Essential thalamic contribution to slow waves of natural sleep

Abstract

Slow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural sleep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75-1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm.

Figure 1.

Properties of high-frequency bursts in VB TC neurons during ketamine-xylazine anesthesia and natural sleep. A, B, Local field potential in VB during anesthesia (A) and natural sleep (B). *Bursts. C, Burst properties of a representative TC neuron in the VB during anesthesia: ISIs for different burst lengths (left), and distribution of interburst intervals (IBI) (right) (n = 5102 bursts). D, Same as C for a different representative TC neuron in the VB during natural sleep (n = 825 bursts). C, D Insets, Representative bursts. E, F, Population data, as in C and D, for n = 85 and n = 9 TC neurons from n = 18 anesthetized and n = 3 naturally sleeping rats, respectively.

Figure 2.

Detection of slow and spindle waves. A, Representative EEG wavelet power spectrum of slow wave frequency band (0.2–4.5 Hz) during ketamine-xylazine anesthesia. B, Representative EEG broadband power spectrum. Dashed lines indicate the frequency band shown in A. C, Expanded EEG trace (black) (from time period marked in A with arrow) illustrating the identification of slow waves (green circles) by means of negative to positive zero-crossing detection on the 0.2–4.5 Hz bandpass-filtered signal (green trace). Waves with peak-to-peak amplitude (red triangles to blue triangles) of <60% of the mean peak-to-peak amplitude were discarded. D, Autocorrelogram of EEG slow waves detected as shown in C from the first 20 min of data shown in A. E, Representative raw (middle), 5–12.5 Hz bandpass-filtered (bottom) EEG traces showing spindle waves (black arrows) and wavelet power spectrum (top) with detected spindle wave episodes (green lines) during anesthesia. F, Average frequency distribution of spindles during anesthesia (n = 7 rats). G, H, Data for spindles during natural sleep are illustrated in the same format as E and F, respectively (n = 6 rats).

Figure 3.

Block of TC neuron firing by TTA-P2 directly applied by reverse microdialysis in the thalamus under anesthesia. A, Coronal brain section showing the position of a microdialysis (DP) (inserted with a 16° angle with respect to the vertical axis, see Materials and Methods) and a silicone probe (SP) in the VB, both stained with a red fluorescent dye. B1, Distance–response curve of TTA-P2-elicited block of high-frequency bursts in VB TC neurons (n = 533 neurons from 37 rats). Burst rate was measured between 50 and 60 min from the start of TTA-P2 or TTX dialysis. Data are normalized to the burst rate measured during the last 10 min of the preceding 1 h of aCSF dialysis (see Materials and Methods). Different TTA-P2 concentrations are color-coded as illustrated and refer to the drug concentration in the inlet dialysis tube. There is similarity in the action of 1 and 3 mm TTA-P2. The effect of TTX is also depicted (n = 33 neurons from 11 rats). B2, Same as B1, but for total TC neuron firing (i.e., high-frequency bursts plus single action potentials). C, Distance dependence of time of half-block of high-frequency bursts by VB microdialysis of 300 μm TTA-P2 (top) and of time of half-block of total firing by VB microdialysis of 50 μm TTX (bottom). Black lines indicate the best fit of a fourth-order parabolic function. D, Schematic brain drawing (from Paxinos and Watson, 2007) showing that the area of burst firing block achieved with the dialysis of 300 μm TTA-P2 (green) (calculated from the data shown in B2) covers almost the entire VB. Only a small increase in the area of block (which now covers a small portion of the NRT) is achieved with 3 mm TTA-P2 (red). This drawing assumes a fully vertical position of the dialysis probe as it was used for all the experiments described in Figures 4, 5, 6, and 7. E, In vivo recovery of TTA-P2 applied by dialysis (n = 6 rats for both concentrations), which was estimated using the formula ([X]_in − [X]_out)/[X]_in, where [X]_in and [X]_out are the TTA-P2 concentration in the inlet and outlet dialysis tubes, respectively (Chan and Chan, 1999).

Figure 4.

