Thalamic control of cortical states

Transcription

1 Supplementary Information Thalamic control of cortical states James F.A. Poulet, Laura M.J. Fernandez, Sylvain Crochet & Carl C.H. Petersen Supplementary Information consists of: 1. Methods 2. Supplementary Table 1 Experimental timeline and recording depths 3. Supplementary Table 2 Thalamic action potential firing rates 4. Supplementary Table 3 Vm during thalamus inactivation and stimulation 5. Supplementary Table 4 LFP during thalamus inactivation and stimulation 6. Supplementary Figure 1 Infraorbital nerve cut blocks the cortical sensory response evoked by whisker deflection 7. Supplementary Figure 2 Thalamic activity after infraorbital nerve cut 8. Supplementary Figure 3 Whole-cell recording from an identified layer 2/3 pyramidal neuron after thalamic inactivation with muscimol 9. Supplementary Figure 4 Cortical LFP after thalamic inactivation with muscimol 10. Supplementary Figure 5 Thalamic inactivation with tetrodotoxin (TTX) 11. Supplementary Figure 6 Thalamic ChR2-stimulation with ramp vs square light pulse stimulus 12. Supplementary Figure 7 Thalamic ChR2-stimulation and cortical LFP 13. Supplementary Figure 8 Thalamic ChR2-stimulation evokes a similar change in cortical state to that observed during active whisking. 1

2 METHODS Surgery All experiments were carried out in accordance with the Swiss Federal Veterinary Office. Mice were implanted with a lightweight metal head holder and a recording chamber under isoflurane anesthesia (see Supplementary Table 1 for details of experimental timeline). A plastic recording chamber was centred over the left barrel cortex. Having recovered from implantation, mice were habituated to head-restraint with daily sessions that increased in duration. The C2 whisker barrel column was functionally located using intrinsic optical imaging. A small craniotomy (<0.5 mm) was drilled above the centre of the intrinsic signal to give access for cortical whole-cell and/or LFP recordings. A second craniotomy was drilled at 1.8 mm posterior to Bregma, 1.5 mm lateral to the midline to give access to thalamus for juxtacellular recording, drug injection or optogenetic stimulation. The brain was then covered with Kwick-Cast (WPI) and the mouse left to recover for 2-4 hours. After the recording session, the mouse was deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). The brain was then removed and postfixed in 4% PFA. Bilateral sectioning of the trigeminal sensory infraorbital nerves (IONs) was performed as previously described (Poulet and Petersen, 2008). The surgery was completed 2 4 h before thalamic juxtacellular recordings. A small incision was made in the skin exposing the IONs where they emerge from the infraorbital fissure (Dörfl, 1985). Sectioning of the IONs was made through the incision starting with the dorsal branches and finishing ventrally. After every recording session, we confirmed complete sectioning of the entire ION on both the left and the right side in a post-mortem examination. The large size of the IONs allows a straightforward verification of the nerve cut. After sectioning of the IONs, the cortical sensory response evoked by whisker stimulation was completely abolished in both intrinsic signal optical imaging and LFP recordings (Supplementary Fig.1). 2

