Net synaptic potentiation in wakefulness and depression in sleep

Transcription

1 Supplementary Figure 1. Features of the LFP response. a. Amplitude and latency of the evoked response (mean ± SEM, n = 7 rats) plotted as a function of linearly-increasing stimulus intensity (5 intensities, determined for each rat from near-threshold up to near-maximal level). The amplitude increases as a function of increasing stimulus intensity, while the latency remains constant. b. Left panel: a representative example of the LFPs evoked by consecutive stimuli delivered every 10 ms (100 Hz; thick vertical bars; different rat from that shown in Figure 2b). Note that each stimulus consistently results in a negative deflection of the signal. Right panel: mean values of the amplitude of the 10 consecutive evoked responses during the 100 Hz stimulation ( ± SEM, n = 6 rats). Note that the amplitude of the response does not show any depression during the stimulation. c. Individual representative evoked responses taken from one rat after low intensity (above threshold, black) and high intensity (near-maximal, grey) stimulation. The left traces depict the early component, which is present in both conditions. The right traces show the emergence, after the high intensity stimulation, of a polysynaptic component with long latency and long duration. d. Left panel: Individual LFPs evoked by paired stimuli delivered with an interval of 10 ms. Middle and right panels: mean values of the amplitude and slope of the response evoked by the first and the second pulse. Note the augmentation of the response after the second pulse ( ± SEM, n = 7 rats). Asterisks denote significant differences (p < 0.05, paired t-test).

2 Supplementary Figure 2. LFP slope distributions after W, S, and EW. a. Upper panel, mean values of the slope of the evoked responses as in Figure 2d. Lower panel, distribution of the slope of the individual evoked responses before and after a consolidated waking episode of ~ 4 hours (W0 and W1 are as in Fig. 2c). The number of responses was computed for 6 bins with linearly increasing slope. Mean values ( ± SEM, n = 13 rats) are plotted as percentage of the total number of responses within the corresponding session. b. Upper panel, mean values of the slope of the evoked responses as in Figure 2d. Lower panel, distribution of the slope of the individual evoked responses before and after a consolidated sleep period of ~ 4 hours (S0 and S1 are as in Fig. 2d). Mean values ( ± SEM, n = 13 rats). c. Upper panel, mean values of the slope of the evoked responses as in Figure 5b. Lower panel, distribution of the slope of the individual evoked responses after 4 hours of sleep or after 4 hours of enforced wakefulness (EW; S and EW are as in Fig. 5b). Mean values ( ± SEM, n = 13 rats). p<0.05, paired t-test.

3 Supplementary Figure 3. LFP slope changes equated for response amplitude/latency. a. Upper panel, mean values of the slope of the evoked responses in W0 and W1 as in Figure 2c. Middle and lower panels, mean difference ( ± SEM, n = 13 rats) between the slopes of evoked responses that were matched based on their amplitude or their latency, respectively. b. Upper panel, mean values of the slope of the evoked responses in S0 and S1 as in Figure 2d. Middle and lower panels, mean difference ( ± SEM, n = 13 rats) between the slopes of evoked responses that were matched based on their amplitude or their latency, respectively. c. Upper panel, mean values of the slope of the evoked responses in S and EW as in Figure 5b. Middle and lower panels, mean difference ( ± SEM, n = 13 rats) between the slopes of evoked responses that were matched based on their amplitude or their latency, respectively. Asterisks denote significant differences (p < 0.05, paired t-test).

4 Supplementary Figure 4. LFP slope changes and preceding REM sleep. Relationship between the slopes of evoked responses and the amount of REM sleep (left panel) or the REM / NREM sleep ratio (right panel) during the preceding 3-hour period (n = 26 rats). Each rat contributed with 3 (n = 23) or 2 (n = 3) values of the light period (0, 4 and 8 h). The slopes for each time point are expressed as % of the mean value between all the time points within an individual. The lines depict a linear regression (Pearson correlation).