Block of thalamic firing decelerates EEG slow waves during anesthesia. A, Spike raster plots (top three traces; *bursts) from 3 VB TC neurons and EEG from S1 (bottom trace) show the effects of 50 μm TTX and 300 μm TTA-P2 dialysis in the VB. The predominant burst firing during aCSF is virtually abolished by TTX and TTA-P2, an effect accompanied by slowing of the EEG rhythm. B, Event-triggered averages of raw EEG traces centered on the middle point of DOWN to UP state transitions were calculated after 1 h of aCSF, TTX, and TTA-P2 dialysis (n = 438, 243, and 222 transitions, respectively). C, Normalized (to predrug period), time-dependent decrease of slow waves by TTX (n = 5) and TTA-P2 (n = 5) (drug dialysis starts at 0). D, EEG power spectra 60 min after start of drug dialysis. E, Normalized (to predrug period), time-dependent reduction of spindle waves by TTX and TTA-P2. C–E, Solid lines indicate the mean; color shadings indicate SEM. In this and the following figures, illustrated drug concentrations during microdialysis are those of the inlet dialysis tube (for brain concentration delivered by dialysis probes, see Fig. 3E). In this experiment and those depicted in Figures 5, 6, and 7, the dialysis probes were inserted in a fully vertical position.

Figure 5.

Systemic injection of TTA-P2 markedly decreases the frequency of slow and spindle waves during anesthesia. A, Spike raster plots (top three traces; *bursts) from 3 different TC neurons in the VB and EEG (bottom trace) from S1 show the effect of two doses of intraperitoneally injected TTA-P2 on neuronal firing and slow waves. B, Time-dependent block of slow waves after 0.3 and 3 mg/kg intraperitoneally of TTA-P2 injected at time 0. C, Power spectra calculated 1 h after TTA-P2 injection. D, Summary data showing the percentage reduction in slow and spindle waves produced by different doses of TTA-P2 (measured 1 h after intraperitoneal injection). Number of animals for saline (Sal) and TTA-P2 0.3, 1, 3, and 10 mg/kg injections are 4, 3, 3, 3, and 1, respectively. Error bars indicate SEM. *p < 0.01 compared with saline injection (Mann–Whitney U test). E, F, Dose–response curve of burst and total spike rate measured 40 min after systemic intraperitoneal injection of TTA-P2 (logistic regression fits, p < 0.05) (ED₅₀ for bursts: 0.18 ± 0.05 mg/kg; ED₅₀ for total spikes: 0.26 ± 0.06 mg/kg). The 3 and 10 mg/kg TTA-P2 abolish bursts (p < 10⁻⁶ compared with saline injection, Mann–Whitney U test, n = 40 TC neurons). B, C, Solid lines indicate the mean; color shadings indicate SEM. Color code in C also applies to B and to the traces in A.

Figure 6.

Block of thalamic firing decreases slow wave frequency during natural sleep. A, Non-REM sleep EEG (bottom) and corresponding wavelet spectra (top) during VB microdialysis of aCSF, 3 mm TTA-P2, and 50 μm TTX. Transient slow waves (white lines) were detected as ridges in the wavelet spectra (see Materials and Methods). B, Slow wave frequency density distribution during aCSF, TTA-P2, and TTX dialysis, 1 h after the start of drug dialysis (arrows indicate the measured peaks). C, Time dependence of TTA-P2 (n = 7 rats) and TTX (n = 5 rats) effects on the normalized peak of the slow wave frequency distribution. D, Raw non-REM EEG power spectra show TTA-P2- and TTX-elicited decrease of power in sleep spindle frequency range and increase of power in slow wave frequency range. E, Time dependence of TTA-P2 and TTX effects on sleep spindles normalized count. B, D, Solid lines indicate the mean; color shading indicates SEM. C, E, Error bars indicate SEM.

Figure 7.