3 Whole-cell and local field potential recordings in barrel cortex The exposed brain was cleaned and covered with Ringer s solution containing (in mm): 135 NaCl, 5 KCl, 5 HEPES, 1.8 CaCl 2, 1 MgCl 2. For whole-cell recordings, glass micropipettes with a resistance of 5-7 MΩ were inserted into the brain under positive pressure until a sudden increase in resistance was observed. Gentle suction would then be applied, a gigaseal formed and finally the membrane broken to enter the whole-cell configuration. Whole-cell pipettes were filled with internal solution containing (in mm): 135 potassium gluconate, 4 KCl, 10 HEPES, 10 sodium phosphocreatine, 4 MgATP, 0.3 Na 3 GTP (adjusted to ph 7.3 with KOH), and 2 mg/ml biocytin. Whole-cell recordings were made with a Multiclamp 700 amplifier (Axon Instruments, Foster City, California, USA). Whole-cell recordings were filtered at 0-10 khz and digitized at 20 khz by an ITC-18 analog-to-digital converter (Instrutech Corporation, Long Island, New York, USA) under the control of IgorPro (Wavemetrics). The membrane potential (Vm) was not corrected for liquid junction potentials. For LFP recordings a glass micropipette filled with Ringer s solution (resistance of 5-7 MΩ) was inserted into the brain under positive pressure until a depth of µ m. The pressure was released after controlling the resistance of the pipette. LFPs were recorded using a Multiclamp 700 amplifier, filtered at 0-10 khz and digitized at 20 khz using an ITC-18 analog-to-digital converter under the control of IgorPro. LFP signals were low-pass filtered (0-100 Hz) for display in the figures. Both LFP and whole-cell recordings were measured with respect to a Ag/AgCl reference electrode in the recording chamber. Juxtacellular recordings in somatosensory thalamus For juxtacellular recordings, 5-7 MΩ glass pipettes (similar to those used for LFP recordings) were filled with Ringer s solution and advanced vertically through the brain (1.8 mm posterior to Bregma, 1.5 mm lateral to the midline) maintaining a high intrapipette pressure ( mbar) until a depth of 3000 µm, at which point the pressure was reduced to 20 mbar. The electrode was slowly advanced and following a sudden increase in resistance, the intrapipette pressure was then released. This recording configuration is considered juxtacellular, since the glass electrode is in direct contact with the 3

4 membrane of the recorded neuron. Juxtacellularly recorded action potentials had positive polarity waveforms with large amplitudes (contrasting with the negative waveforms and lower amplitudes obtained by extracellular recordings sampling neuronal activity over a larger volume). Recordings were included only if spikes were detected before and after data collection. Juxtacellular action potentials were recorded using the Multiclamp 700 amplifier in current clamp mode, band pass filtered between 300 Hz and 10 khz and digitized at 20 khz using an ITC18 under the control of IgorPro. For identification of the recording sites, a fine tungsten electrode (10-12 MΩ, FHC) was lowered following the same penetration from the surface to a depth of 3500 µm and then a small electrolytic lesion was made. Quantification of whisker movement We filmed the C2 whisker during the experiment using a high speed (500 Hz) camera (MotionPro, Redlake) in sweeps of 20 s duration. The mouse was illuminated from below with infrared light (850 nm). Behavioral images were synchronized to the electrophysiological recording through TTL pulses. Custom written routines running within ImageJ or IgorPro were used to automatically determine the whisker angle off-line. Pharmacological inactivation of somatosensory thalamus Pharmacological injections into thalamus were made with thin glass micropipettes (tip diameter µm). Muscimol (1 mm) or TTX (50 µm) and Pontamine Sky Blue (5%) were dissolved in Ringer s solution and backfilled into the injection pipette. The pipette was positioned vertically just above the craniotomy (1.8 mm posterior to Bregma, 1.5 mm lateral to the midline) at the start of the experiment. During the experiment the pipette was slowly inserted into the thalamus to a subpial depth of 3300 µm. Approximately 100 nl of muscimol or TTX was then slowly pressure injected. The pipette was then moved ventrally by 200 µ m and a second 100 nl injection was made. The injection pipette was left in the brain for 3-5 minutes to allow the drug to spread and then gradually retracted out of the brain. Whole-cell and LFP recordings were carried out directly before and after thalamic inactivation in the same mice. The location of the injection site labeled with Pontamine Sky 4

