Coincident development and synchronization of sleep-dependent delta in the cortex and medulla.

In early development, active sleep is the predominant sleep state before it is supplanted by quiet sleep. In rats, the developmental increase in quiet sleep is accompanied by the sudden emergence of the cortical delta rhythm (0.5-4 Hz) around postnatal day 12 (P12). We sought to explain the emergence of the cortical delta by assessing developmental changes in the activity of the parafacial zone (PZ), a medullary structure thought to regulate quiet sleep in adults. We recorded from the PZ in P10 and P12 rats and predicted an age-related increase in neural activity during increasing periods of delta-rich cortical activity. Instead, during quiet sleep, we discovered sleep-dependent rhythmic spiking activity-with intervening periods of total silence-phase locked to a local delta rhythm. Moreover, PZ and cortical delta were coherent at P12 but not at P10. PZ delta was also phase locked to respiration, suggesting sleep-dependent modulation of PZ activity by respiratory pacemakers in the ventral medulla. Disconnecting the main olfactory bulbs from the cortex did not diminish cortical delta, indicating that the influence of respiration on delta at this age is not mediated indirectly through nasal breathing. Finally, we observed an increase in parvalbumin-expressing terminals in the PZ across these ages, supporting a role for local GABAergic inhibition in the PZ's rhythmicity. The unexpected discovery of delta-rhythmic neural activity in the medulla-when cortical delta is also emerging-provides a new perspective on the brainstem's role in regulating sleep and promoting long-range functional connectivity in early development.

Developmentally Unique Cerebellar Processing Prioritizes Self- over Other-Generated Movements.

Animals must distinguish the sensory consequences of self-generated movements (reafference) from those of other-generated movements (exafference). Only self-generated movements entail the production of motor copies (i.e., corollary discharges), which are compared with reafference in the cerebellum to compute predictive or internal models of movement. Internal models emerge gradually over the first three postnatal weeks in rats through a process that is not yet fully understood. Previously, we demonstrated in postnatal day (P) 8 and P12 rats that precerebellar nuclei convey corollary discharge and reafference to the cerebellum during active (REM) sleep when pups produce limb twitches. Here, recording from a deep cerebellar nucleus (interpositus, IP) in P12 rats of both sexes, we compared reafferent and exafferent responses with twitches and limb stimulations, respectively. As expected, most IP units showed robust responses to twitches. However, in contrast with other sensory structures throughout the brain, relatively few IP units showed exafferent responses. Upon finding that exafferent responses occurred in pups under urethane anesthesia, we hypothesized that urethane inhibits cerebellar cortical cells, thereby disinhibiting exafferent responses in IP. In support of this hypothesis, ablating cortical tissue dorsal to IP mimicked the effects of urethane on exafference. Finally, the results suggest that twitch-related corollary discharge and reafference are conveyed simultaneously and in parallel to cerebellar cortex and IP. Based on these results, we propose that twitches provide opportunities for the nascent cerebellum to integrate somatotopically organized corollary discharge and reafference, thereby enabling the development of closed-loop circuits and, subsequently, internal models.

Developmentally unique cerebellar processing prioritizes self-over other-generated movements.

Animals must distinguish the sensory consequences of self-generated movements (reafference) from those of other-generated movements (exafference). Only self-generated movements entail the production of motor copies (i.e., corollary discharges), which are compared with reafference in the cerebellum to compute predictive or internal models of movement. Internal models emerge gradually over the first three postnatal weeks in rats through a process that is not yet fully understood. Previously, we demonstrated in postnatal day (P) P8 and P12 rats that precerebellar nuclei convey corollary discharge and reafference to the cerebellum during active (REM) sleep when pups produce limb twitches. Here, recording from a deep cerebellar nucleus (interpositus, IP) in P12 rats of both sexes, we compared reafferent and exafferent responses to twitches and limb stimulations, respectively. As expected, most IP units showed robust responses to twitches. However, in contrast with other sensory structures throughout the brain, relatively few IP units showed exafferent responses. Upon finding that exafferent responses occurred in pups under urethane anesthesia, we hypothesized that urethane inhibits cerebellar cortical cells, thereby disinhibiting exafferent responses in IP. In support of this hypothesis, ablating cortical tissue dorsal to IP mimicked the effects of urethane on exafference. Finally, the results suggest that twitch-related corollary discharge and reafference are conveyed simultaneously and in parallel to cerebellar cortex and IP. Based on these results, we propose that twitches provide opportunities for the nascent cerebellum to integrate somatotopically organized corollary discharge and reafference, thereby enabling the development of closed-loop circuits and, subsequently, internal models.

