rapid whisker movements in sleeping newborn rats...whiskers (black circles); also shown are the two...

Current Biology 22, 1–6, November 6, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.09.009

ReportRapid Whisker Movementsin Sleeping Newborn Rats

Alexandre Tiriac,1 Brandt D. Uitermarkt,1

Alexander S. Fanning,1 Greta Sokoloff,1

and Mark S. Blumberg1,*1Department of Psychology, Program in Behavioral andCognitive Neuroscience, The University of Iowa, Iowa City,IA 52242, USA

Summary

Spontaneous activity in the sensory periphery drives infantbrain activity and is thought to contribute to the formationof retinotopic and somatotopic maps [1–3]. In infant ratsduring active (or REM) sleep, brainstem-generated sponta-neous activity triggers hundreds of thousands of skeletalmuscle twitches each day [4]; sensory feedback from the re-sulting limb movements is a primary activator of forebrainactivity [1]. The rodent whisker system, with its preciseisomorphic mapping of individual whiskers to discrete brainareas, has been a key contributor to our understanding ofsomatotopic maps and developmental plasticity [5–7]. Butalthough whisker movements are controlled by dedicatedskeletal muscles [8, 9], spontaneous whisker activity hasnot been entertained as a contributing factor to the develop-ment of this system [10]. Herewe report in 3- to 6-day-old ratsthat whiskers twitch rapidly and asynchronously duringactive sleep; furthermore, neurons in whisker thalamusexhibit bursts of activity that are tightly associated withtwitches but occur infrequently during waking. Finally, weobserved barrel-specific cortical activity during periods oftwitching. This is the first report of self-generated, sleep-related twitches in the developingwhisker system, a sensori-motor system that is unique for the precision with whichit can be experimentally manipulated. The discovery ofwhisker twitching will allow us to attain a better under-standing of the contributions of peripheral sensory activityto somatosensory integration and plasticity in the devel-oping nervous system [11–13].

Results and Discussion

Newborn Rats Exhibit Whisker Twitches duringActive SleepPrevious studies of whisker movements in infant ratshave focused primarily on the emergence of synchronizedmovements of the whiskers (i.e., whisking) in awake animals[14–16]. Because whisking does not develop until the end ofthe second postnatal week, self-generated whisker move-ments have been overlooked as possible contributors tothe perinatal development of whisker system morphology orplasticity. Here we asked whether asynchronous whiskermovements (i.e., twitching) occur during active sleep in 3- to6-day-old rats (n = 5), similar to the twitches that occur in otherlimbs controlled by skeletal muscles [17].

Using high-speed videography (200 frames/s), we assessedmovements of the whiskers and mystacial pad as pups cycled

between sleep and wakefulness (Figure 1A). During periods ofactive wakefulness when high-amplitude limb movements(e.g., kicking, stretching) were seen, whisker and mystacialpad movements were noisy and seemingly haphazard. Allmovements subsided during the transition from wakefulnessto quiet sleep. Then, with the onset of active sleep as the distallimbs and tail began to twitch, contemporaneous bouts ofwhisker twitcheswere observed.We saw a diversity of whiskermovements (Figure 1B; see Movie S1 available online), in-cluding independent twitches of single whiskers, simulta-neous twitches of adjacent or nonadjacent whiskers, andcomplex movements comprising various subsets of whiskersmoving in variable directions. Independent whisker twitcheswere easily distinguished from events involving pronouncedmovements of the mystacial pad in which all whiskers movedin unison. This is the first demonstration of whisker twitching insleeping infant rats, although they have been noted anecdot-ally in sleeping adults [18].Working from a catalog of 51 whisker twitches, we calcu-

lated mean maximum whisker displacements from rest of0.13 6 0.01 mm (range: 0.02–0.27 mm) and mean angularvelocities of 121 6 7 deg/s (range: 34–255 deg/s). The meanlatency from twitch onset to maximum displacement was65.4 6 3.4 ms (range: 30–150 ms). Although the majority ofthese whisker movements were in the protraction and retrac-tion directions, movements in a diversity of other directionswere also observed (Figure S1). The patterns of movementsobserved here are consistent with the known anatomy of thewhisker muscle system [8, 9], as well as findings in adultsfrom whisker muscle and facial nucleus stimulation studies[19, 20] and observations of adjacent whisker movements [21].