5 Blue was confirmed to be the somatosensory thalamus in each experiment. Optogenetic stimulation of somatosensory thalamus Virus injections were targeted to somatosensory thalamus (1.8 mm posterior to Bregma; 1.5 mm lateral to the midline; vertical depth 3500 µm from pial surface) under deep ketamine/xylazine anesthesia. Approximately 1 µ l of adeno-associated virus encoding ChR2-Venus (AAV2/1.CAG.ChR2- Venus.W.SV40; Addgene plasmid 20071; Petreanu et al., 2009) was injected with a thin glass pipette (diameter ~10 µm). After injection mice were returned to their home cages for at least 3 weeks to allow time for expression. At the start of the recording session a 200 µm diameter optical fiber coupled to a 470 nm blue LED (Luxeon) light source was slowly inserted into thalamus to a vertical depth of 3300 µm. Light stimulation was performed in a ramp from minimal ( mw) to maximal ( mw) light intensity over a 5 second period (Supplementary Fig. 6). Data analysis Data were analysed using IgorPro and Excel (Microsoft). Based on the whisker behavior, recording segments were classified as quiet waking (no whisker movement) or active whisking (continuous rhythmic whisker movements without object contacts). Three second time windows were used to compute mean, variance and frequency spectra. The spectral analysis was done using a Fast Fourier Transform (FFT) procedure under IgorPro. The FFT magnitude was computed after subtraction of the mean and it was normalized by the number of samples (n/2). All data are presented as mean ± standard deviation. Non-parametric statistical tests were used to evaluate statistical significance (Wilcoxon-Mann-Whitney two-sample rank test or Wilcoxon Signed Rank test). Linear correlations with t statistics were used to assess the relationship between thalamic AP rates and low frequency (1-5 Hz) cortical activity. 5

6 References Dörfl, J. J. Anat. 142, (1985). Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. Nature 457, (2009). Poulet, J.F.A. & Petersen, C.C.H. Nature 454, (2008). 6

7 Supplementary Table 1 Mice Age implantation Age virus injection Age recording Recordings Cell depth from pia Experiment (n) range (weeks) range (weeks) range (weeks) (n) range mean ± SD (µm) Thalamus juxtacellular recordings control NA ± 334 Thalamus juxtacellular recordings ION cut NA ± 315 Thalamus inactivation (Mus) WC 12 4 NA ± 108 Thalamus inactivation (Mus) LFP NA NA Thalamus inactivation (TTX) WC 1 4 NA Thalamus inactivation (TTX) LFP NA NA Thalamus optogenetic WC 8* ± 105 Thalamus optogenetic LFP 19* NA * Optogenetic stimulation of the thalamus was performed in a total of 19 mice, from which we obtained 19 LFP recordings. We additionally obtained 8 WC recordings from the 19 mice. Supplementary Table 1 For each type of experiment carried out in this study, the table gives: the number of mice used; the range of ages at the time of implantation, virus injection and recording; the number of recordings; and the depth of the recorded cells from pia (range and mean ± SD). Mus and TTX indicate inactivation of the thalamus by muscimol and tetrodotoxin injection, respectively. LFP and WC indicate experiments conducted with local field potential and whole-cell recording, respectively. NA, not applicable. 7

8 Supplementary Table 2 Condition No. of cells (n) AP rate (Hz) LFP 1-5 Hz vs AP rate correlation Quiet Whisking r IONs intact ± ± ± 0.17 IONs cut ± ± ± 0.15 Supplementary Table 2 Thalamic action potential firing rates. Data are presented as mean ± SD. 8

9 Supplementary Table 3 Vm Vm FFT Condition Mean (mv) Variance (µv²) 1-5 Hz (mv) Hz (mv) Control (n = 12) Quiet ± ± ± ± 1.3 Whisking ± ± ± ± 1.5 Thalamus inactivation (Muscimol) (n = 12) Quiet ± ± ± ± 0.8 Whisking ± ± ± ± 1.2 Thalamus stimulation (ChR2) (n = 8) Quiet ± ± ± ± ChR ± ± ± ± 1.2 Supplementary Table 3 Vm during thalamus inactivation and stimulation. Data are presented as mean ± SD. 9

10 Supplementary Table 4 LFP LFP FFT Condition Variance (nv²) 1-5 Hz (µv) Hz (µv) Muscimol (n = 9) Control Thalamus inactivated Quiet 23.8 ± ± ± 48.6 Whisking 8.7 ± ± ± 48.0 Quiet 42.7 ± ± ± 41.2 Whisking 4.1 ± ± ± 19.9 TTX (n = 6) Control Thalamus inactivated Quiet 22.6 ± ± ± 24.2 Whisking 7.3 ± ± ± 30.1 Quiet 54.0 ± ± ± 43.0 Whisking 6.0 ± ± ± 16.7 ChR2 (n = 19) Control Thalamus stimulation Quiet 18.7 ± ± ± 46.0 Whisking 8.8 ± ± ± 44.6 Quiet 8.6 ± ± ± 55.6 Supplementary Table 4 LFP during thalamus inactivation and stimulation. Data are presented as mean ± SD. 10