DELTA-RHYTHMIC ACTIVITY IN THE MEDULLA DEVELOPS COINCIDENT WITH CORTICAL DELTA IN SLEEPING INFANT RATS.

In early development, active sleep is the predominant sleep state before it is supplanted by quiet sleep. In rats, the developmental increase in quiet sleep is accompanied by the sudden emergence of the cortical delta rhythm (0.5-4 Hz) around postnatal day 12 (P12). We sought to explain the emergence of cortical delta by assessing developmental changes in the activity of the parafacial zone (PZ), a medullary structure thought to regulate quiet sleep in adults. We recorded from PZ in P10 and P12 rats and predicted an age-related increase in neural activity during increasing periods of delta-rich cortical activity. Instead, during quiet sleep we discovered sleep-dependent rhythmic spiking activity-with intervening periods of total silence-phase-locked to a local delta rhythm. Moreover, PZ and cortical delta were coherent at P12, but not at P10. PZ delta was also phase-locked to respiration, suggesting sleep-dependent modulation of PZ activity by respiratory pacemakers in the ventral medulla. Disconnecting the main olfactory bulbs from the cortex did not diminish cortical delta, indicating that the influence of respiration on delta at this age is not mediated indirectly through nasal breathing. Finally, we observed an increase in parvalbumin-expressing terminals in PZ across these ages, supporting a role for GABAergic inhibition in PZ's rhythmicity. The discovery of delta-rhythmic neural activity in the medulla-when cortical delta is also emerging-opens a new path to understanding the brainstem's role in regulating sleep and synchronizing rhythmic activity throughout the brain.

Neural decoding reveals specialized kinematic tuning after an abrupt cortical transition.

The primary motor cortex (M1) exhibits a protracted period of development, including the development of a sensory representation long before motor outflow emerges. In rats, this representation is present by postnatal day (P) 8, when M1 activity is "discontinuous." Here, we ask how the representation changes upon the transition to "continuous" activity at P12. We use neural decoding to predict forelimb movements from M1 activity and show that a linear decoder effectively predicts limb movements at P8 but not at P12; instead, a nonlinear decoder better predicts limb movements at P12. The altered decoder performance reflects increased complexity and uniqueness of kinematic information in M1. We next show that M1's representation at P12 is more susceptible to "lesioning" of inputs and "transplanting" of M1's encoding scheme from one pup to another. Thus, the emergence of continuous M1 activity signals the developmental onset of more complex, informationally sparse, and individualized sensory representations.

Cortical Representation of Movement Across the Developmental Transition to Continuous Neural Activity.

Primary motor cortex (M1) exhibits a protracted period of development that includes the establishment of a somatosensory map long before motor outflow emerges. In rats, the sensory representation is established by postnatal day (P) 8 when cortical activity is still "discontinuous." Here, we ask how the representation survives the sudden transition to noisy "continuous" activity at P12. Using neural decoding to predict forelimb movements based solely on M1 activity, we show that a linear decoder is sufficient to predict limb movements at P8, but not at P12; in contrast, a nonlinear decoder effectively predicts limb movements at P12. The change in decoder performance at P12 reflects an increase in both the complexity and uniqueness of kinematic information available in M1. We next show that the representation at P12 is more susceptible to the deleterious effects of "lesioning" inputs and to "transplanting" M1's encoding scheme from one pup to another. We conclude that the emergence of continuous cortical activity signals the developmental onset in M1 of more complex, informationally sparse, and individualized sensory representations.

Movements during sleep reveal the developmental emergence of a cerebellar-dependent internal model in motor thalamus.

