The neural substrates of infant sleep in rats

Abstract

Sleep is a poorly understood behavior that predominates during infancy but is studied almost exclusively in adults. One perceived impediment to investigations of sleep early in ontogeny is the absence of state-dependent neocortical activity. Nonetheless, in infant rats, sleep is reliably characterized by the presence of tonic (i.e., muscle atonia) and phasic (i.e., myoclonic twitching) components; the neural circuitry underlying these components, however, is unknown. Recently, we described a medullary inhibitory area (MIA) in week-old rats that is necessary but not sufficient for the normal expression of atonia. Here we report that the infant MIA receives projections from areas containing neurons that exhibit state-dependent activity. Specifically, neurons within these areas, including the subcoeruleus (SubLC), pontis oralis (PO), and dorsolateral pontine tegmentum (DLPT), exhibit discharge profiles that suggest causal roles in the modulation of muscle tone and the production of myoclonic twitches. Indeed, lesions in the SubLC and PO decreased the expression of muscle atonia without affecting twitching (resulting in "REM sleep without atonia"), whereas lesions of the DLPT increased the expression of atonia while decreasing the amount of twitching. Thus, the neural substrates of infant sleep are strikingly similar to those of adults, a surprising finding in light of theories that discount the contribution of supraspinal neural elements to sleep before the onset of state-dependent neocortical activity.

Figure 1. DiI Infusion Sites

(A) Halos from 20 nl medial (n = 5) and lateral (n = 5) DiI infusions recreated from fluorescent photomicrographs on a coronal section of the medullary inhibitory area of a P8 rat. The largest (black) and smallest (gray) halos are shown. (B) Light photomicrograph of a representative DiI infusion site in the medial medulla. 4V, fourth ventricle; H, hypoglossal nucleus; Gi, nucleus gigantocellularis; IO, inferior olive; ST, spinal trigeminal nucleus

Figure 2. Representative Labeling of Cell Bodies in Selected Areas after a DiI Infusion into the Medullary Inhibitory Area

The arrows indicate examples of labeled neurons in (A) nucleus gigantocellularis (Gi), (B) subcoeruleus (SubLC), (C) dorsal raphé (DR), and (D) laterodorsal tegmental nucleus (LDT). Inset in (B) depicts enlarged view of the boxed area. 4V, fourth ventricle; AQ, cerebral aqueduct; DT, dorsal tegmental nucleus; Me5, mesencephalic trigeminal nucleus; ST, spinal trigeminal nucleus

Figure 3. Schematic Representation of the Classes of Neurons Identified in the Current Study and Their Prevalence

(A) Summary of the classes of discharge profiles described in the present study in relation to nuchal EMG activity. (B) Venn diagram depicting the interrelation of the classes of state-dependent neurons. Values indicate the number of neurons in each class found in the present study.

Figure 4. State-Dependent Neural Activity within Nucleus Gigantocellularis

(A) Recording sites of state-dependent neurons reconstructed on a coronal section of the medulla of a P8 rat. Note the anatomical overlap of the different classes of neurons. Arrow corresponds to top arrow in (B). (B) Photomicrograph depicting two marking lesions in nucleus gigantocellularis (the two lesions are approximately 80 μm apart). The white arrow indicates the location of the recording site identified by the arrow in (A). (C) Lower trace: single unit activity of a representative Gi EMG-on neuron. Upper trace: concurrently recorded nuchal EMG. Far right: Averaged waveform of representative EMG-on neuron. (D) Lower trace: single unit activity of a representative Gi atonia-on neuron. Upper trace: concurrently recorded nuchal EMG. Far right: Averaged waveform of representative atonia-on neuron. Gi, nucleus gigantocellularis; Pyr, pyramids; ST, spinal trigeminal nucleus; V, vestibular nucleus