5 Supplementary Figure 5. LFP slope and brain temperature. Top panels. Time course of the changes in LFP slope (left) and brain temperature (right) during a continuous 4-hour waking period. Mean values ( ± SEM; n = rats) of the slope of evoked responses are shown for hour 0 (starting min after arousal, see Methods), and the following 0.5, 1, 2 and 3-4 hours of wakefulness. Mean values ( ± SEM; n = 7 rats) of brain temperature are shown for hour 0 (starting min after arousal, as for the evoked responses), and the following 4 hours of wakefulness. Values are expressed as the difference from the mean 2-min value calculated during sleep immediately before each rat woke up (determined by LFPs and EMG recordings). Brain temperature is plotted in consecutive 10- min intervals during the 4-hour waking period. Horizontal lines depict linear regressions (least square method). Corresponding r2- and p-values are provided within each panel. Bottom panel. Dissociation between the time course of the LFP slopes during the 4 hours of wakefulness and brain temperature. The individual values of the slopes of the LFPs recorded after 0.5, 1, 2 and 3-4 hours of wakefulness (as depicted on the top left panel; % of hour 0) are plotted against brain temperature values during the corresponding 10-min intervals (as in the top right panel). Pearson product-moment correlation coefficient r = 0.03, n.s.

6 Supplementary Figure 6. Relationship between the slope of evoked responses at light onset after 12 hours of continuous waking during the dark period and SWA in the subsequent NREM sleep. In 5 rats baseline evoked responses were collected every 4 hours during the light period. Animals were then constantly observed during the 12-hour dark period and presented with novel objects whenever they exhibited signs of drowsiness. After evoked responses were recorded at the end of the 12-hour waking period, the animals were allowed to obtain undisturbed sleep for ~ 4 hours. At the end of the 12-hour period of waking slopes were 15.8 ± 3.7% above the mean over the baseline light period (average of 0, 4, 8 and 12 h), when the animals predominantly slept (p = 0.014, paired t-test). Left panel. Relationship between mean SWA of the first hour of recovery NREM sleep after the 12-hour waking period (as % of mean SWA in NREM sleep during the first 4 hours of baseline light period) and the slope of evoked responses recorded at the end of the 12- hour waking period before recovery sleep (expressed as % of the slopes obtained at hour 8 of baseline light period, when sleep pressure was at the lowest level). Right panel. Peak SWA (maximal 10-min value reached during the first hour of recovery NREM sleep) after the 12-hour waking period (as % of mean SWA in NREM sleep during the first 4 hours of baseline light period). Slopes are as in the left panel.

7 Supplementary Figure 7. Relationship between the decline of the slope of evoked responses and amount of REM sleep (in min, left panel), and theta power (6-9 Hz) during REM sleep in the corresponding 4- hour interval (S0-S1, circles, n = 23 rats; EW-R, triangles, n = 13 rats). The line depicts a linear regression (Pearson correlation).

