electrodes Protection To protect the electrode wire, we used the technique of tethered cable, similar to that used in electrophysiological studies in cats and rats. All wires were joined as a braid and isolated with plastic adhesive tape; this prevents the animal from biting or pulling them, damaging the PSG montage (Fig. 3d). To protect the electrodes, a cotton‐lycra cap was designed and placed on the monkey’s head (Fig. 3e and f). We used a standard measure based on the average extent of the head of a young‐adult spider monkey. The cap was adjusted at the time of placement by sewing with needle and thread through the back and basement, allowing the braid out through the top of the cap. Two caps were placed on each monkey to give a greater resistance to the PSG montage. It is necessary to sew the braid to the two caps. Scissors were used to make orifices for the eyes, mouth, and nose (Fig. 3g). Habituation Period After the placement of electrodes, the monkey was introduced into the sleeping cage (9:00 AM, approximately), and the habituation period began, which included the recovery time from anesthesia (approximately 1 hr). Animals were habituated to PSG montage discomfort and restriction of sleep cage
Fig. 3. Method and sleep postures: (a) anesthesia; (b, c) surface electrode placement; (d) wire protection; (e) electrode protection, (f, g) adjustment cap; (h) recovered active and eating monkey; (i) freedom of movement; (j) N‐REM posture; (k) REM posture; and (l) safe removal of electrodes.
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for 22 continuous hours, from the time they were put in the habituation cage until the next day. It was necessary to monitor, clean and assist the animal continuously to achieve complete habituation (Fig. 3h and i). PSG Recording The PSG recordings were performed following the 22 hr period of habituation. PSG paper recordings were obtained during 24 continuous hours under light/dark cycles from 07:00 AM to 07:00 AM of the next day. Recordings were carried out on a Grass Model 7‐D polygraph at a speed of 12 mm/s. A band‐ passing filter was used for an EEG of 1–25 Hz, EOG of 3–10 Hz, and EMG of 10–100 Hz. Electrode Removals Using the movable horizontal grid, the animal was immobilized and anesthetized with 1.2 mg/kg intramuscular Zoletil. The animal was then extracted from the monkey sleeping cage, caps were snipped off and the electrodes were detached by mean cotton soaked in acetone (Fig. 3l). The monkey was then was returned to their home cage. Data Analysis The effectiveness of the habituation period was evaluated, and pulling the tether and caps behavior across 22 hr was quantified; two‐tailed Student’s t‐tests were performed to compare the number of pulling the tether and caps events between the first 3 hr versus the last 3 hr. The different sleep stages were visually appraised in continuous epochs of 12 s
using the Rechtschaffen and Kales, [1968], qualitative description of PSG traces made concomitantly with natural sleep postures exhibited by animals. The temporal distribution of wake‐sleep stages was analyzed across a continuous 24 hr light–dark cycle. Sleep efficiency (EFFIC), total time of sleep (TTS), sleep latency (SLA), and percentage of total time occupied by each sleep stage (TT) were obtained. The average duration of N‐REM/REM sleep cycle (calculated as the cumulative amount of sleep time from the start of one REM sleep episode to the start of the next) and average duration of REM sleep were calculated. To differentiate between the light and dark periods over sleep stage parameters, two‐tailed Student’s t‐tests were performed. For both the effectiveness of the habituation period and the temporal distribution of wake‐sleep, a P‐value less than 0.05 was considered to be statistically significant. RESULTS Habituation Period Exemplars of spider monkeys showed an adequate habituation to condition recordings because attempted cap‐pulling behavior decreased significantly when compared to the first 3 hr (36.50 SD 12.69 attempt numbers) with the last 3 hr (3 SD 2 attempt numbers) of the habituation period (t ¼ 6.38, P ¼
