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Copy pathPCAanalysis3_pupil.m
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PCAanalysis3_pupil.m
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% Run this script to perform PCA analyses.
%% LOAD PRE-PROCESSED DATA
load(dataFile);
%% INITIALISE PARAMETERS
lists
pcaParams
%% LOAD RECORDINGS AND EXTRACT LFP MEASURES
% LOOP THROUGH DB ENTRIES
fnsData = fieldnames(dataStruct.seriesData);
for dbCount = dbStart:numel(fnsData)
if dbCount > dbEnd
break
end
dbStruct = dataStruct.seriesData.(fnsData{dbCount});
% TEST FOR EYE DATA
if isfield(dbStruct, 'lfpPowerData') && ~isfield(dbStruct.lfpPowerData, 'areaInterpFilt')
continue
end
% TEST FOR EXCEPTIONS
[seriesName, animal] = seriesFromEntry(dbStruct.db(dbCount).entryName);
if numel(seriesName) <= 14
continue
elseif numel(seriesName) == 15 &&...
(strcmpi(seriesName(15), '3') || strcmpi(seriesName(15), '4') || strcmpi(seriesName(15), '5'))
continue
elseif numel(seriesName) >= 16
continue
end
% TEST FOR RIPPLES
ripplesExist = rippleTest(fnsData{dbCount}, fullSeries);
% IDENTIFY AREA
area = determineArea(seriesName);
if ~area
continue
end
% PICK UP LFP CHANNEL
if ~(numel(seriesName) == 16)
chOI = dbStruct.lfpPowerData.chOIDB;
iCh = pickChan(area, animal, chOI);
if isempty(iCh)
continue
end
end
% LOAD THE CONTENTS OF THE DB STRUCTURE VARIABLE
params = dbStruct.conf.params;
probe = dbStruct.conf.probe;
if strcmpi(probe, 'Neuropixels')
strInd = strfind(dbStruct.io.baseFilename, 'ap');
baseFilename = [dbStruct.io.baseFilename(1:strInd-1) 'lf.bin'];
if ~exist(baseFilename, 'file')
baseFilename = [dbStruct.io.baseFilename(1:strInd-1) 'lf.dat'];
end
else
baseFilename = [dbStruct.io.baseFilename '.dat'];
end
if ~exist(baseFilename, 'file')
error(['The supplied LFP file does not exist. Please check the location folder: ' dbStruct.io.dataDir]);
end
chN = dbStruct.db(dbCount).chN;
entryName = dbStruct.db(dbCount).entryName;
% LOAD EXISTING LFP ANALYSIS DATA
if strcmpi(seriesName(15), '1')
neuralActivity = [];
LFPs = [];
PRs = [];
areas = [];
entryNames = {};
end
if isfield(dbStruct,'lfpPowerData')
if (area == 5 || area == 10) && medianSubtracted % It's ok to use the existing ripple rate because it is based on median subtracted LFP
rippleRate = dbStruct.lfpPowerData.rippleRate;
else
rippleRate = {};
rippleRate{iCh} = []; %#ok<*SAGROW>
end
theta2deltaRatio = dbStruct.lfpPowerData.theta2deltaRatio;
chOIDB = dbStruct.lfpPowerData.chOIDB;
dssrLFPinit = dbStruct.lfpPowerData.dssrLFPinit;
lfpTimes = dbStruct.lfpPowerData.lfpTimes;
rippleDuration = dbStruct.lfpPowerData.rippleDuration;
sdGaussian = dbStruct.lfpPowerData.sdGaussian;
wGaussian = dbStruct.lfpPowerData.wGaussian;
else
error(['No LFP analysis data exist for series: ' fnsData{dbCount} '. Please run lfpLoad.m']);
end
% RESAMPLE EXISTING LFP DATA IF NECESSARY
if params.srData ~= dssrLFPinit
interpTimes = 1/params.srData:1/params.srData:lfpTimes(end);
if (area == 5 || area == 10) && medianSubtracted
rippleRate{iCh} = interp1(lfpTimes, rippleRate{iCh}, interpTimes)';
end
theta2deltaRatio{iCh} = interp1(lfpTimes, theta2deltaRatio{iCh}, interpTimes)';
else
interpTimes = lfpTimes;
end
% RECALCULATE RIPPLE RATE IF EXISTING ONE IS NOT BASED ON MEDIAN-SUBTRACTED LFP
if (area == 5 || area == 10) && ~medianSubtracted && ripplesExist
srRippleRate = 1000;
if strcmpi(probe, 'Neuropixels')
[LFPbandPower] = lfpFileLoad(baseFilename, 1, chunkSize, chN, chOIDB(iCh), LFPbands, params.srRecordingLFP, srRippleRate, true, deleteChans, false);
else
[LFPbandPower] = lfpFileLoad(baseFilename, 1, chunkSize, chN, chOIDB(iCh), LFPbands, params.srRecording, srRippleRate, true, deleteChans, false);
end
rippleBandPower{iCh} = LFPbandPower{1}(end,:);
rippleRate{iCh} = rippleRateCalculator({rippleBandPower{iCh}}, rippleDuration, sdGaussian, wGaussian, chOIDB(iCh), bandNames, srRippleRate); %#ok<CCAT1>
rippleRate{iCh} = rippleRate{iCh}{1};
rippleRateTimes = 1/srRippleRate:1/srRippleRate:numel(rippleRate{iCh})/srRippleRate;
rippleRate{iCh} = interp1(rippleRateTimes, rippleRate{iCh}, interpTimes)';
elseif (area == 5 || area == 10) && ~ripplesExist
rippleRate{iCh} = zeros(size(theta2deltaRatio{iCh}));
end
% LOAD, DOWN-SAMPLE LFP DATA, AND CALCULATE WAVELET TRANSFORMS
if strcmpi(probe, 'Neuropixels')
[LFPbandPower, ~, ~, LFP] = lfpFileLoad(...