8 SUPPLEMENTARY METHODS Surgery and chronic recording of sleep and wakefulness Male WKY rats (Harlan, week old at time of surgery) were used. Under deep isoflurane anesthesia (1.5-2% volume), rats used in electrophysiological experiments were implanted in the frontal cortex (B: mm, L: 2-3 mm) with bipolar concentric local field potential (LFP) electrodes (outer electrode: length: 1.5 mm, diameter: 0.8 mm; inner electrode projection: 1 mm, diameter: 0.28 mm; PlasticsOne, Inc) for stimulation and chronic electroencephalographic (EEG) recordings. Electrodes were fixed to the skull with dental cement. Two stainless steel wires (diameter 0.4 mm) inserted into the neck muscles served to record the electromyogram (EMG). In rats used for molecular studies, to avoid damage to the left cortical hemisphere (used to prepare synaptoneurosome preparations, see below), the EEG was recorded using epidural screw electrodes implanted over the right parietal and occipital cortex, and cerebellum (reference electrode). After surgery all rats were housed individually in transparent Plexiglas cages (36.5 x 25 x 46 cm), and kept in sound-proof recording boxes for the duration of the experiment. Lighting and temperature were kept constant (LD 12:12, lights on at 10 am, 23 ± 1 C; food and water were available ad libitum and replaced daily at 10 am). Seven-ten days were allowed for recovery after surgery, and experiments were started only after the sleep/waking cycle had fully normalized. The rats were connected by means of a flexible cable to a commutator (Airflyte, Bayonne, NJ) and recorded continuously for 2-4 weeks using a Grass electroencephalograph (mod. 15LT, Astro-Med. Inc., West Warwick, RI). Video recordings were performed continuously with infrared cameras (OptiView Technologies, Inc) and stored in real time (AVerMedia Technologies, Inc). EEG (screw or LFP electrodes) and EMG signals were amplified (amplification factor 2000), and filtered [attenuation 50 % amplitude ( - 6 db)] as follows: EEG: high-pass filter at 0.1 Hz; low-pass filter at 30 Hz; EMG: high-pass filter at 10 Hz; low-pass filter at 100 Hz. All signals were sampled and stored at 128 Hz resolution. EEG power spectra were computed by a Fast Fourier Transform (FFT) routine for 4-s epochs (0.25 Hz resolution). Sleep stages were scored off-line by visual inspection of 4-s epochs (SleepSign, Kissei). Wakefulness was characterized by a low-voltage, high-frequency EEG pattern and phasic EMG-activity. Non-rapid eye movement (NREM) sleep was characterized by the occurrence of high-amplitude slow waves, spindles and low tonic EMG activity. During REM sleep, instead, the EEG was similar to that during waking, but only heart beats and occasional twitches were evident in the EMG signal. Epochs containing artifacts, predominantly during active waking, were excluded from spectral analysis. Vigilance state could always be determined. All animal procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and facilities were reviewed and approved by the IACUC of the University of Wisconsin-Madison, and were inspected and accredited by AAALAC. Enforced wakefulness (EW) Each day from 10 to 10:30 am all rats were gently handled and exposed to a new object to become familiar with the EW procedure (these objects were not reused during EW). Novel objects included nesting and bedding material from other rat cages, wooden blocks, small rubber balls, plastic, metallic, wooden, or paper boxes and tubes of different shape and color. The boxes often had holes through which the rat could reach for a palatable food pellet or a paper towel. During the EW experiment, a new object was introduced into the cage whenever the rat appeared drowsy and slow waves became apparent on the EEG. The rats were never disturbed when they were spontaneously awake, feeding or drinking.

