REM sleep twitches rouse nascent cerebellar circuits: Implications for sensorimotor development

Abstract

The cerebellum is critical for sensorimotor integration and undergoes extensive postnatal development. During the first postnatal week in rats, climbing fibers polyinnervate Purkinje cells and, before granule cell migration, mossy fibers make transient, direct connections with Purkinje cells. Activity-dependent processes are assumed to play a critical role in the development and refinement of these and other aspects of cerebellar circuitry. However, the sources and patterning of activity have not been described. We hypothesize that sensory feedback (i.e., reafference) from myoclonic twitches in sleeping newborn rats is a prominent driver of activity for the developing cerebellum. Here, in 6-day-old rats, we show that Purkinje cells exhibit substantial state-dependent changes in complex and simple spike activity-primarily during active sleep. In addition, this activity increases significantly during bouts of twitching. Moreover, the surprising observation of twitch-dependent increases in simple spike activity at this age suggests a functional engagement of mossy fibers before the parallel fiber system has developed. Based on these and other results, we propose that twitching comprises a unique class of self-produced movement that drives critical aspects of activity-dependent development in the cerebellum and other sensorimotor systems.

Keywords: cerebellum; corollary discharge; myoclonic twitching; reafference; sleep.

Figure 1

Purkinje cell activity in 6-day-old rats during sleep and wake. (A) The location of the recording sites (white dots) in this study. All sites were located in the Purkinje cell layer and typically yielded more than one unit. The yellow arrow indicates the track of a silicon electrode in cerebellar cortex, traversing multiple cell layers in simplex. Inset: an expanded view of the location of that track. The track was visualized by coating the electrode with fluorescent DiI. dcn, deep cerebellar nuclei; pl, Purkinje layer; egl, external granular layer. (B) Representative sample of data showing occurrences of behaviorally scored twitches (vertical green ticks), nuchal EMG, and Purkinje cell activity during sleep and wake. The neural activity (recorded from vermis) is expanded at bottom to highlight instances of complex spike (CS) and simple spike (SS) activity. (C) Frequency distribution of the number of individual spikes comprising complex spikes across all 30 Purkinje cells. Most, but not all, complex spikes were “doublets,” consistent with previous reports in infant rats (Crepel, 1971; Puro and Woodward, 1977a). Inset: Complex spikes were defined on the basis of action potentials exhibiting interspike intervals (ISIs) ≤15 ms. The example shown here has an ISI of 9 ms.

Figure 2

(A) Interspike intervals (ISIs) for all action potentials across all pups. Complex spikes were defined as 2 or more action potentials with ISIs ≤ 15 ms (red bars). All other unit activity was classified as simple spikes (black bars). (B) Representative Purkinje cell recording from a 6-day-old rat to show a train of simple spikes. (C) Perievent histogram plotting simple spike firing rate per 10-ms bin in relation to complex spikes in a sleeping 6-day-old rat. The red bar designates the period when, by our definition of complex spikes, a simple spike could not occur. Note the suppression of simple spike activity after complex spikes as well as the presence of significantly increased simple spike activity before, but not after, complex spikes. * significant increase in relation to jittered values, p < 0.05; † significant decrease in relation to jittered values, p < 0.05.

Figure 3

Complex and simple spike activity is state-dependent. (A) Firing rate (Hz) of complex spikes (top) and simple spikes (bottom) during wake, quiet sleep (QS), and active sleep (AS) in Purkinje cells characterized as AS-On and AS/Wake-On (data for Sleep-On, Wake-On, and State-Independent categories not shown). * significantly different from the other two groups (p < 0.017). (B) Percentage of complex and simple spikes (n=30 in each group) characterized as AS-On, AS/Wake-On, Sleep-On, Wake-On, and State-Independent. (C) Firing rate (Hz) of complex spikes (top) and simple spikes (bottom) during wake, quiet sleep, and active sleep in Purkinje cells regardless of state. * significantly different from the other two groups (p < 0.017).

Figure 4

Complex and simple spike activity increases immediately after twitches. (A) Perievent histogram plotting the number of complex spikes per 10-ms bin in relation to myoclonic twitching in sleeping 6-day-old rats. Data were pooled across all 30 units. The vertical black bar is the last bin before a twitch and the horizontal red bars indicate statistical significance (p < 0.05), as determined using a jitter protocol (see Materials and Methods). (B) Same as in (A) except for simple spikes (p < 0.05). (C) Perievent line histograms for complex spikes broken down for the W1 (red), W2 (green), and W3 (blue) units, as well as the units that showed no window preference (NP, black). For a small subset (10%) of all twitches analyzed, a unit that fired in one window fired again in one or both of the other windows. There was no clear relationship between a unit’s window assignment and its state-dependency. The data were smoothed using a 3-bin moving window. * significant peak within window (p < 0.0125). (D) Same as in (C) except for simple spikes. * significant peak within window (p < 0.0125).

Figure 5

Twitch-related complex and simple spike activity for AS-On units. Perievent line histograms plotting complex (A) and simple spike (B) counts per 10-ms bin in relation to myoclonic twitching in sleeping 6-day-old rats. Each plot is broken down for the W1 (red), W2 (green), and units that showed no window preference (NP, black). All data were smoothed using a 3-bin moving window. * significant peak within window (p < 0.017). (C) Representative data showing occurrences of behaviorally scored twitches from all visible limbs and tail (vertical green ticks), complex spikes (vertical purple ticks), nuchal EMG, and Purkinje cell activity (recorded from crus I) during periods of active sleep and active wake. Note the increased Purkinje cell and complex spike activity during periods of active sleep.

Figure 6

Complex spike auto-rhythmicity in sleeping 6-day-old rats. For three individual units, autocorrelograms are shown at left and spectral density plots are shown at right. (A) A unit exhibiting strong 10-Hz rhythmicity. This unit was Sleep-On and did not exhibit a window preference. (B) A unit exhibiting strong 8-Hz rhythmicity. This unit was State-Independent and exhibited a preference for Window 2 (W2). (C) A unit exhibiting weaker rhythmicity at 11.5 Hz. This unit was As-On and exhibited a preference for Window 1 (W1 unit). These three units were recorded from two pups.

Figure 7

Hypothesized framework relating self-produced and other-produced movements with the accompaniment of corollary discharge. Conventionally, self-produced movements generate reafference that is accompanied by corollary discharge, whereas other-produced movements generate exafference that is not accompanied by corollary discharge (green cells). For obvious reasons, other-produced movements cannot be accompanied by corollary discharge (n/a: not applicable). Here we propose a third possible category whereby sleep-related twitches are self-produced movements that are not accompanied by corollary discharge (red cell).