11 Supplementary Figure 1 Supplementary Figure 1 Infraorbital nerve (ION) cut blocks the cortical sensory response evoked by whisker deflection. (a) Intrinsic signal optical imaging of the barrel cortex during C2 whisker stimulation before ION section showed a localized signal over the C2 barrel column (left). The intrinsic signal was completely suppressed after ION section in the same mouse (right). Similar results were obtained from two further mice. (b) The sensory-evoked response measured in the cortical local field potential (LFP) was also abolished after ION section. Averaged responses before (red trace) and after (blue trace) ION section (n=60 trials; reversed polarity). Same mouse as in panel a. (c) The cortical sensory LFP response evoked by C2 whisker stimulation was completely suppressed after ION cut in all three mice tested. Mean peak amplitude of sensory response before and after ION cut. 11

12 Supplementary Figure 2 Supplementary Figure 2 Thalamic action potential firing rates increase in ventrobasal nuclei (VB) comparing active whisking to quiet wakefulness in both control mice and mice with cut sensory infraorbital nerves (IONs). (a) Brightfield image of a coronal section showing the electrolytic lesion site (red arrow) at 3500 µ m below the pial surface and schematic drawing adapted 12

13 from the Paxinos & Franklin Mouse Brain Stereotaxic Atlas showing anatomical structures at 1.82 mm posterior to Bregma. Ventrobasal nuclei of the thalamus (VB, yellow shading); primary somatosensory cortex (S1, blue shading). (b) Whisker position (green trace) together with simultaneous juxtacellular recording (black trace) of action potential firing in the thalamus of control mice. The neuron in the upper example (depth 3630 µm) is from the same mouse as shown in panel a and this neuron had a mean quiet firing rate of 3.8 Hz which increased during whisking to 19.4 Hz. The neuron in the lower example (depth 3490 µ m) had a mean quiet firing rate of 5.0 Hz which increased during whisking to 13.6 Hz (from a different mouse). (c) Whisker position together with simultaneous juxtacellular recording in the thalamus from mice with bilaterally sectioned infraorbital nerves. The neuron in the upper example (depth 4050 µm) had a mean quiet firing rate of 6.2 Hz which increased during whisking to 23.3 Hz. The neuron in the lower example (from a different mouse, depth 3413 µ m) had a mean quiet firing rate of 8.9 Hz which increased during whisking to 16.5 Hz. 13

14 Supplementary Figure 3 Supplementary Figure 3 Whole-cell recording from an identified layer 2/3 pyramidal neuron after thalamic inactivation by muscimol. (a) Anatomical reconstruction of the recorded layer 2/3 pyramidal neuron (soma and dendrites shown in black and the main descending axon shown in red). (b) Membrane potential recording of the neuron shown in panel a. The thalamus was inactivated by injection of muscimol. During whisking (whisker angle shown in green) the slow membrane potential fluctuations are suppressed and the neuron remains in a hyperpolarized state with low variance. These membrane potential dynamics recorded in an identified layer 2/3 pyramidal neuron were similar to those found in recordings from unidentified cortical neurons (see Fig. 2). 14

15 Supplementary Figure 4 Supplementary Figure 4 Cortical local field potential (LFP) dynamics during quiet wakefulness and active whisking, before and after thalamic inactivation with muscimol. (a) The cortical LFP shows slow fluctuations during quiet wakefulness that are replaced by high frequency fluctuations during whisker movements. Green trace shows whisker position and black traces the LFP with reversed polarity. (b) After thalamic inactivation the slow cortical activity is enhanced during quiet wakefulness, but both low and high frequency fluctuations are suppressed during active whisking (same recording as panel a). (c) The Fast Fourier Transform (FFT) averaged across 9 LFP recordings from 9 mice, before and after thalamic inactivation during quiet wakefulness 15