With our eyes closed, we can track a limb's moment-to-moment location in space. If this capacity relied solely on sensory feedback from the limb, we would always be a step behind because sensory feedback takes time: for the execution of rapid and precise movements, such lags are not tolerable. Nervous systems solve this problem by computing representations-or internal models-that mimic movements as they are happening, with the associated neural activity occurring after the motor command but before sensory feedback. Research in adults indicates that the cerebellum is necessary to compute internal models. What is not known, however, is when-and under what conditions-this computational capacity develops. Here, taking advantage of the unique kinematic features of the discrete, spontaneous limb twitches that characterize active sleep, we captured the developmental emergence of a cerebellar-dependent internal model. Using rats at postnatal days (P) 12, P16, and P20, we compared neural activity in the ventral posterior (VP) and ventral lateral (VL) thalamic nuclei, both of which receive somatosensory input but only the latter of which receives cerebellar input. At all ages, twitch-related activity in VP lagged behind the movement, consistent with sensory processing; similar activity was observed in VL through P16. At P20, however, VL activity no longer lagged behind movement but instead precisely mimicked the movement itself; this activity depended on cerebellar input. In addition to demonstrating the emergence of internal models of movement, these findings implicate twitches in their development and calibration through, at least, the preweanling period.

Sensory Coding of Limb Kinematics in Motor Cortex across a Key Developmental Transition.

Primary motor cortex (M1) undergoes protracted development in mammals, functioning initially as a sensory structure. Throughout the first postnatal week in rats, M1 is strongly activated by self-generated forelimb movements-especially by the twitches that occur during active sleep. Here, we quantify the kinematic features of forelimb movements to reveal receptive-field properties of individual units within the forelimb region of M1. At postnatal day 8 (P8), nearly all units were strongly modulated by movement amplitude, especially during active sleep. By P12, only a minority of units continued to exhibit amplitude tuning, regardless of behavioral state. At both ages, movement direction also modulated M1 activity, though to a lesser extent. Finally, at P12, M1 population-level activity became more sparse and decorrelated, along with a substantial alteration in the statistical distribution of M1 responses to limb movements. These findings reveal a transition toward a more complex and informationally rich representation of movement long before M1 develops its motor functionality.SIGNIFICANCE STATEMENT Primary motor cortex (M1) plays a fundamental role in the generation of voluntary movements and motor learning in adults. In early development, however, M1 functions as a prototypical sensory structure. Here, we demonstrate in infant rats that M1 codes for the kinematics of self-generated limb movements long before M1 develops its capacity to drive movements themselves. Moreover, we identify a key transition during the second postnatal week in which M1 activity becomes more informationally complex. Together, these findings further delineate the complex developmental path by which M1 develops its sensory functions in support of its later-emerging motor capacities.

Twitches emerge postnatally during quiet sleep in human infants and are synchronized with sleep spindles.

In humans and other mammals, the stillness of sleep is punctuated by bursts of rapid eye movements (REMs) and myoclonic twitches of the limbs.1 Like the spontaneous activity that arises from the sensory periphery in other modalities (e.g., retinal waves),2 sensory feedback arising from twitches is well suited to drive activity-dependent development of the sensorimotor system.3 It is partly because of the behavioral activation of REM sleep that this state is also called active sleep (AS), in contrast with the behavioral quiescence that gives quiet sleep (QS)-the second major stage of sleep-its name. In human infants, for which AS occupies 8 h of each day,4 twitching helps to identify the state;5-8 nonetheless, we know little about the structure and functions of twitching across development. Recently, in sleeping infants,9 we documented a shift in the temporal expression of twitching beginning around 3 months of age that suggested a qualitative change in how twitches are produced. Here, we combine behavioral analysis with high-density electroencephalography (EEG) to demonstrate that this shift reflects the emergence of limb twitches during QS. Twitches during QS are not only unaccompanied by REMs, but they also occur synchronously with sleep spindles, a hallmark of QS. As QS-related twitching increases with age, sleep spindle rate also increases along the sensorimotor strip. The emerging synchrony between subcortically generated twitches and cortical oscillations suggests the development of functional connectivity among distant sensorimotor structures, with potential implications for detecting and explaining atypical developmental trajectories.