9 Data analysis and statistics The software package MATLAB (The Math Works, Inc., Natick, MA, USA) was used for signal and data analysis and statistics. Differences were tested with one-way ANOVAs and paired t- tests. The relationship between the changes in LFPs slopes, brain temperature and sleep variables was determined by Pearson linear correlations. Molecular studies Animals Three groups of rats were used. Sleeping rats (S) were sacrificed during the light hours (around 4pm) at the end of a long period of sleep (at least 45 min, interrupted by periods of waking no longer than 2 min) after spending at least 75% of the previous 6 hours asleep. Spontaneously awake rats (W) were sacrificed during the dark phase (around 4am) after a long period of continuous waking (1 hour, interrupted by periods of sleep not longer than 4 min), and after spending at least 75 % of the previous 6 hours awake. Rats subjected to enforced wakefulness (EW) were sacrificed during the light period at the same time of day as S rats (around 4pm) after 6 hours of EW. Rats were deeply anesthetized with isoflurane (within 1 min) and decapitated. The head was cooled in liquid nitrogen and the whole brain was removed. Left and right cerebral cortex, left and right hippocampus, and cerebellum were dissected, while the rest of the brain was left intact. Samples were immediately frozen on dry ice and stored at - 80ºC. Synaptoneurosome preparation This protocol was optimized according to suggestions from Dr. Mark F. Bear and his laboratory, and is essentially as described in 10. Cortical samples (entire left hemisphere) were homogenized in ice-cold homogenization buffer (10 mm Hepes (Sigma) / 1.0 mm EDTA (Promega) / 2.0 mm EGTA (Fisher Scientific, Ithasca, IL)/0.5 mm DTT (Invitrogen) / 0.1 mm PMSF (Fluka) / 10 mg/l leupeptin (Sigma) / 50 mg/l soybean trypsin inhibitor (Roche) / 100 nm microcystin (Alexis) using a glass/glass tissue homogenizer (Kontes). A fraction ( ~ 10%) of the homogenate from each sample was boiled in 10% SDS for 10 min and stored unprocessed at - 80 C. The other fraction of the homogenate was passed through two 105-μm-pore nylon mesh filters (Small Parts Inc.), then through a 5-μm-pore filter (Millipore), and centrifuged at 1000 x g for 10 min at 4 C. Pellets were resuspended in boiling 1% SDS, boiled for 10 min and stored at 80 C. Protein concentration was determined by the bicinchonic acid assay (BCA, Pierce). Quantitative immunoblotting The protocol was essentially as described in 10, except that a within-lane IgY signal, instead than a within-lane actin signal, was used as loading control. This is because anti-actin antibodies do not distinguish between polymeric F-actin and monomeric G-actin, and it has been shown that protein levels of F-actin increase in the dendritic regions (spines) where synaptic potentiation has occurred. After protein concentration was determined by BCA, equal amounts (10 μg) of synaptoneurosomes from each animal were first separated by Tris-HCl gel electrophoresis (Bio- Rad) in 1X Tris / Glycine/SDS Buffer (BioRad), transferred to 0.45 μm pore size nitrocellulose membranes (BioRad) in 1X Tris base / Glycine / Methanol blotting buffer, and immunoblotted with anti-psd-95 (1 : 250, BD Biosciences Pharmingen) and / or anti-tubulin (1 : 1000, Chemicon) antibodies to confirm the enrichment for synaptic proteins in synaptoneurosomes (Fig. 1a). Afterwards, equal amounts of synaptoneurosomes from each animal were spiked with nonimmune chicken IgY (0.03 μg / lane, Promega), separated by 4-15% Tris-HCl gel electrophoresis and transferred to 0.45 μm pore size nitrocellulose membranes as above, and immunoblotted with one of the following antibodies: anti-phospho-glur1p845 (1 : 100; UpState), anti-glur1 (1