16 and active whisking. Low frequency (1-5 Hz) fluctuations markedly decreased during whisking both before (P=0.004) and after (P=0.004) thalamic inactivation. Low frequency (1-5 Hz) fluctuations during quiet wakefulness were significantly larger after thalamic inactivation compared to control (P=0.01). High frequency ( Hz) dynamics were slightly increased comparing whisking with quiet wakefulness in control conditions (P=0.02), but strongly decreased after thalamic inactivation (P=0.004). (d) LFP variance was higher during quiet wakefulness following thalamic inactivation compared to control (P=0.012), but reduced to almost zero during whisking compared to quiet wakefulness (P=0.004). (e) Thalamic inactivation removes the sensory response evoked by C2 whisker deflection and measured by a LFP recording in the C2 barrel column. The traces show the evoked sensory responses averaged across 9 mice before (red) and after (blue) thalamic inactivation. 16

17 Supplementary Figure 5 Supplementary Figure 5 Thalamic inactivation with tetrodotoxin (TTX) has a similar effect to thalamic inactivation with muscimol. (a) Whole-cell Vm (black trace) recording of a layer 2/3 cortical neuron (300 µ m below pia) during 17

18 quantified whisker behavior (whisker position, green trace) before (Preinjection, top) and after (Post-injection, bottom) thalamic inactivation with TTX injection. Slow large-amplitude Vm fluctuations during quiet wakefulness are replaced by faster and smaller-amplitude fluctuations during whisking in the control pre-injection condition. After thalamus inactivation slow Vm fluctuations are enhanced during quiet wakefulness but both slow and fast Vm fluctuations are suppressed during active wakefulness, leaving the membrane potential hyperpolarised. (b) A similar effect was observed using LFP recordings in the barrel cortex before (Pre-injection, top) and after (Postinjection, bottom) thalamic inactivation with TTX injection. (c) Quantified across all LFP recordings (n=6 mice) thalamic inactivation with TTX (compared to the control pre-injection period) resulted in a significant increase in slow (1-5 Hz) cortical activity during quiet wakefulness (P=0.03) and a significant decrease in high frequency ( Hz) activity during whisking (P=0.03). (d) LFP variance was increased during quiet wakefulness after thalamic inactivation compared to the control pre-injection period (P=0.03) and reduced to almost zero during whisking. (e) Thalamic inactivation with TTX completely blocked the sensory response evoked by C2 whisker deflection and measured by a LFP recording in the C2 barrel column. The traces show the evoked sensory responses averaged across 4 mice before (red) and after (blue) thalamic inactivation. 18

19 Supplementary Figure 6 Supplementary Figure 6 Thalamic ChR2 stimulation with ramp vs square light pulse stimulus. Thalamic ChR2 stimulation was performed using an optic fiber coupled to an LED (blue light power shown in blue, below) while whisker behavior was filmed. Square light pulse stimulation (left) of 0.5 mw in amplitude consistently evoked whisker movements (whisker angle, green traces) immediately after the onset of the light pulse. Ramp light pulse (right) from 0.08 to 0.5 mw did not evoke whisker movements. 19

20 Supplementary Figure 7 Supplementary Figure 7 Effect of optogenetic stimulation of somatosensory thalamus upon cortical activity measured with local field potential (LFP) recordings. (a) Whisker position (green) together with simultaneous LFP recording (black, reversed polarity) in the barrel cortex of an awake mouse during quiet wakefulness and active whisking behavior. (b) Whisker position and simultaneous LFP recording in the barrel cortex of the same mouse during quiet wakefulness before, during and after optogenetic stimulation of the thalamus (blue shading). (c) Fast Fourier Transform (FFT) of LFP averaged across 19 recordings from 19 mice during quiet wakefulness (Quiet, green), free whisking (Whisk, red) and during quiet wakefulness with thalamus stimulation (ChR2, blue). Compared to quiet wakefulness, low frequency (1-5 Hz) LFP fluctuations were significantly reduced during both free whisking (P=4x10-6 ) and thalamic stimulation (P=4x10-6 ), but high frequency 20