Parallel and Serial Sensory Processing in Developing Primary Somatosensory and Motor Cortex.

It is generally supposed that primary motor cortex (M1) receives somatosensory input predominantly via primary somatosensory cortex (S1). However, a growing body of evidence indicates that M1 also receives direct sensory input from the thalamus, independent of S1; such direct input is particularly evident at early ages before M1 contributes to motor control. Here, recording extracellularly from the forelimb regions of S1 and M1 in unanesthetized rats at postnatal day (P)8 and P12, we compared S1 and M1 responses to self-generated (i.e., reafferent) forelimb movements during active sleep and wake, and to other-generated (i.e., exafferent) forelimb movements. At both ages, reafferent responses were processed in parallel by S1 and M1; in contrast, exafferent responses were processed in parallel at P8 but serially, from S1 to M1, at P12. To further assess this developmental difference in processing, we compared exafferent responses to proprioceptive and tactile stimulation. At both P8 and P12, proprioceptive stimulation evoked parallel responses in S1 and M1, whereas tactile stimulation evoked parallel responses at P8 and serial responses at P12. Independent of the submodality of exafferent stimulation, pairs of S1-M1 units exhibited greater coactivation during active sleep than wake. These results indicate that S1 and M1 independently develop somatotopy before establishing the interactive relationship that typifies their functionality in adults.SIGNIFICANCE STATEMENT Learning any new motor task depends on the ability to use sensory information to update motor outflow. Thus, to understand motor learning, we must also understand how animals process sensory input. Primary somatosensory cortex (S1) and primary motor cortex (M1) are two interdependent structures that process sensory input throughout life. In adults, the functional relationship between S1 and M1 is well established; however, little is known about how S1 and M1 begin to transmit or process sensory information in early life. In this study, we investigate the early development of S1 and M1 as a sensory processing unit. Our findings provide new insights into the fundamental principles of sensory processing and the development of functional connectivity between these important sensorimotor structures.

THE DEVELOPING BRAIN REVEALED DURING SLEEP.

Given the prevalence of sleep in early development, any satisfactory account of infant brain activity must consider what happens during sleep. Only recently, however, has it become possible to record sleep-related brain activity in newborn rodents. Using such methods in rat pups, it is now clear that sleep, more so than wake, provides a critical context for the processing of sensory input and the expression of functional connectivity throughout the sensorimotor system. In addition, sleep uniquely reveals functional activity in the developing primary motor cortex, which establishes a somatosensory map long before its role in motor control emerges. These findings will inform our understanding of the developmental processes that contribute to the nascent sense of embodiment in human infants.

Self-Generated Whisker Movements Drive State-Dependent Sensory Input to Developing Barrel Cortex.

Cortical development is an activity-dependent process [1-3]. Regarding the role of activity in the developing somatosensory cortex, one persistent debate concerns the importance of sensory feedback from self-generated movements. Specifically, recent studies claim that cortical activity is generated intrinsically, independent of movement [3, 4]. However, other studies claim that behavioral state moderates the relationship between movement and cortical activity [5-7]. Thus, perhaps inattention to behavioral state leads to failures to detect movement-driven activity [8]. Here, we resolve this issue by associating local field activity (i.e., spindle bursts) and unit activity in the barrel cortex of 5-day-old rats with whisker movements during wake and myoclonic twitches of the whiskers during active (REM) sleep. Barrel activity increased significantly within 500 ms of whisker movements, especially after twitches. Also, higher-amplitude movements were more likely to trigger barrel activity; when we controlled for movement amplitude, barrel activity was again greater after a twitch than a wake movement. We then inverted the analysis to assess the likelihood that increases in barrel activity were preceded within 500 ms by whisker movements: at least 55% of barrel activity was attributable to sensory feedback from whisker movements. Finally, when periods with and without movement were compared, 70%-75% of barrel activity was movement related. These results confirm the importance of sensory feedback from movements in driving activity in sensorimotor cortex and underscore the necessity of monitoring sleep-wake states to ensure accurate assessments of the contributions of the sensory periphery to activity in developing somatosensory cortex.