10 μg / ml;upstate), anti-phospho-glur1p831 (1 : 500, Upstate), anti-glur2 (0.5 μg / ml, Chemicon), anti-camkii (1 : 2000, Abcam), anti-phosphot286-camkii (1 : 200, Abcam), anti-nr2a (2 μg / ml, Upstate), anti-gsk3β (1 : 200, SantaCruz), and anti-phospho-gsk3β (Ser9; 1 : 300, Cell Signaling). All blots were first blocked for one hour at room temperature in freshly prepared 3% nonfat dry milk (Biorad) in 1 X PBS (Fisher) with 0.05% Tween 20 (Sigma) (PBST- MLK), incubated with the primary antibody overnight with agitation at 4 C, rinsed four times in ddh 2 O, and then incubated with the secondary antibody (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies, Chemicon) for one hour with agitation at room temperature in PBST-MLK. After the final washes (two times in PBST, 4 times in ddh 2 O), the immunoreactive bands were visualized using autoradiographic enhanced chemiluminescence (ECL-Plus, Amersham) captured by Typhoon 9410 Variable Mode Imager (Amersham). Blots were re-probed with anti-igy (for one hour), or, after incubation with anti-phospho-glur1p845 or anti-phospho-glur1p831, re-stripped and probed with anti-glur1, which recognizes both phosphorylated and non-phosphorylated GluR1. ECL signal intensities were quantified using the ImageQuant software (Amersham). Optical densities were calculated for each band of interest after performing background-correction (by subtracting the value of a band immediately above the band of interest in the same lane) and normalization (by dividing for the within-lane IgY signal used as loading control). The signal for phospho-specific antibodies was normalized to the within-lane total GluR1 signal and expressed as ratio (e.g. GluR1-s845 / GluR1). The linear range for each antibody was determined using a 5-point dilution series of rat cortical synaptoneurosomes prior to running the experimental samples. During the entire procedure, from synaptoneurosome preparation to signal intensity quantification, the experimenter was blind to the origin of the samples. Most experiments that used IgY as loading control were subsequently repeated using actin, and gave similar results (data not shown; due to the limited amount of proteins in synaptoneurosomes, not all Western blots could be performed using both controls). Electrophysiological studies LFP evoked responses In all rats LFP and EMG signals were continuously recorded during a 24-h baseline period, sleep deprivation, and recovery after sleep deprivation. The collection of LFP evoked responses occurred at ~ 4-hour intervals, usually starting at around light onset (10 am) of the baseline day, and in some animals continued during sleep deprivation and the following 4 hours of recovery. Responses were collected from the left frontal cortex (M1, B: mm, L: 2-3 mm) after electrical stimulation of the contralateral homotopic area. The transcallosal stimulation paradigm was chosen because the corpus callosum is the major brain commissure, and is composed of well organized, uniform excitatory fibers that establish direct monosynaptic excitatory cortical connections between homotopic areas, increasing the likelihood of obtaining monosynaptic responses. The electrode configuration was optimized for callosal connections, which terminate in the superficial (I - III) and deep (V - Vi) layers. In these experimental conditions, negative deflections of the field potential reflect current sinks related to inward currents along the dendrites of pyramidal cells, as a result of excitatory synaptic input. Evoked potentials obtained in the LFP signal looked similar to the responses obtained from the deep electrode referenced to the cerebellum. The responses obtained by referencing the superficial electrode to the cerebellum rarely had a well defined early monosynaptic component, and therefore were not analyzed. Prior to the experiment, input-output tests were performed in each rat. First, stimulus intensity was varied in the range between 0.1 and 7 ma (in most cases < 2 ma) to obtain a clearly identifiable early component. Evoked responses were then collected for each of 4-7 different intensities (10-15 responses / intensity), ranging from sub-threshold intensity to a level below

