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Extraocular muscle activity, rapid eye movements and the development of active and quiet sleep
Extraocular muscle activity, rapid eye movements
and the development of active and quiet sleep
Adele M. H. Seelke, Karl �. Karlsson, Andrew J. Gall and Mark S. Blumberg
Program in Behavioural and Cognitive Neuroscience, Department of Psychology, University of Iowa, Iowa City, IA 52242, USA
Keywords: atonia, electroencephalogram, myoclonic twitching, rat, REM
Abstract
Rapid eye movements (REMs), traditionally measured using the electrooculogram (EOG), help to characterize active sleep in adults.
In early infancy, however, they are not clearly expressed. Here we measured extraocular muscle activity in infant rats at 3 days of age
(P3), P8 and P1415 in order to assess the ontogeny of REMs and their relationship with other forms of sleep-related phasic activity.
We found that the causal relationship between extraocular muscle twitches and REMs strengthened during the rst two postnatal
weeks, reecting increased control of the extraocular muscles over eye movements. As early as P3, however, phasic bursts of
extraocular muscle twitching occurred in synchrony with twitching in other muscle groups, producing waves of phasic activity
interspersed with brief periods of quiescence. Surprisingly, the tone of the extraocular muscles, invisible to standard EOG measures,
uctuated in synchrony with the tone of other muscle groups; focal electrical stimulation within the dorsolateral pontine tegmentum,
an area that has been shown to contain wake-on neurons in P8 rats, resulted in the simultaneous activation of high tone in both
nuchal and extraocular muscles. Finally, when state-dependent neocortical electroencephalographic activity was observed at P14, it
had already integrated fully with sleep and wakefulness as dened using electromyographic criteria alone; this nding is not
consistent with the notion that active sleep in infants at this age is half-activated. All together, these results indicate exquisite
temporal organization of sleep soon after birth and highlight the possible functional implications of homologous activational states in
striated muscle and neocortex.
Introduction
Sleep and wakefulness are discriminated in adult placental mammals
using measures of eye movements, muscle tone and neocortical
activity. This trio of indicators is thought to provide necessary and
sufcient information for categorizing sleepwake states in adults, but
its usefulness for categorizing these states in infants is limited
(Rechtschaffen & Kales, 1968). Indeed, although sleep predominates
during infancy (Roffwarg et al., 1966; Jouvet-Mounier et al., 1970),
the idiosyncratic nature of infant sleep poses unique challenges
(Blumberg et al., 2005). For example, before the end of the second
postnatal week, infant rats do not exhibit state-dependent electroen-
cephalographic (EEG) activity (Gramsbergen, 1976; Corner &
Mirmiran, 1990; Frank & Heller, 1997), a feature of infant sleep that
has contributed to its characterization as disorganized, diffuse and
produced by distinct neurophysiological mechanisms (Adrien &
Lanfumey, 1984; Frank & Heller, 1997, 2003).
Work from our laboratory has shown that nuchal electromyography
(EMG) alone is sufcient for dening sleepwake states during early
infancy (Karlsson & Blumberg, 2002; Karlsson et al., 2004; Seelke &
Blumberg, 2005). Most recently, it was shown in week-old rats that sleep
and wake states identied in this way are governed by neural
mechanisms that are similar to those that govern sleepwake states in
adults (Karlsson & Blumberg, 2005; Karlsson et al., 2005). Thus, of
the trio of established indicators of adult sleepwake states, only the
mechanisms underlying rapid eye movements (REMs) have yet to be
examined adequately in infants.
Although work in rats, cats and guinea pigs (Shimizu & Himwich,
1968; Jouvet-Mounier et al., 1970; McGinty et al., 1977; Van
Someren et al., 1990) has addressed the development of REMs using
electrooculographic (EOG) techniques, such techniques may not be
adequate for assessing the developmental precursors of REMs in very
young animals. Thus, although it was reported that rats, in which the
eyelids open on postnatal day (P)15, rst exhibit REMs around P6
(Jouvet-Mounier et al., 1970), it is unclear whether the extraocular
muscles that control REMs are active during sleep at earlier ages. If,
however, REMs are produced by twitches of the extraocular muscles,
as has been suggested (Chase & Morales, 1983, 1990), and if these
extraocular muscle twitches are phenomenologically similar to the
twitches produced by other striated muscles, then direct measurement
of extraocular muscle activity might reveal ontogenetic precursors of
REMs that have heretofore gone unnoticed.