Extrinsic Whisker Muscles Twitch during Active SleepWhiskers are controlled by a complex system of extrinsicand intrinsic muscles [8, 9]. To ensure that the whiskertwitches observed using high-speed videography were notdue to movement artifact, we recorded electromyographic(EMG) activity from two extrinsic whisker muscles in 4- to6-day-old rats (n = 4), m. maxillolabialis and m. nasolabialis(Figure 2A). We also recorded EMG activity from the nuchalmuscle, the primary elevator of the head, which provides areliable measure of behavioral state [22]. Both extrinsicwhisker muscles twitched during periods of active sleepwhen twitches occurred in the nuchal muscle (Figure 2B).Twitching in nuchal and extrinsic muscles exhibited strongand significant cross-correlations (Figure 2C). Similar cross-correlations were observed in the three other subjects. Ingeneral, these extrinsic whisker muscles exhibited sleep-wake profiles and patterns of twitching similar to those foundin other skeletal muscles [23, 24].

Whisker Thalamus Exhibits Twitch-Dependent ActivitySensory feedback from twitching limbs increases neuralactivity in the somatosensory thalamus and cortex of infantrats [1], as well as hippocampus [24]. Moreover, in the whiskersystem, thalamic and cortical mechanisms are responsive tomechanical whisker stimulation soon after birth [25, 26]. Wefound that whisker twitches result in sensory feedback to the*Correspondence: [email protected]

Please cite this article in press as: Tiriac et al., Rapid Whisker Movements in Sleeping Newborn Rats, Current Biology (2012), http://dx.doi.org/10.1016/j.cub.2012.09.009

http://dx.doi.org/10.1016/j.cub.2012.09.009


mailto:[email protected]

ventral posteromedial nucleus (VPM), a primary thalamic inputof the whisker system. Overall, 7 VPM units were isolated from3- to 6-day-old rats (n = 7) (Figure 3A). For one representativeunit (Figures 3B–3E), firing rate increased during periods ofactive sleep (Figure 3B) and exhibited a significant increasein firing rate 100 ms after a twitch, peaking at approximately250 ms (Figure 3C). Similarly significant relationships betweenunit activity and twitching were documented in 5 otherVPM units (one subject’s EMG record was too noisy for thisanalysis).

All 7 VPM units exhibited bursts of neural activity. We usedfrequency histograms of interspike interval (ISI; Figure 3D) todefine a VPM burst as the occurrence of two or more spikeswith ISIs % 150 ms. Across all subjects, the number of spikesper burst varied widely and, for some subjects, more than tenspikes per burst was not uncommon. Bursts predominatedduring sleep (Figure 3E). Moreover, their rate of occurrencewas significantly higher during active sleep both withineach subject individually (ps < 0.05) and across all subjects(t6 = 7.4, p < 0.001; Figure 3F). The state-dependent thalamicbursting observed here is consistent with previous observa-tions in neonatal cortex and hippocampus [1, 24] and may

Figure 1. Diverse Patterns of Whisker Move-ments during Active Sleep in 4- to 6-Day-Old Rats

(A) Left shows one side of the snout, highlighting11 marked whiskers (boxed area). Right showslabels and relative locations of the 11 markedwhiskers (black circles); also shown are the twomarkings on the skin surface of the mystacialpad (red circles).(B) Quiver plots depicting four types of whiskerand mystacial pad movements observed usinghigh-speed videography. The locations of thewhiskers (black circles) and the mystacial padmarkings (red circles) correspond to those in(A). The lines emanating from the circles areproportional to the whisker or pad displacementover the previous 25 ms (five frames); the direc-tion of movement is also indicated. The fourpatterns depicted correspond to Movie S1. SeeFigure S1 for additional examples of the diversityof whisker trajectories.