11 that necessary to induce a motor response. Stability and reproducibility of the shape and intensity-dependence of the responses were confirmed 2-3 days prior to the beginning of the experiment. For the final analysis one intensity was selected, corresponding to ~ 50% of the maximal intensity. The early component of the LFP evoked response consisted of a depth-negative wave with a latency to the peak of ~ 4-5 ms. The slope of the component was determined in each individual trial, and computed as mean first derivative of the first down-going segment. In addition to the short latency, the monosynaptic nature of the response was indicated by the fact that 1) the response could follow high frequency stimulation (100 Hz; Fig. 2b); 2) its latency did not change with increasing intensity of the stimulation. Mean slopes were computed per each recording session (e.g. W0, W1 etc). A normalization procedure was applied to eliminate differences among individual animals in the absolute values of the slopes. Thus, for each rat, the values of the slopes for each experimental condition (e.g. W0 and W1) are expressed as % of the mean between them prior to averaging among animals. S88 Dual Output Square Pulse Stimulator and stimulus isolating unit (PSIU-6; Grass- Telefactor, AstroMed, Inc.) were used for electrical stimulation, which consisted of monophasic squared pulse of 0.1 ms duration. Each recording session lasted approximately 1-3 min, during which a sufficient number of artifact-free responses was collected (on average ~ 40, at irregular intervals ranging from 3 sec to 1 min). In most experiments LFP evoked responses were collected in quiet waking. To standardize this behavioral state as much as possible, behavior, EEG, and EMG activity of each rat were under constant visual observation by one investigator for the entire stimulation session and for the preceding 10 minutes, during which a second investigator interacted in a standardized manner with each rat ensuring that it was awake (rats had been habituated to this protocol for at least one week). Direct contact with the animals to induce arousal was always avoided. Stimuli were delivered manually only when the rat was quietly awake (immobile, eyes open, low EMG tone, low-voltage, high-frequency cortical EEG activity). We did not attempt to record evoked responses continuously for several hours in freely behaving rats because it is impossible to maintain the animals in a standard quiet wakefulness for more than a few minutes. Moreover, even if successful, such procedure is likely to be stressful, because it affects the spontaneous behavior of the rats. Furthermore, we always tried to minimize the number of stimuli per session, to avoid potential damage to the tissue or the induction of long-lasting changes in excitability. We did not find any consistent response change within each recording session (Fig. 2g), suggesting that our stimulation protocol per se did not lead to significant plastic changes. In some experiments (described in Fig. 3) evoked responses for the high and low sleep pressure conditions were also collected during active waking, NREM sleep, and REM sleep. After each session, all individual responses were visually screened by an experimenter blind to their origin, and only those that were recorded on a background of stable low-amplitude, high-frequency cortical activity were used for further analysis. LFP evoked responses show substantial and predictable changes in shape and/or amplitude with changes in behavioral state, and in some individuals a few responses (less than 5%) were discarded on that basis (largely due to brief movements immediately before the investigator triggered the stimulation). Responses were recorded with 5 khz sampling rate, analog filters: high-pass filter: attenuation 50 % amplitude at 0.1 Hz; low-pass filter: attenuation 50% amplitude at 1 khz. In a pilot study, we recorded two consecutive sessions of LFP evoked responses at the light-dark transition (10 pm) and at the dark-light transition (10 am). In the morning the first session was performed ~ 5 min before lights went on, and the second session ~ 5 min after lights went on; in the evening the first session occurred ~ 5 min before lights went off on and the second session ~ 5 min after lights went off. We found no consistent changes in the slope of LFPs between the 'light' and 'dark' session at either 10 am or 10 pm. For LTP induction experiments, in 7 rats transcallosal evoked responses were collected as described above (always in quiet wakefulness) at light onset, after a spontaneous waking pe-

12 riod, and after ~ 4 hours of undisturbed sleep. LFPs were collected immediately before and after a set of high-frequency trains was delivered to the frontal cortex to induce LTP. The stimulation paradigm consisted of sixty 24-ms trains at 300 Hz delivered every 10 sec (=60 trains in total). Each rat was subjected to the LTP-inducing protocol twice, at light onset and after 4 hours of sleep (the two conditions were counter-balanced, and spaced at least 2 days apart). To measure brain temperature, a calibrated 10-kOhm thermistor (model 44008; Omega Engineering Inc., USA) was implanted through a hole in the skull above the parietal cortex. The data were acquired with the LabJack device and LabView software and stored on PC with 50 Hz resolution. Consistent with previous published studies, we found that brain temperature was, on average, 0.6 C ± 0.1 higher during the dark period than during the light period (p < 0.01). In addition, we found that brain temperature during wakefulness was 0.35 C ± 0.05 higher than during sleep during the light period, and 0.4 C ± 0.06 higher than during sleep during the dark period. Histological verification Upon completion of the experiments the position of the LFP electrodes was verified by histology in all animals. Perfusion was performed under deep isoflurane anesthesia (3% in oxygen), with saline followed by a 4% solution of paraformaldehyde (PFA) in 0.1 M sodium phosphate, ph 7.2. Brains were post-fixed overnight in 4% PFA, cryoprotected in increasing concentration of sucrose (15, 20, 30%) in phosphate buffered saline, rapidly frozen on dry ice, cut into 50 µm serial coronal sections, and subjected to cresyl-violet (Nissl) staining. In all cases the deep LFP electrode was located within layer V, whereas the superficial LFP electrode was in layers I - II.