Sleep cycles consist of rapid alterations between arousal states, including transient perturbation of sleep rhythms, microarousals, and full-blown awake states. Here we demonstrate that the calretinin (CR)-containing neurons in the dorsal medial thalamus (DMT) constitute a key diencephalic node that mediates distinct levels of forebrain arousal. Cell-type-specific activation of DMT/CR+ cells elicited active locomotion lasting for minutes, stereotyped microarousals, or transient disruption of sleep rhythms, depending on the parameters of the stimulation. State transitions could be induced in both slow-wave and rapid eye-movement sleep. The DMT/CR+ cells displayed elevated activity before arousal, received selective subcortical inputs, and innervated several forebrain sites via highly branched axons. Together, these features enable DMT/CR+ cells to summate subcortical arousal information and effectively transfer it as a rapid, synchronous signal to several forebrain regions to modulate the level of arousal.
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We thank Z.J. Huang (CSHL, NY, USA) for providing us with the Calb2-Cre mice and C. Smerdou and C. Ballesteros (CIMA, University of Navarre, Spain) for synthesizing the Sindbis-Pal-eGFP RNA construct. The technical help of K. Faddi, K. Varga, A. Jász and E. Szabo-Egyud is acknowledged. The authors thank the Nikon Microscopy Center at IEM, Nikon Austria GmbH, and Auro-Science Consulting Ltd for kindly providing microscopy support and thank the Human Brain Research Laboratory (IEM/HAS) for the preparation of human material. The authors thank J. Poulet, B. Hangya, and H. Bokor for comments and discussions on the manuscript. This work was supported by the National Office for Research and Technology (NKTH-ANR-09-BLAN-0401, Neurogen to L.A; K119650 to P.B.; FK124434 to F.M.; PD124034 to B.B.), “Lendület” Program of the Hungarian Academy of Sciences (LP2012-23; B.B.), Hungarian Korean Joint Laboratory Program, Hungarian Brain Research Program (grants no. KTIA_NAP_13-2-2015-0010 to F.M., KTIA_NAP_13-2-2014-0016 to P.B. and KTIA_13_NAP-A-I/1 to L.A.), ERC (FRONTHAL, 742595 to L.A.), and HBP-FLAG-ERA (118886 to L.A.).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Activity of DMT cells before and after spontaneous arousals in freely sleeping conditions.
(a-c), Low power confocal images of a representative injection site at three coronal levels following a single injection of AAV-DIO-ChR2(H134)-eYFP into DMT of CR-Cre mice (n = 4 mice). (d), High power confocal images of the co-localization of CR immunostaining and eYFP in DMT 6 weeks after the viral injection. (e), An example of an optically tagged DMT/CR+ cell which increased firing activity together with (but not before) the sleep/wake transition in freely moving conditions. From left-to-right, waveforms (WF, top) and autocorrelogram (ACG, bottom), peri-event time histogram upon optogenetic tagging and change of firing rate of the sample DMT/CR+ cell. (f), Population data for the activity of those DMT/CR+ units which increased their firing only at the onset of sleep/wake transition (0/+; n = 8/31) but not before. (g), Population data for the activity of those DMT/CR+ units which displayed anticipatatory firing before the onset of the sleep/wake transition (+/all; n = 20/31). 1 s bins indicate the averages of z-scores, while green line shows its variance (SD). Red dashed lines represent the significance levels for P < 0.05 Z-score values (1.96). (h), Pie chart shows the distribution of firing rate changes for all 31 DMT/CR+ neurons before and after the sleep/wake transition. (i), Pie chart shows the distribution of firing rate changes for all 34 DMT/CR- before and after sleep/wake transition. Activation before EMG ON vs. others, DMT/CR+ vs. DMT/CR- Fisher’s exact test, two-tailed, P = 0.0012.
a1-a3, Overlap between foot-shock activated c-Fos-positive neurons and CR expression in DMT (n = 4 mice).