Figure 5. State-Dependent Neural Activity within the Mesopontine Region

(A) Recording sites of state-dependent neurons reconstructed on a coronal section at the mesopontine level of a P8 rat. Note the predominance of atonia-on neurons. (B) Averaged waveform of a representative atonia-on neuron. (C) Upper trace: multiunit activity. Lower trace: concurrently recorded nuchal EMG. Spike sorting revealed two units that are easily distinguished by their amplitudes. The higher-amplitude unit is atonia-on; note its tonic discharge throughout the atonia period. (D) Upper trace: multiunit activity. Lower trace: concurrently recorded nuchal EMG. Spike sorting revealed two units that are easily distinguished by their amplitudes. The higher-amplitude unit is AS-on; note the absence of multiunit activity at the onset of the atonia period and then the increase in activity coinciding with the appearance of nuchal twitches. (E) Mean discharge rates of a representative AS-on neuron during bouts of AS and QS as defined, respectively, by the presence or absence of phasic nuchal twitches during periods of atonia. The arrowhead indicates the midpoint of the AS and QS bouts. 4V, fourth ventricle; LC, locus coeruleus; PC, nucleus pontis caudalis; SubLC, subcoeruleus;

Figure 6. Activity of LC Neurons across the Sleep-Wake Cycle in a P8 Rat

(A) Coronal section of the LC indicating the recording site (double arrows). (B) Averaged waveform of unit 2 in (C). (C) Upper trace: LC multiunit activity. Middle traces: Activity of two isolated units derived from the multiunit activity. Bottom trace: concurrently recorded nuchal EMG. 4V, fourth ventricle; LC, locus coeruleus

Figure 7. State-Dependent Neuronal Discharges within the Pontine Tegmentum

(A) Recording sites of state-dependent neurons reconstructed on a coronal section of the brainstem. Note the predominance of EMG-on neurons. (B) Averaged waveform of a representative EMG-on neuron. (C) Upper trace: multiunit activity. Lower trace: concurrently recorded nuchal EMG. One EMG-on neuron was isolated from the multiunit record; note its tonic discharge during the period of high muscle tone. (D) Upper trace: multiunit activity. Lower trace: concurrently recorded nuchal EMG. (E) Expanded view of the boxed area from (D). Note how multiunit activity precedes the twitch. Asterisks identify a single isolated unit. (F) Peristimulus histogram and raster plot for the twitch-on neuron identified in (E) during a 10-min recording session in a P7 rat (83 total twitches). Inset depicts 55 superimposed action potential waveforms for this unit. This unit's mean discharge rate peaks 5–10 ms before the twitch (red line). (G) Averaged nuchal EMG for all 83 twitches represented in (F). AQ, cerebral aqueduct; DT, dorsal tegmental nucleus; LDT, laterodorsal tegmental nucleus; PO, nucleus pontis oralis

Figure 8. Effects of Brainstem Lesions on the Expression of Nuchal Muscle tone in P8 Rats

(A) Representations of lesions of the dorsolateral pontine tegmentum (DLPT; blue), pontis oralis (PO; green), and subcoeruleus (SubLC; red) on coronal sections. Outlined areas indicate the extent of all lesions in a group, and filled areas indicate the smallest lesion in a group. (B) Mean ± standard error bout durations of atonia and high muscle tone for sham (n = 7; black), PO lesion (n = 5; green), and SubLC lesion (n = 4; red) groups. † Significant difference from sham group, p < 0.05. Pie charts beneath each bar graph indicate percentage of time spent in atonia or high muscle tone. * Significant difference from sham group, p < 0.05. (C) Mean ± standard error bout durations of atonia and high muscle tone for sham (n = 7; black) and DLPT lesion (n = 6; blue) groups. † Significant difference from sham group, p < 0.05. Pie charts beneath each bar graph indicate percentage of time spent in atonia or high muscle tone. * Significant difference from sham group, p < 0.05. (D) Representative 4-min recordings of nuchal EMG for a sham pup and for pups with PO, SubLC, and DLPT lesions. DT, dorsal tegmental nucleus; IC, inferior colliculus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; PC, nucleus pontis caudalis; PO, nucleus pontis oralis