reflect the action of corollary dischargemechanisms [27, 28]. (This thalamicbursting should not be conflated withthat observed in awake adult rats duringso-called ‘‘whisker twitching,’’ definedin that study as rhythmic 7–12 Hzwhisker movements [29].)The temporal relationship between

whisker twitching and VPM activity issimilar to that reported previouslybetween limb twitching and forebrainactivity, for which a causal role fortwitching has been established [1, 24].We were able to assess causalitybetweenwhisker twitching and thalamicactivity in one subject before, during,and after peripheral sensory blockadewhile recording a twitch-dependentVPM unit (Figure S2). Within 11 min oflidocaine (1%) injection into the mysta-cial pad, the rate of VPMbursts declined

substantially as sleep-related twitching continued, consistentwith peripheral sensory blockade [30]. As the effects of thelidocaine dissipated, the rate of VPM bursts returned to prein-jection levels. Therefore, whisker twitches can drive VPMactivity during active sleep.

Barrel Cortex Exhibits Spontaneous Activity duringActive SleepAs early as the day of birth, VPMactivity is sufficient to activatebarrel cortex in a precise topographic manner [26]. In light ofthe finding above that VPMunits increase their firing ratewithin100 ms of a twitch, we predicted that periods of twitchingwould also be accompanied by barrel-specific activationpatterns. We used voltage-sensitive dye (VSD) imaging tomonitor barrel cortex activity during bouts of active sleep (Fig-ure 4A). We imaged three subjects that exhibited significantcontralateral VSD responses to whisker stimulation (Fig-ure S3A). In each subject during active sleep, we identifiedthe first ten events inwhich therewasEMGevidence of a twitchalong with behavioral confirmation of twitching. We nextexamined barrel activity within a 500 ms window of eachEMG-defined twitch. In total, 26/30 (86.7%) of these twitches

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were followed by clear barrel activity. Varying levels of corticalactivation were found, from single to multiple barrels (Fig-ure 4B; Movie S2), mirroring the diversity of whisker move-ments observed using high-speed videography.

ConclusionsThe rodent whisker system affords many advantages to inves-tigators interested in understanding the mechanisms thatgive rise to somatotopic maps, sensorimotor integration, anddevelopmental plasticity [5, 11–13]. After several decades ofresearch, however, important questions remain as to theinstructive role played by the sensory periphery in early devel-opment. The present findings suggest that past assessmentsof the importance of the sensory periphery for the developingwhisker system were handicapped by incomplete knowledgeregarding the sources and timing of sensory input. Thus,contrary to the view that, in infants before the onset of whisk-ing, whisker stimulation only arises from passive interactionswith the mother and littermates [10, 12], we have shown herethat self-generated, asynchronous whisker twitches drivebrain activity during active sleep in a manner that is strikinglysimilar to the brain activation produced by twitches elsewherein the body [1, 17, 24].

We focused here onwhisker twitching in 3- to 6-day-old rats,an age when thalamocortical projections to layer 4 are estab-lished, when the sensitive period for structural cortical plas-ticity is ending, but when other aspects of cortical plasticityremain [5, 12, 13, 31]. Given that limb twitches occur in uteroand exhibit continuity with postnatal twitching [32], it is likelythat whisker twitches also commence in utero and therefore

may influence subcortical and cortical development in variousways throughout early prenatal and postnatal life.As with retinal waves [33], the developmental and spatio-

temporal features of twitching may provide clues to its func-tions. For example, a twitch is characterized by discrete motoroutput, precisely timed sensory feedback, and high signal-to-noise ratio afforded by the background of muscle atonia [17].These characteristics may make twitches better suited thanother forms of motor activity (such as that produced duringwaking) to produce the isomorphic mapping and multilevelsensorimotor integration that characterizes the whiskersystem [34–36]. Also, because whisker twitches are diversein form and direction (see Figure 1B, Figure S1, andMovie S1), they provide a range of experiences beyond thatprovided by whisking, which is largely limited to synchronouswhisker movements along the protraction-retraction axis [19].Accordingly, it is tempting to suggest that this diversity ofwhisker movements during twitching aids in establishing the‘‘pinwheel’’ map of whisker directional movement [37, 38].These and perhaps other features of whisker twitch move-ments, which predominate during the active-sleep-rich periodof early infancy [39], may ultimately help us to better under-stand the functional value of that sleep state for the developinginfant [17].