(a), Experimental design to monitor the effects of tail pinch on the DMT/CR+ cells firing along with the prelimbic cortical (PrL) LFP. (b-c), Individual responses of single DMT/CR+ cells which increased (b) or decreased (c) their firing to tail pinch. Black and red traces indicate the corresponding raw and filtered (1–3 Hz) PrL LFP, respectively. (d), Top, population data for responses to tail-pinch of DMT/CR+ (n = 13 cells). 6/13 DMT/CR+ neurons responded with elevated activity (PRE 1.26 ± 0.62 Hz; TAIL 4.96 ± 3.43 Hz;) which persisted long after the termination of the stimulus (3.39 ± 2.73Hz in the 40 s post stimulus period). One-way ANOVA, F(2, 15) = 5.3735, P = 0.0174; Fisher’s LSD, Pre vs. Tail, P = 0.0053; Pre vs Post, P = 0.0748; Tail vs. Post, P = 0.1978. The remaining neurons (7/13) decreased their activity during tail pinch (Pre 0.91 ± 0.31 Hz; Tail 0.17 ± 0.26 Hz; Post 0.12 ± 0.2 Hz,). One-way ANOVA, F(2, 18) = 50.533, P < 0.0001; Fisher’s LSD, Pre vs. Tail, P < 0.0001; Pre vs Post, P < 0.0001; Tail vs. Post P = 0.7262. Bottom, changes of PrL delta power underlying tail pinch. Black and grey dots indicate individual cells which increase or decrease their firing, respectively, during tail pinch [group (increase vs. decrease)-wise comparison: Repeated measures of ANOVA, F(2, 16) = 0.187, P = 0.8314; Effect (Pre-Tail-Post)-wise comparison: one-way ANOVA, F(2, 27) = 61.372, P < 0.0001; Fisher’s LSD, Pre vs. Tail, P < 0.0001; Pre vs Post, P < 0.0001; Tail vs. Post P = 0.8692; while light and dark green colored bars represent their mean ± SD, respectively. #P < 0.1; *P < 0.05; **P < 0.01, ***P < 0.001; n.s., non-significant.
a, Experimental design to validate SwichR-mediated silencing of DMT/CR+ cells. b-d, Juxtacellular recording of a DMT cell using 0.5 s (a), 1 s (b) and 2 s (c) long blue laser light under urethane anesthesia.
Supplementary Figure 5 Quantification of arousal events and features of the EMG ON states evoked by stimulations with different durations.
(a), Definitions of EMG ON and OFF (active/inactive) states for quantitative purposes (onset, duration, probability) based on the raw EMG signal. Black, 300–600 Hz filtered EMG; purple, standard deviation of EMG; green, threshold for EMG active state. Three criteria were used. 1: standard deviation (+2.1–5 SD of baseline) above threshold; 2: EMG ON states shorter than 0.5 s are discarded since they represent muscle twitches rather than arousals or microarousals. 3: Gaps shorter than 2 s between EMG ON states were filled with ON state, since it is unlikely that mice go back to sleep for 2 s. (b), Cumulative distribution of the latency of spontaneously occurring (black) or optogenetically evoked arousals (1s; red, 10s blue) in a 60 s window after stimulation onset. Shaded area represents ± s.e.m. (c), Average durations of spontaneous and evoked arousals, using different stimulus durations (n = 225 trials in 5 mice, spontaneous (sp), 2.67 ± 0.77; 0.5 s, 2.08 ± 0.78 s; 1 s, 3.22 ± 0.8 s; 2 s, 9.59 ± 4.02 s; 10 s, 56.41 ± 2 s; Repeated measures of ANOVA, F(3,12) = 836.88, P < 0.0001. Pairwise comparison with Bonferroni correction shows significant difference only for 0.5 s vs 10 s, P < 0.0001).The horizontal lines in the box plots indicate medians, the box limits indicate first and third quantiles, and the vertical whisker lines indicate minimum and maximum values.
Supplementary Figure 6 Alteration of EEG, not EMG activity, is the primary response after DMT/CR+ stimulation.