Spatiotemporal organization of myoclonic twitching in sleeping human infants.

During the perinatal period in mammals when active sleep predominates, skeletal muscles twitch throughout the body. We have hypothesized that myoclonic twitches provide unique insight into the functional status of the human infant's nervous system. However, assessments of the rate and patterning of twitching have largely been restricted to infant rodents. Thus, here we analyze twitching in human infants over the first seven postnatal months. Using videography and behavioral measures of twitching during bouts of daytime sleep, we find at all ages that twitching across the body occurs predominantly in bursts at intervals of 10 s or less. We also find that twitching is expressed differentially across the body and with age. For example, twitching of the face and head is most prevalent shortly after birth and decreases over the first several months. In addition, twitching of the hands and feet occurs at a consistently higher rate than does twitching elsewhere in the body. Finally, the patterning of twitching becomes more structured with age, with twitches of the left and right hands and feet exhibiting the strongest coupling. Altogether, these findings support the notion that twitches can provide a unique source of information about typical and atypical sensorimotor development.

Active Sleep Promotes Coherent Oscillatory Activity in the Cortico-Hippocampal System of Infant Rats.

Active sleep (AS) provides a unique developmental context for synchronizing neural activity within and between cortical and subcortical structures. In week-old rats, sensory feedback from myoclonic twitches, the phasic motor activity that characterizes AS, promotes coherent theta oscillations (4-8 Hz) in the hippocampus and red nucleus, a midbrain motor structure. Sensory feedback from twitches also triggers rhythmic activity in sensorimotor cortex in the form of spindle bursts, which are brief oscillatory events composed of rhythmic components in the theta, alpha/beta (8-20 Hz), and beta2 (20-30 Hz) bands. Here we ask whether one or more of these spindle-burst components are communicated from sensorimotor cortex to hippocampus. By recording simultaneously from whisker barrel cortex and dorsal hippocampus in 8-day-old rats, we show that AS, but not other behavioral states, promotes cortico-hippocampal coherence specifically in the beta2 band. By cutting the infraorbital nerve to prevent the conveyance of sensory feedback from whisker twitches, cortical-hippocampal beta2 coherence during AS was substantially reduced. These results demonstrate the necessity of sensory input, particularly during AS, for coordinating rhythmic activity between these two developing forebrain structures.

Genome-Wide Association Study in Two Cohorts from a Multi-generational Mouse Advanced Intercross Line Highlights the Difficulty of Replication Due to Study-Specific Heterogeneity.

There has been extensive discussion of the "Replication Crisis" in many fields, including genome-wide association studies (GWAS). We explored replication in a mouse model using an advanced intercross line (AIL), which is a multigenerational intercross between two inbred strains. We re-genotyped a previously published cohort of LG/J x SM/J AIL mice (F34; n = 428) using a denser marker set and genotyped a new cohort of AIL mice (F39-43; n = 600) for the first time. We identified 36 novel genome-wide significant loci in the F34 and 25 novel loci in the F39-43 cohort. The subset of traits that were measured in both cohorts (locomotor activity, body weight, and coat color) showed high genetic correlations, although the SNP heritabilities were slightly lower in the F39-43 cohort. For this subset of traits, we attempted to replicate loci identified in either F34 or F39-43 in the other cohort. Coat color was robustly replicated; locomotor activity and body weight were only partially replicated, which was inconsistent with our power simulations. We used a random effects model to show that the partial replications could not be explained by Winner's Curse but could be explained by study-specific heterogeneity. Despite this heterogeneity, we performed a mega-analysis by combining F34 and F39-43 cohorts (n = 1,028), which identified four novel loci associated with locomotor activity and body weight. These results illustrate that even with the high degree of genetic and environmental control possible in our experimental system, replication was hindered by study-specific heterogeneity, which has broad implications for ongoing concerns about reproducibility.

Behavioral states modulate sensory processing in early development.