Experimental Procedures

All experiments were approved by the Institutional Animal Care and UseCommittee of The University of Iowa. All surgeries were performed underisoflurane anesthesia and all recording was performed in unanesthetized

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(A) Locations of EMG electrodes (black asterisks) in m. maxillolabialis andm. nasolabialis (also referred to asm. levator labii superioris in [8], fromwhich thisdrawing is adapted).(B) Raw EMG record depicting twitching in the two extrinsic muscles as well as the nuchal muscle. Sharp spikes in the EMG records indicatemyoclonic twitches.(C) Cross-correlograms showing highly correlated bouts of twitch activity for each pair of muscles. The horizontal dashed line indicates statisticalsignificance.

Rapid Whisker Movements in Sleeping Newborns3


subjects. For analysis of whisker movements (n = 5), whiskers were trimmedto 1mmand the tips weremarked with fluorescent paint for recording underultraviolet-light illumination. A high-speed digital video camera (IDT,

Tallahassee, FL) recorded whisker movements, which were tracked andanalyzed offline using ProAnalyst software (Xcitex, Cambridge, MA). EMGactivity in two extrinsic whisker muscles and nuchal muscle (n = 4) was

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(A) Coronal sections through the thalamus to show the recording sites. VPM, ventral posteromedial thalamus; VPL, ventral posterolateral thalamus; Po,posterior thalamus.(B) Representative data depicting multiunit activity (MUA), single unit activity, and nuchal EMG. Twitch and wake movements (black and red asterisks,respectively) are also shown. Recording site is the yellow circle in (A).(C) Perievent histogram for the VPM unit in (B). Vertical line at 0ms denotes time of nuchal muscle twitch and horizontal line indicates statistical significance.The twitch-following properties of another VPM unit were further investigated by anesthetizing the whisker pad (see Figure S2).(D) Frequency histogram of interspike intervals (ISI) for the VPM unit in (B).(E) VPM unit firing rate (spikes/5 s), unit activity, and burst activity over a 15 min recording session.(F) Mean (+SE) bursts/min during active sleep and wake across all seven subjects. * represents significant difference from wake, p < 0.001.



recorded and analyzed using established methods [19, 24]. For recording ofneural activity in the VPM of head-fixed pups (n = 7), the apparatus,methods, and analyses have been described previously [4, 24]. Twitch-trig-gered perievent histograms were constructed and a randomization proce-dure was used to test the relationship between VPM activity and twitching[24]. State-related differences in mean VPM burst rates were tested withinsubjects (Wilcoxonmatched-pairs signed-ranks test) and between subjects(paired t test). For voltage-sensitive dye (VSD) imaging of barrel cortex (n =3), pups were prepared and recorded similarly as above with the addition ofa unilateral craniotomy over the right hemisphere (Figure 4A). A voltage-sensitive dye (RH1838; Optical Imaging, Inc., Rehovot, Israel) was appliedtopically to the dura and a window was created for imaging (MiCAM Ultima,SciMedia Costa Mesa, CA). Imaging trials consisted of contralateral andipsilateral whisker stimulations (Figure S3A) and spontaneous behavior.dF/F0 was calculated and analyzed offline using custom-written scripts inMATLAB (MathWorks, Natick, MA) and procedures similar to thosedescribed previously [26].

Supplemental Information

Supplemental Information includes three figures, Supplemental Experi-mental Procedures, and twomovies and can be foundwith this article onlineat http://dx.doi.org/10.1016/j.cub.2012.09.009.