21 fluctuations ( Hz) were not significantly affected during whisking (P=0.9) and increased slightly during thalamus stimulation (P=0.005). No significant differences were found between free whisking and thalamus stimulation (1-5 Hz FFT, P=0.9; Hz FFT P=0.16). (d) The LFP variance was significantly decreased for both free whisking (P=4x10-6 ) and thalamus stimulation (P=4x10-6 ) compared to quiet wakefulness. No significant difference in LFP variance was found between free whisking and thalamus stimulation (P=0.9). 21

22 Supplementary Figure 8 Supplementary Figure 8 Optogenetic stimulation of somatosensory thalamus during quiet wakefulness evokes a cortical state, which is very similar to the active cortical state during whisking. The data are compiled from Figures 2 and 3. (a) Cortical slow membrane potential fluctuations were quantified by integrating the area of the FFT from 1 Hz to 5 Hz (data compiled from Figures 2c and 3d). Thalamic stimulation with ChR2 induces a reduction in cortical slow membrane potential fluctuations compared to quiet wakefulness, which is similar to the reduction observed in control mice during whisking. In contrast slow membrane potential fluctuations are very different in mice with muscimol-inactivated thalamus. (b) Cortical high frequency membrane potential fluctuations were quantified by integrating the area of the FFT from 30 Hz to 100 Hz (data compiled from Figures 2c and 3d). Thalamic stimulation with ChR2 does not alter high frequency membrane potential fluctuations compared to quiet wakefulness, which is very similar to the absence of any effect during whisking in control mice. In contrast high frequency membrane potential fluctuations are strongly suppressed during 22

23 whisking in thalamic-inactivated mice. (c) The mean membrane potential is affected little by thalamic ChR2 stimulation, similar to the membrane potential during whisking compared to quiet wakefulness. However, after thalamic inactivation the membrane potential hyperpolarizes during whisking compared to quiet wakefulness. Data compiled from Figures 2d and 3e. (d) Optogenetic stimulation of the thalamus evokes a decrease in membrane potential variance, which is very similar to the decrease in membrane potential variance during whisking compared to quiet wakefulness. In contrast, cortical membrane potential variance is very different in mice after thalamic inactivation. Data compiled from Figures 2e and 3f. We conclude that the cortical state induced by thalamic ChR2 stimulation is very similar to that observed during whisking in control mice. There was no significant difference between these two experimental conditions for: i) the area of the FFT from 1 Hz to 5 Hz comparing whisking in control mice with thalamic ChR2 stimulation P=0.34. ii) the area of the FFT from 30 Hz to 100 Hz comparing whisking in control mice with thalamic ChR2 stimulation P=0.62. iii) the membrane potential during whisking in control mice compared to during thalamic ChR2 stimulation P=0.43. iv) the membrane potential variance during whisking in control mice compared to during thalamic ChR2 stimulation P=0.73. In contrast the cortical state during whisking after thalamic inactivation with muscimol is significantly different for: i) the area of the FFT from 1 Hz to 5 Hz comparing whisking after thalamic inactivation with whisking in control mice P= ii) the area of the FFT from 1 Hz to 5 Hz comparing whisking after thalamic inactivation with thalamic ChR2 stimulation P= iii) the area of the FFT from 30 Hz to 100 Hz comparing whisking after thalamic inactivation with whisking in control mice P= iv) the area of the FFT from 30 Hz to 100 Hz comparing whisking after thalamic inactivation with thalamic ChR2 stimulation P=

24 v) the membrane potential during whisking after thalamic inactivation compared to during whisking in control mice P= vi) the membrane potential during whisking after thalamic inactivation compared to during thalamic ChR2 stimulation P= vii) the membrane potential variance during whisking after thalamic inactivation compared to during whisking in control mice P= viii) the membrane potential variance during whisking after thalamic inactivation compared to during thalamic ChR2 stimulation P=