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Selective optical drive of thalamic reticular nucleus generates thalamic bursts & cortical spindles

The thalamic reticular nucleus (TRN) is hypothesized to regulate neocortical rhythms and behavioral state. Using optogenetics and multi-electrode recording in behaving mice, we found that brief selective drive of TRN switched thalamocortical firing mode from tonic to bursting and generated state-dependent neocortical spindles. These findings provide causal support for the TRN in state-regulation in vivo and introduce a new model for addressing the role of this structure in behavior.

In this study, we sought to selectively and causally test the role of the TRN in thalamic bursting and the generation of neocortical spindles. Several thalamocortical and corticothalamic fibers pass through the TRN, making selective electrical stimulation of this structure impossible. To manipulate TRN function in vivo, we used a transgenic animal in which the light-activated ion channel, channelrhodospin-2 (ChR2), was expressed under the control of the vesicular GABA transporter (VGAT2). Histological characterization of brain sections derived from VGAT-ChR2 animals revealed expression of ChR2 in GABAergic neurons of several brain regions. Somatosensory thalamic expression of ChR2 was limited to TRN ( Fig. S1A-C) and was several-fold higher than that in the neocortex (p < 0.001) (Fig. S1D-E). When recording extracellularly from putative TRN units in these mice, we observed short latency, high-reliability spiking in response to brief pulses of 473 nm light (Fig. S2).
We isolated 11 TC neurons, and recorded cortical local field potential (LFP) and surface electroencephalography (EEG) across six sessions in three freely behaving VGAT-ChR2 mice using integrated optical fiber/dual site multielectrode implants (Fig. 1a). Thalamic stereotrodes yielded well-isolated putative TC single units (Fig. S2), whose firing was altered by delivering 20 msec blue laser pulses to the somatosensory section of the TRN (Fig. 1b, c). Though the overall firing rate in the 1 second period post stimulation did not significantly change (pre vs. post, p = 0.4131), following a brief suppression period, TC units exhibited "bursts" (Fig 1d, green arrows) characterized by smaller interspike intervals (ISI) than the pre-stimulus period (Fig. 1e). Enhanced probability of bursting (as defined by two or more spikes of ISI < 4 msec, preceded by 100 msec of silence 11 ) was sustained for hundreds of milliseconds following stimulation, across all units (Fig. 1f, Fig. S3; pre vs. post, p<0.0028, Wilcoxon signed-rank test). The probability of bursting in the second following a light pulse for single units was 26% (vs. 9% in the second prior to the light pulse; p < 0.05, Wilcoxon signed-rank test; Fig. S4 shows the distribution across cells). The probability that at least one neuron in our recorded sample (either 3 or 4 units/session) would burst following each light pulse was 72% (55-89%; N = 4 sessions). Burst probability peaked in the bin 100-200 msec post-stimulation, consistent with 'rebound' bursting of TC cells following inhibition generated by TRN activation.
Single 20 msec optical pulses delivered to the TRN were occasionally followed by neocortical spindle oscillations that were indistinguishable from spontaneous spindle oscillations recorded in the same animals (Fig. 2a). Evoked spindles were detected at the level of neocortical LFP and EEG and were preceded by thalamic activity (Fig. 2a). Spectrograms of thalamic LFP, cortical LFP and surface EEG showed a pronounced increase in power following TRN stimulation that was maximal in the spindle frequency range (Fig. 2b). Spontaneous and evoked neocortical spindles were not time-locked to bursting or LFP activity in the spindle range at the site of recording electrodes in thalamus (Fig. 2a, Fig. S5). These results in naturally sleeping mice are in apparent contrast to prior observations of sustained spindles and widely synchronous bursting in thalamic relay centers during neocortical spindles in barbiturate anesthetized cats 12 , but are in agreement with recent intracranial human recordings in sleep showing localized spindle expression 13 .
Spontaneous spindle events are observed in non-rapid eye movement (NREM) sleep 9 . To test whether optically-driven spindle generation was state dependent, we used video recording to monitor behavior in addition to electrophysiological recording. As described for other mammalian species 14 , mice in this study showed state-dependent changes in TC neural firing rate that paralleled behavioral and EEG changes (Fig. 2c). Based on monitoring gross behavioral changes (activity vs. inactivity), we observed that brief optical activation of the TRN resulted in state-dependent changes in EEG spindle power increase (example session, Fig. 2d). A significant negative correlation between firing rate and probability of spindle induction was observed (Fig. S6). Using polysomnography with combined EEG and EMG in two additional VGAT-ChR2 mice to fully characterize this state-dependence, we found that spindle induction was largely limited to NREM sleep: Only 7% of all induced spindles occurred in electrographically identified wake epochs, and none occurred in REM sleep (spindles/laser trials: NREM 19.56%, Wake 1.27%, REM 0%, p<0.001; Fig. S7).
Our findings support the hypothesis that the TRN controls TC neural output by switching its firing mode (Fig. 1). Whether burst firing is used in the brain to enhance transmission of sensory input is unknown and is a key point of debate in sensory neuroscience. Several preparations across different modalities have shown that sensory drive can induce bursts, suggesting these events could differentially impact processing 1 . The ability to record and control thalamocortical relay cell activity described here during free behavior is a crucial step toward answering this important question. Combining selective optical drive of TRN with the precise control of sensory input afforded by head-fixed preparations can address the role of sensory-driven burst activity in stimulus detection.
Our findings provide the first demonstration that TRN activation drives neocortical spindle oscillations in the intact mouse brain (Fig. 2). These spindles were largely limited to NREM sleep, suggesting that optical stimulation was utilizing endogenous physiological mechanisms of spindle generation. Selective silencing of TRN, by optical or other means, will be required to establish a full causal relation between TRN activity and spindle generation. The ability to control spindle generation with high temporal precision will allow for causally testing the role of these oscillations in processes such as sleep-dependent memory consolidation 9 . Given that disrupted sleep spindles are a characteristic of diseases such as schizophrenia 15 , our approach can be applied to animal models of neurological and psychiatric illness to investigate diagnostic and therapeutic questions.  Example of a VGAT-ChR2 mouse exhibiting two states of activity, delineated by visual inspection of the EEG, TC firing rate, and high resolution video recording (d) Trial sorting based on visual inspection of these measures reveals a more robust optical induction of spindle oscillations in the EEG when the animal is quiet. Spectrogram of one animal. Statedependent spindle power increase after optical stimulation of the TRN shows a more robust impact when mice are quiet (N = 3 animals; p<0.001)