Acknowledgments

For technical support and guidance we thank Bob McMurray, CassieColeman, Ashlynn Gerth, William Todd, Josh Weiner, Jian-Young Wu, and

Weifeng Xu. Jenq-Wei Yang and Heiko Luhmann generously provided theircustom-written VSD imaging analysis software. Thisworkwas supported bygrants from the National Institutes of Health (HD63071, MH66424) to M.S.B.

Received: July 29, 2012Revised: August 28, 2012Accepted: September 6, 2012Published online: October 18, 2012

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Figure 4. Voltage-Sensitive Dye (VSD) Imagingof Barrel Cortex Activity during Active Sleep in4-Day-Old Rats

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Current Biology, Volume 22

Supplemental Information

Rapid Whisker Movements

in Sleeping Newborn Rats Alexandre Tiriac, Brandt D. Uitermarkt, Alexander S. Fanning, Greta Sokoloff, and Mark S. Blumberg

Supplemental Inventory

1. Supplemental Figures and Tables

Figure S1, related to Figure 1



2. Supplemental Experimental Procedures

3. Supplemental References

1. Figure S1. Representative whisker-twitch events in 4-6-day-old rats to show the diversity of whisker trajectories Six patterns are shown, each color-coded to match the whisker labels in the upper left-hand corner of each plot. The origin of each plot represents the resting position for each whisker. Positive values on the x-axis represent protraction (P) and negative values represent retraction (R). Positive values on the y-axis represent the dorsal direction (D) and negative values represent the ventral direction (V). Values on the x and y axes are in mm.

Figure S2. Sensory Blockade of Whiskers Decreases Peripheral Sensory Feedback to VPM in a 4-Day-Old Rat (A) Twenty min before lidocaine (1%) injection into the contralateral mystacial pad, the VPM unit exhibits strong activation in response to contralateral whisker stimulation using a fine brush (denoted by horizontal bars). Ipsilateral whisker stimulation does not evoke a response. Both multiunit activity (MUA) and single unit activity are shown. (B) Perievent histogram for the same VPM unit in (A). The firing rate increases significantly 100 ms after a nuchal muscle twitch, denoted by the vertical line at 0 ms. The horizontal line indicates statistical significance. (C) The activity of the VPM unit is depicted before and after lidocaine injection. Behavioral state is indicated on the top row, with sleep denoted by light gray and wake denoted by dark gray. Nuchal muscle twitch activity, VPM unit activity, burst activity, and bursts/min are also shown. The number of bursts/min decreases steadily between 11 and 25 min post-injection, before returning to pre-injection levels as the lidocaine wears off. At the end of the recording session, we confirmed that stimulation of the contralateral and ipsilateral whiskers yielded results identical to those in (A) (data not shown). The location of the recording site is indicated by the green circle in Figure 3A.

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Figure S3. Barrel Cortex Responses to Evoked Whisker Stimulation in 4-Day-Old Rats (A) Left: Voltage-sensitive dye (VSD) optical signals to single trials of contralateral (black) and ipsilateral (red) whisker stimulation for one subject with all whiskers intact. Right: Mean (+SE) VSD responses over the entire ROI across 7 pairs of contralateral and ipsilateral trials for the same subject. * significant difference from ipsilateral, P< 0.05. (B) VSD images for an additional subject in which all contralateral whiskers except C2 and E2 were plucked and C2 was stimulated. As expected, the C2 barrel (dashed circle) is differentially activated by the whisker stimulation. With this procedure, we were able to orient a map of barrel cortex within the ROI. Color bar at right indicates the range of values for dF/F0. In the optical signal presented below, red arrows indicate acquisition times of VSD images.