(a), Average peri-stimulus distribution of the probability of EMG ON states (black), normalized delta power (red) and raw EMG activity (gray) during microarousals following 1 s DMT/CR+ stimulations in NREM sleep (n = 5 mice). Microarousals with longer than 2 s latencies are included. Note the instantaneous drop in delta activity followed by the onset of EMG activity after several seconds. (b), In this figure in the top the evoked EMG ON states are grouped according to onset latencies (0–1 s, 1–2 s, etc, n = 521 trials in 5 mice) and labeled with different colors and in the bottom the corresponding average peristimulus EEG delta powers data are shown with the same colors. Note, that the drop in the delta power is tightly linked to the stimulation regardless of the onset of EMG ON state demonstrating the primacy of EEG response. (c), Distribution of time differences between the drop in delta activity and the onset of EMG ON states in case of individual evoked arousal events. Bin size is 0.25 s. In the vast majority of the cases (89%) the EEG response occurs first.
Supplementary Figure 7 State dependency of DMT/CR+ responses and characterization of evoked VB responses.
(a), Spike triggered EEG averages of the cortical evoked responses during DMT/CR+ stimulations resulting in microarousals (red) or sleep throughs (blue) in NREM sleep (n = 5 mice). The traces are triggered by the first stimulation of the 1 s long 10 Hz train. No difference in cortical response can be observed in these two conditions. (b-c), Normalized, average delta (b) and sigma (c) powers preceding DMT/CR+ stimulations resulting in microarousals (red) or sleep throughs (blue) in NREM sleep (n = 5 mice). No systematic difference is present between the two conditions. Light blue and red lines represents ± s.e.m. (d-f), Average peristimulus distribution of EMG ON states (d), delta (e) and sigma (f) power during microarousals (MA, red) and sleep-through (ST, blue) in case of 1 s VB stimulations n = 274 trials in 4 unilateral stimulations. Note prolonged disturbance of sigma activity even in case of sleep-throughs. Light blue and red lines represents ± s.e.m.
Supplementary Figure 8 Stimulation intensity vs. arousal (EMG ON) probability curves for individual DMT/CR+ and VB mice.
(a), DMT/CR+ mice (n = 5) (b), VB mice (n = 4). Black curves, moving averages of 30 points; red curves, sigmoid fitted on data; cyan horizontal lines, probability of spontaneous arousal (EMG ON) within 10s (using the same criteria as for evoked). Blue dots indicate trials of laser intensities at which the stimulation evoked microarousal (1) or sleep-through (0). Each panel contains the laser intensity range used for the given animal.
(a–k), AAV-DIO-ChR2-eYFP labeled axonal processes (green) in a CR-Cre mouse arising from DMT. DMT/CR+ fibers can be found in all layers (L1-L6) of the prelimbic (PrL, a) and insular cortex (IC; b), in L6 of the primary somatosensory cortex (S1; c), deep layers of the temporal association cortex (TeA; d), nucleus accumbens (NAc; e), lateral septum (LS; f), dorsomedial part of the caudate putamen (dmCPu) and bed nucleus of the stria terminalis (BNST; g); interstitial nucleus of the posterior limb of the anterior commissure (IPAC), olfactory tubercle (Tu), substantia innominata (SI) and ventral pallidum (VP; h); amygdalostriatal transition area (Astr), centrolateral amygdala (CeL), basolateral amygdala (BLA) but not lateral amygdala (LA; i); lateral hypothalamus (LH; j) and thalamic reticularis nucleus (TRN; k). Note the varying density of DMT/CR+ axonal arbor around the cholinergic (Chat; red in h) and orexinergic cells (Orx, red in j). The territory of TRN is labeled by parvalbumin (PV, red) in k. LV, lateral ventricle. (l–n), Schematic drawings of the experiments (top) and representative low power confocal image of a CR-immunostained (red) sections of DMT (bottom). Yellow dots indicate the position of DMT neurons retrogradely labeled from the prelimbic cortex (l; PrL; 891/922 cell, 96.64 %; n = 5 mice), amygdala (m; AMY; 416/438 cells, 94.98 %, n = 4 mice) and nucleus accumbens (n; NAc; 2081/2114 cell, 98.44 %, n = 5 mice). Graphs in the left corner of the images show the proportion of CR+/CTB-labeled cells in the DMT and represent means ± SD.