PURPOSE OF REVIEW: Sleep-wake states modulate cortical activity in adults. In infants, however, such modulation is less clear; indeed, early cortical activity comprises bursts of neural activity driven predominantly by peripheral sensory input. Consequently, in many studies of sensory development in rodents, sensory processing has been carefully investigated, but the modulatory role of behavioral state has typically been ignored. RECENT FINDINGS: In the developing visual and somatosensory systems, it is now known that sleep and wake states modulate sensory processing. Further, in both systems, the nature of this modulation shifts rapidly during the second postnatal week, with subcortical nuclei changing how they gate sensory inputs. SUMMARY: The interactions among sleep and wake movements, sensory processing, and development are dynamic and complex. Now that established methods exist to record neural activity in unanesthetized infant animals, we can provide a more comprehensive understanding of how infant sleep-wake states interact with sensory-driven responses to promote developmental plasticity.

Corollary discharge in precerebellar nuclei of sleeping infant rats.

In week-old rats, somatosensory input arises predominantly from external stimuli or from sensory feedback (reafference) associated with myoclonic twitches during active sleep. A previous study suggested that the brainstem motor structures that produce twitches also send motor copies (or corollary discharge, CD) to the cerebellum. We tested this possibility by recording from two precerebellar nuclei-the inferior olive (IO) and lateral reticular nucleus (LRN). In most IO and LRN neurons, twitch-related activity peaked sharply around twitch onset, consistent with CD. Next, we identified twitch-production areas in the midbrain that project independently to the IO and LRN. Finally, we blocked calcium-activated slow potassium (SK) channels in the IO to explain how broadly tuned brainstem motor signals can be transformed into precise CD signals. We conclude that the precerebellar nuclei convey a diversity of sleep-related neural activity to the developing cerebellum to enable processing of convergent input from CD and reafferent signals.

Wakefulness suppresses retinal wave-related neural activity in visual cortex.

In the developing visual system before eye opening, spontaneous retinal waves trigger bursts of neural activity in downstream structures, including visual cortex. At the same ages when retinal waves provide the predominant input to the visual system, sleep is the predominant behavioral state. However, the interactions between behavioral state and retinal wave-driven activity have never been explicitly examined. Here we characterized unit activity in visual cortex during spontaneous sleep-wake cycles in 9- and 12-day-old rats. At both ages, cortical activity occurred in discrete rhythmic bursts, ~30-60 s apart, mirroring the timing of retinal waves. Interestingly, when pups spontaneously woke up and moved their limbs in the midst of a cortical burst, the activity was suppressed. Finally, experimentally evoked arousals also suppressed intraburst cortical activity. All together, these results indicate that active wake interferes with the activation of the developing visual cortex by retinal waves. They also suggest that sleep-wake processes can modulate visual cortical plasticity at earlier ages than has been previously considered.NEW & NOTEWORTHY By recording in visual cortex in unanesthetized infant rats, we show that neural activity attributable to retinal waves is specifically suppressed when pups spontaneously awaken or are experimentally aroused. These findings suggest that the relatively abundant sleep of early development plays a permissive functional role for the visual system. It follows, then, that biological or environmental factors that disrupt sleep may interfere with the development of these neural networks.

Theta Oscillations during Active Sleep Synchronize the Developing Rubro-Hippocampal Sensorimotor Network.

Neuronal oscillations comprise a fundamental mechanism by which distant neural structures establish and express functional connectivity. Long-range functional connectivity between the hippocampus and other forebrain structures is enabled by theta oscillations. Here, we show for the first time that the infant rat red nucleus (RN)-a brainstem sensorimotor structure-exhibits theta (4-7 Hz) oscillations restricted primarily to periods of active (REM) sleep. At postnatal day 8 (P8), theta is expressed as brief bursts immediately following myoclonic twitches; by P12, theta oscillations are expressed continuously across bouts of active sleep. Simultaneous recordings from the hippocampus and RN at P12 show that theta oscillations in both structures are coherent, co-modulated, and mutually interactive during active sleep. Critically, at P12, inactivation of the medial septum eliminates theta in both structures. The developmental emergence of theta-dependent functional coupling between the hippocampus and RN parallels that between the hippocampus and prefrontal cortex. Accordingly, disruptions in the early expression of theta could underlie the cognitive and sensorimotor deficits associated with neurodevelopmental disorders such as autism and schizophrenia.