Supplemental Experimental Procedures All experiments were approved by the Institutional Animal Care and Use Committee of The University of Iowa. Subjects Sprague-Dawley Norway rats (Rattus norvegicus) at 3-6 days of age were used in all experiments. Males and females were used and littermates were always assigned to different experimental groups. Litters were culled to 8 pups within 3 days of birth. Mothers and their litters were housed and raised in standard laboratory cages (48 x 20 x 26 cm). Food and water were available ad libitum. All animals were maintained on a 12-h light-dark schedule with lights on at 0700 h. High-Speed Videography of Whisker Movements Five 4- to 6-day-old rats were used. Under isoflurane anesthesia, the pup’s whiskers were trimmed to approximately 1 mm from the skin surface so that all whisker tips were in the same focal plane. The tips were marked with paint that fluoresces under ultraviolet-light illumination (Figure 1A). Several additional marks were placed on the mystacial pad. The skin overlying the skull was removed and a custom-built head-fix device was attached to the skull with cyanoacrylate adhesive [4]. The pup was then transferred to a humidified incubator maintained at thermoneutrality (35 ˚C). The head-fixed pup was supported on a narrow platform and its limbs dangled freely on both sides of the platform. A high-speed digital video camera (IDT, Tallahassee, FL) with a 105 mm micro lens (Nikon, Tokyo, Japan) recorded whisker movements at 200 frames/s. Two LEDs were positioned on either side of the whiskers to allow simultaneous documentation of sleep and wake limb movements. Motion Studio software (IDT, Tallahassee, FL) was used to record videos. A calibration image was acquired by placing a ruler next to the whiskers. When the pup was cycling between sleep and wakefulness, multiple 10-s recordings of whisker activity were obtained. An experimenter observed the subject and marked the occurrence of myoclonic twitching of the distal limbs and tail (indicative of active sleep) and high-amplitude limb movements (indicative of wakefulness) [1, 2].

In total, 70 videos were recorded. Two independent and blind behavioral scorers reviewed a subset of video clips and produced a catalogue of 29 events comprising 51 whisker movements. These 51 movements were tracked using ProAnalyst. Tracked data for each whisker movement were exported to Excel (Microsoft, Redmond, WA) and mean maximum displacement and angular velocity were calculated [3]. Electromyographic Analysis of Extrinsic Whisker Muscle Activity Four 4- to 6-day-old rats were used. Under isoflurane anesthesia, pups were prepared for EMG recording from two extrinsic whisker muscles [3] (see Figure 2A) and nuchal muscle (the elevator muscle of the head). After surgery and recovery, the pup was secured in a stereotaxic apparatus. The 3 sets of EMG electrodes were connected to a differential amplifier (A-M Systems, Carlsborg, WA; amplification: 10,000x; filter setting: 300-5000 Hz). EMG signals were sampled at 1 kHz using a digital interface and Spike2 software (Cambridge Electronic Design, Cambridge, UK). Recordings were 15 min in duration and consisted of continuous EMG monitoring of the pup as it cycled freely between sleep and wakefulness. The experimenter, blind

to the EMG signals, marked the occurrence of sleep and wake movements. After the recording session, pups were overdosed with sodium pentobarbital (1.5 mg/g) and the region around the mystacial pad was dissected to confirm EMG placements.