Materials and methods
All experiments were performed under National Institutes of Health
guidelines for the care of animals in research and were approved by the
Institutional Animal Care and Use Committee of the University of Iowa.
Subjects
A total of 21 rats from 21 litters were used: 18 P3, P8 and P1415 rats
for behavioural experiments and three P79 rats for electrical
Correspondence: Dr Mark S. Blumberg, E11 Seashore Hall, University of Iowa, Iowa
City, IA 52242, USA
E-mail: mark-blumberg@uiowa.edu
Received 16 March 2005, revised 17 April 2005, accepted 10 May 2005
European Journal of Neuroscience, Vol. 22, pp. 911920, 2005
� Federation of European Neuroscience Societies
doi:10.1111/j.1460-9568.2005.04322.x stimulation. Weights ranged from 8.6 to 10.1 g at P3, 19.820.7 g at
P79 and 37.041.1 g at P1415. All pups were born to Harlan
Sprague-Dawley rats housed in the animal colony at the University of
Iowa. The pups were raised in litters that were culled to eight pups
within 3 days of birth (day of birth was day 0). Litters and mothers
were raised in standard laboratory cages (48 cm long
� 20 cm
wide
� 26 cm high), in which food and water were available
ad libitum. All rats were maintained on a 12-h lightdark schedule
with lights on at 07.00 h.
Behavioural recording
Test environment
Pups were tested inside an electrically shielded double-walled glass
chamber (height, 17.0 cm; i.d., 12.5 cm) with a Plexiglas lid. Air
temperature inside the chamber was regulated using a temperature-
controlled water circulator. Access holes in the side and lid of the
chamber allowed for passage of air through the chamber as well as the
passage of EMG and EEG electrodes. A round platform constructed of
polyethylene mesh was tted inside the chamber and a perforated felt
pad was placed on top of the mesh.
Procedure
Eighteen P3, P8 and P1415 rats from 18 litters were used. On the day
of testing a P3 or P8 rat with a visible milkband was removed from the
litter, weighed and anaesthetized with isourane. An incision was
made in the scalp, which was then retracted to expose the top half of
each eyeball. One unipolar stainless steel hook electrode (50 lm
diameter; California Fine Wire, Grover Beach, CA, USA) was inserted
between the eyeball and orbit on both the nasal and temporal sides of
the eye with the aim of implanting in the medial and lateral rectus
muscles, respectively. This procedure was repeated for the other eye.
The electrodes were secured to the skull with cyanoacrylate adhesive
and the scalp was closed. Two bipolar stainless steel hook electrodes
(California Fine Wire) were then placed bilaterally in the nuchal
muscle and secured with collodion. The subject was lightly restrained
on a felt-covered platform, placed in an incubator maintained at
thermoneutrality (i.e. 35 C) and allowed to recover for 1 h. It should
be noted that restraint does not interfere with sleepwake cycling at
this age; Karlsson et al. (2004). The subject was then infused with
warm milk (3% body weight, commercial half-and-half) and allowed
to acclimatise to the testing chamber (also maintained at 35 C) for
1 h. Testing consisted of collecting 1 h of both behavioural (using
digital video) and electrographic data. After testing the subject was
killed using an overdose of Nembutal.
A similar protocol was followed for P1415 subjects. On the day of
testing a pup was removed from the litter, weighed and anaesthetized
using isourane. An incision was made in the scalp, which was then
retracted to expose the skull. Holes were drilled over the cerebellum, left
parietal and left frontal cortices, and electrodes, consisting of skull
screws (0096
� 1 16; Plastics One, Roanoke, VA, USA) attached to
insulated silver wires (AG 10T; Medwire, Mt. Vernon, NY, USA), were
placed in each of the holes to a depth of 1.5 mm. The cerebellar
electrode served as the ground. The scalp was then retracted further,
exposing the top half of each eyeball, and electrodes were implanted into
the extraocular muscles as described above. The scalp was then closed
and bipolar stainless steel hook electrodes were implanted bilaterally
into the nuchal muscle and secured with collodion. The subject was
placed into the testing chamber, maintained at thermoneutrality (i.e.
32 C), for 24 h to recover and acclimatise. At this age, pups were
tested while freely moving. Electrographic (including extraocular and
nuchal EMG and cortical EEG) data were collected for 2 h, including
1 h in which behaviour was recorded to digital videotape. After testing,
the subject was killed using an overdose of Nembutal.
Nuchal