Systemic injection of TTA-P2 markedly decreases the frequency of slow waves and abolishes spindles during natural sleep. A, Non-REM sleep EEG (middle), corresponding wavelet spectra (top), and EMG (bottom) after intraperitoneal injection of saline (left) and 10 mg/kg TTA-P2 (right). Transient slow waves (white lines) were detected as ridges in the wavelet spectra as in Figure 2. B, Slow wave frequency distribution after saline and TTA-P2 injection. There is a shift of the peak (arrows) from ∼2 Hz to ∼0.7 Hz. C, Time dependence of TTA-P2 (n = 4 rats) and saline (n = 6 rats) effects on the normalized peak of the slow wave frequency distribution. D, Raw non-REM sleep EEG power spectra show TTA-P2-elicited decrease of power in sleep spindle frequency range and increase of power in slow wave frequency range compared with saline injection. E, Time dependence of TTA-P2 effects on sleep spindles normalized count. F, G, Dose–response curve of burst (F) and total spike (G) rate measured 40 min after systemic intraperitoneal injection of TTA-P2 (logistic regression fits, p < 0.05) (ED₅₀ for bursts: 0.55 ± 0.03 mg/kg; ED₅₀ for total spikes: 1.71 ± 0.11 mg/kg). The 3 and 10 mg/kg TTA-P2 abolish bursts recorded during natural sleep (p < 10⁻⁶ compared with saline injection, Mann–Whitney U test, n = 42 TC neurons). B, D, Solid lines indicate the mean; color shading indicates SEM. C, E–G, Error bars indicate SEM.

Figure 8.

Channelrhodopsin-2 expression in VB TC neurons. A, Immunostaining of channelrhodopsin-2-mCherry (red) showing that the expression of the channelrhodopsin-2 protein is restricted to VB TC neurons and some TC axons passing through the NRT (top). Arrow indicates the putative site of the virus injection. NeuN staining (green) is evident in both VB and NRT somata (middle). Merged images (bottom) demonstrate colocalization of channelorhodopsin-2 and NeuN in TC, but not NRT, neurons. B, Higher magnification of a portion of the respective panels in A. Scale bars, A, B: 300 μm.

Figure 9.

Thalamic entrainment of EEG slow waves during anesthesia. A, EEG trace (bottom) and wavelet transform (top) showing the effect of 20 ms, 473 nm light pulses at 1, 1.5, and 2 Hz. Dashed white line indicates the start of the first pulse. B, Event-triggered EEG averages centered on the 20 ms light pulses (blue vertical bars) for the illustrated stimulation frequencies. Gray areas indicate SEM. A total of 10 s of EEG was used for each average. C, Raster plots of firing of 4 VB TC neurons in responses to two consecutive 20 ms pulses (at 1 Hz) (blue vertical lines). *Bursts. The corresponding EEG trace is superimposed as a black line. D, EEG power spectra in response to 20 ms light pulses at the illustrated frequencies (indicated by arrows). There are frequency-dependent amplification and the shift of the peak of the power spectra compared with control (i.e., without light stimulation, black line).

Figure 10.

Thalamic entrainment of EEG slow waves requires thalamic T-type calcium channels during anesthesia. A, EEG traces showing the effect of 20 ms, 473 nm light pulses (vertical blue lines) at 1 and 2 Hz during aCSF and TTA-P2 dialysis in VB. B, Raster plots of firing of 4 VB TC neurons in responses to 20 ms pulses (at 1 Hz) during aCSF (B1) and TTA-P2 dialysis (B2). *Bursts. The corresponding EEG trace is superimposed. B3, Light pulse-triggered spike rates (red trace is right-shifted for clarity) from 41 stimulation epochs. C, EEG power spectra in response to 20 ms light stimulation at 1 (C1) and 2 (C2) Hz (arrows) during thalamic aCSF (blue, n = 7 rats) and TTA-P2 (red, n = 3 rats) dialysis compared with power spectra without light stimulation. Black represents aCSF; green represents TTA-P2. D, The power values at a given stimulation frequency (obtained from spectra as in C) for different light stimulation frequencies are plotted during aCSF and TTA-P2 dialysis. Error bars indicate SEM. *p < 0.05 for 5 and 20 ms pulses (Mann–Whitney U test).