Sleep and wake periods were defined by calculating mean EMG activity, as described previously [4]. Briefly, each nuchal EMG record was rectified and smoothed (τ = 0.05 s). The mean amplitude of high muscle tone and atonia was calculated from five 1-s representative EMG segments, and the midpoint between the 2 means was determined. The EMG signal was then dichotomized into periods of high tone (indicative of wake) and atonia (indicative of sleep). To identify and quantify twitch events [5], wake periods were removed from the EMG records. Next, using a threshold set to 2x the baseline mean EMG value for periods of atonia, twitch events were marked. The temporal correlations among twitches occurring in the nuchal and whisker muscles were analyzed using cross-correlograms [5]. We considered the correlation between twitches in any pair of muscle groups to be significant when at least 1 of the 40 bins in the cross-correlogram had a value greater than the mean of 5 randomized cross-correlograms plus 3 standard deviations (P < 0.003, two-tailed). Thalamic Neurophysiology A total of 358 3- to 6-day-old rats were used. We identified clear units in 88 subjects and, of these, there were 7 subjects with 7 units for which electrode placement within VPM was confirmed and nuchal EMG records were adequate for analysis. The apparatus and methods used here for recording state-dependent thalamic activity in head-fixed infant rats are similar to those used previously to examine activity in other brain areas [5-8]. Platinum/iridium tetrodes and single-ended electrodes (Thomas Recording, Giessen, Germany), with impedances ranging from 1-2 MΩ, were connected to a data acquisition system (Tucker-Davis Technologies, Alachua, FL; amplification: 10,000x; filter setting: 1-3000 Hz). A chlorinated silver (Ag/AgCl) electrode (MedWire, Mt. Vernon, NY, 0.25 mm diameter), placed into the cerebellum, served as ground. EMG electrodes were connected to a differential amplifier (amplification: 10,000x; filter setting: 300-5000 Hz). Neural and nuchal EMG signals were sampled at 12.5 kHz and 1 kHz, respectively, using a digital interface and Spike2. Brain temperature (36-37 ºC) was measured using a fine-wire thermocouple (Omega Engineering, Stamford, CT) placed in the contralateral cortex. After the electrode was lowered into the thalamus (coordinates: 1.8-2.0 mm caudal to bregma, 1.5-1.6 mm lateral to midline) and state-dependent units were identified, neural, EMG, and behavioral sleep-wake data were recorded for 15 min at a time. As sleep-wake behavior was scored, the experimenter was blind to the EMG and neural signals. After the last recording session, a marking lesion was produced by passing a 50 µA anodal current through the recording electrode for 5 s using a linear stimulus isolator (World Precision Instruments, Sarasota, FL). The pup was then overdosed with sodium pentobarbital (1.5 mg/g) and prepared for histology. Electrode position was verified at 4X magnification using a Leica microscope (Leica Microsystems, Buffalo Grove, IL).

Spike sorting was performed using principal component analysis in Spike2. Signal artifacts were identified and removed manually. A cluster was judged to contain a single unit only if the autocorrelation analysis indicated a refractory period of at least 2 ms. To quantitatively determine the relationship between thalamic activity and twitching, twitch events were first identified and marked as described above. Then, twitch-triggered perievent histograms of thalamic unit activity were constructed and a randomization procedure was used for statistical

testing [5]. Based on the frequency histograms of interspike interval (ISI) for each VPM unit, a burst was defined as two or more action potentials with ISIs≤150 ms. State-related differences in mean VPM burst rates were tested using a Wilcoxon matched-pairs signed-ranks test for within-subjects comparisons and a paired t test for the between-subjects comparison with alpha set at 0.05. Voltage-Sensitive Dye (VSD) Imaging of Barrel Cortex Activity Three 4-day-old rats were used and, under isoflurane anesthesia, were prepared similarly to those described above for thalamic recording. In addition, a unilateral craniotomy was performed over the right hemisphere (Figure 4A). The skull was scored with a handheld drill along midline from lambda to bregma and approximately 5 mm lateral to midline and was removed in one piece while leaving the dura intact. A voltage-sensitive dye (RH1838; Optical Imaging, Inc., Rehovot, Israel), dissolved at 2 mg/ml in ACSF (pH 7.4), was applied topically to the dura for 90 min while the pup was maintained at thermoneutrality. Every 20-30 min the dye was removed and reapplied to ensure even coverage. After 90 min, the cortex was washed with ACSF for 10 min to remove unbound dye. The craniotomy was then covered in 1% agarose dissolved in ACSF. A coverslip was placed over the agarose to stabilize the cortex and provide a window for VSD imaging (MiCAM Ultima, Scimedia, Costa Mesa, CA).