(a), The magnitude of evoked multi-unit activity (eMUA) in PrL, NAc and BLA by DMT/CR+ stimulation at 10 Hz depends on laser intensity. (b), Quantification of the peak amplitude for the 1st, 2nd, 5th and 10th pulses of an 1 s long 10 Hz optical stimulation eMUA display depression in PrL (top; n = 6 animals; One-way ANOVA, F(3, 20)=14.788, P < 0.0001; Newman-Keuls test, 1st vs. 2nd, P = 0.4790; 1st vs. 5th, P = 0.0236; 1st vs. 10th, P = 0.0002) and NAc (middle; n = 3; One-way ANOVA, F(3, 16)=15.254, P < 0.0001; Newman-Keuls test, 1st vs. 2nd, P = 0.1322; 1st vs. 5th, P = 0.0008; 1st vs. 10th, P = 0.0003) and less prominently in BLA (bottom; n = 3, One-way ANOVA, F(3, 8)=1.7067, p = 0.2424). (c), Representative evoked LFP signal in PrL by 1 (left) and 10 Hz (right) optical stimulation of DMT/CR+ neurons (dashed lines). Note the lack of augmenting responses. (d), Population data (n = 6) show no changes in evoked LFP amplitude at 1 Hz (One-way ANOVA, F(3, 20) = 0.30281, p = 0.823) but significant depression at 10 Hz (F(3, 20)=38.227, P < 0.0001; Newman-Keuls test, 1st vs. 2nd, P = 0.2326; 1st vs. 5th, P = 0.0002; 1st vs. 10th, P = 0.0002). Data are means ± SD; *P < 0.05, ***P < 0.001.
(a-l), Distribution of single (red and green dots) and double labeled DMT cells (yellow triangles) from PrL and NAc (top; a-d), BLA and PrL (middle; e-h), and BLA and NAc (bottom; i-l) at two AP levels. (m-o), A single cell labelling of a DMT/CR+ neuron with Pal-eGFP-Sindbis. Confocal images in m show the colocalization of GFP (green, top) with CR (red, middle). Note the neighboring CR+ (*) and a CR- (#) cell. n, A low magnification confocal image indicates the location of the same labeled cell in DMT/CR+ region. o, A partial reconstruction of the same DMT/CR+ cell (green) shows its axon branching (black) in many forebrain sites including cortical [insular (IC) and piriform cortices (Pir) as well as basolateral amygdala (BLA)] and subcortical [nucleus reticular thalami (nRT), nucleus basalis (NB), nucleus accumbens (NAc), anterior cortical amygdaloid nucleus (ACo), central amygdala (CeA), interstitial nucleus of the posterior limb of the anterior commissure (IPAC) and medial amygdala (MeA)] structures. (p-r), Following double retrograde tracings using Fluorogold (FG) and choleratoxin B (CTB) injected into any combination of the three main DMT/CR+ targets (PrL, NAc or AMY) revealed largely non-overlapping population of projecting neurons in any other brain region examined beside DMT (see above). This indicates that only DMT/CR+ neurons provide significant amount of branching collaterals linking these regions. In this example, FG is injected into PrL (a, green) and CTB into into NAc (b, red). In the two brain regions which contained neurons projecting to both sites, (the supramammilary nucleus, SUM, p and the ventral tegmental area, VTA, q) to ratio of dual projecting cells were extremely low (9/700 neurons, 1.3%; n = 4 mice) (r).
Supplementary Figures 1–11
Ten seconds long stimulation of DMT/CR+ cells evokes full blown arousal. 10 s activation of DMT/CR+ cells during natural sleep. Green lamp on the left (red circle) indicates the start of the laser stimulus. Note normal awakening behavior, including stretching.
One second long stimulation of DMT/CR+ cells evokes microarousal. Similar conditions but 1 s optogenetic activation of DMT/CR+ neurons as in Supplementary Video 1. Note only short head and neck movements.
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Mátyás, F., Komlósi, G., Babiczky, Á. et al. A highly collateralized thalamic cell type with arousal-predicting activity serves as a key hub for graded state transitions in the forebrain. Nat Neurosci 21, 1551–1562 (2018). https://doi.org/10.1038/s41593-018-0251-9
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