Each pup was imaged while head-fixed as described above. EMG signals and sleep-wake behaviors were acquired using a differential amplifier (amplification: 10,000x; filter setting: 300-5000 Hz) and digital acquisition system (MP150, Biopac Systems, Goleta, CA). The voltage-sensitive dye was excited with a 150 W halogen light, band-pass filtered through a 650 nm dichroic mirror and focused onto the cortex through a Leica Planapo 2X lens (Leica Microsystems, Wetzlar, Germany). The emitted fluorescence was captured, long-pass filtered at >665 nm, and focused using a Leica Planapo 1X lens onto the detector chip of the camera. The detector chip was 100x100 pixels and received optical signals from a 4x4 mm region of cortex. Placement of the camera over barrel cortex was standardized using previously reported coordinates [9]. The camera was focused on the surface of the cortex and then adjusted to focus no more than 250 μm below the surface. Mechanical stimulation of contralateral whiskers was used to confirm barrel cortex location within the imaging window (Figure S3A).

Stimulation trials consisted of mechanical stimulation of the contralateral or ipsilateral whiskers. Stimulation trials were 4 s in duration and the onset of stimulation was marked with a button press. Behavioral trials were 16 s in duration, during which time the experimenter scored sleep and wake behaviors as VSD images were collected at 500 frames/s. dF/F0 was calculated and analyzed offline using custom-written procedures in MATLAB (MathWorks, Natick, MA). For offline analysis, a 2.5 x 2.5 mm region-of-interest (ROI) was constructed. For stimulation trials, dF/F0 values exceeding 1.5% were excluded from the analysis to minimize noise. The dF/F0 signal of the ROI was considered significant if it exceeded 7 times the standard deviation of the first 8 reference frames [9]. Only if activity in the ROI was significantly higher for contralateral stimulation trials (one-tailed t test; P < 0.05) were spontaneous behavior trials also analyzed.

For one additional 4-day-old rat, all whiskers except C2 and E2 were plucked on the side contralateral to the craniotomy. When the C2 whisker was stimulated, we observed evoked activity isolated to the C2 barrel (Figure S3B). Single whisker stimulation allowed us to orient a map of barrel cortex within the ROI.

Supplemental References 1. Gramsbergen, A., Schwartze, P., and Prechtl, H. (1970). The postnatal development of

behavioral states in the rat. Dev Psychobiol 3, 267–280. 2. Blumberg, M. S., and Seelke, A. M. H. (2010). The form and function of infant sleep: From

muscle to neocortex. In The Oxford Handbook of Developmental Behavioral Neuroscience, M. S. Blumberg, J. H. Freeman, and S. R. Robinson, eds. (New York: Oxford University Press), pp. 391–423.

3. Hill, D. N., Bermejo, R., Zeigler, H. P., and Kleinfeld, D. (2008). Biomechanics of the vibrissa motor plant in rat: rhythmic whisking consists of triphasic neuromuscular activity. J Neurosci 28, 3438–3455.

4. Seelke, A. M. H., and Blumberg, M. S. (2008). The microstructure of active and quiet sleep as cortical delta activity emerges in infant rats. Sleep 31, 691–699.

5. Mohns, E. J., and Blumberg, M. S. (2010). Neocortical activation of the hippocampus during sleep in newborn rats. J Neurosci 30, 3438–3449.

6. Marcano-Reik, A. J., and Blumberg, M. S. (2008). The corpus callosum modulates spindle-burst activity within homotopic regions of somatosensory cortex in newborn rats. Eur J Neurosci 28, 1457–1466.

7. Karlsson, K. Æ., Gall, A., Mohns, E. J., Seelke, A. M. H., and Blumberg, M. S. (2005). The neural substrates of infant sleep in rats. PLoS Biol 3, e143.

8. Karlsson, K. Æ., and Blumberg, M. S. (2005). Active medullary control of atonia in week-old rats. Neurosci 130, 275–283.

9. Yang, J.-W., An, S., Sun, J.-J., Reyes-Puerta, V., Kindler, J., Berger, T., Kilb, W., and Luhmann, H. J. (2012). Thalamic network oscillations synchronize ontogenetic columns in the newborn rat barrel cortex. Cereb Cortex.

rapid whisker movements in sleeping newborn rats...whiskers (black circles); also shown are the two...

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