baseFilename, min(LFPbands{1}), chunkSize, chN, chOIDB, LFPbands, params.srRecordingLFP, params.srData, medianSubtracted, deleteChans, false);
else
[LFPbandPower, ~, ~, LFP] = lfpFileLoad(...
baseFilename, min(LFPbands{1}), chunkSize, chN, chOIDB, LFPbands, params.srRecording, params.srData, medianSubtracted, deleteChans, false);
end
% LOAD SPIKING DATA
PR = sum(dbStruct.popData.MUAsAll,1);
% CONCATENATE DATA
PR = torow(PR);
LFP{iCh} = torow(LFP{iCh});
rippleRate{iCh} = torow(rippleRate{iCh});
theta2deltaRatio{iCh} = torow(theta2deltaRatio{iCh});
dataDuration = max([size(neuralActivity,2) size(LFPbandPower{iCh},2) numel(rippleRate{iCh}) numel(theta2deltaRatio{iCh})...
numel(LFP{iCh}) size(LFPs,2) numel(PR) size(PRs,2)]);
if isempty(PR)
PR = zeros(1,dataDuration);
end
if ~isempty(neuralActivity) && size(neuralActivity,2) < dataDuration
zeroPad = zeros(size(neuralActivity,1), dataDuration - size(neuralActivity,2));
neuralActivity = [neuralActivity zeroPad];
end
if size(LFPbandPower{iCh},2) < dataDuration
zeroPad = zeros(size(LFPbandPower{iCh},1), dataDuration - size(LFPbandPower{iCh},2));
LFPbandPower{iCh} = [LFPbandPower{iCh} zeroPad];
end
if (area == 5 || area == 10) && size(rippleRate{iCh},2) < dataDuration
zeroPad = zeros(size(rippleRate{iCh},1), dataDuration - size(rippleRate{iCh},2));
rippleRate{iCh} = [rippleRate{iCh} zeroPad];
end
if size(theta2deltaRatio{iCh},2) < dataDuration
zeroPad = zeros(size(theta2deltaRatio{iCh},1), dataDuration - size(theta2deltaRatio{iCh},2));
theta2deltaRatio{iCh} = [theta2deltaRatio{iCh} zeroPad];
end
if size(LFP{iCh},2) < dataDuration
zeroPad = zeros(size(LFP{iCh},1), dataDuration - size(LFP{iCh},2));
LFP{iCh} = [LFP{iCh} zeroPad];
end
if size(PR,2) < dataDuration
zeroPad = zeros(size(PR,1), dataDuration - size(PR,2));
PR = [PR zeroPad];
end
if (area == 5 || area == 10)
neuralActivity = [neuralActivity; LFPbandPower{iCh}; rippleRate{iCh}; theta2deltaRatio{iCh}; LFP{iCh}; PR];
else
neuralActivity = [neuralActivity; LFPbandPower{iCh}; theta2deltaRatio{iCh}; LFP{iCh}; PR];
end
addedVectors = size(LFPbandPower{iCh},1) + ~isempty(rippleRate{iCh}) + size(theta2deltaRatio{iCh},1) + size(LFP{iCh},1) + ~isempty(PR);
neuralActivity(isnan(neuralActivity)) = 0;
neuralActivity(isnan(neuralActivity)) = 0;
if size(LFPs,2) < dataDuration
zeroPad = zeros(size(LFPs,1), dataDuration - size(LFPs,2));
LFPs = [LFPs zeroPad];
end
LFPs = [LFPs; LFP{iCh}];
if size(PRs,2) < dataDuration
zeroPad = zeros(size(PRs,1), dataDuration - size(PRs,2));
PRs = [PRs zeroPad];
end
PRs = [PRs; PR];
areas = [areas; ones(addedVectors,1)*area];
entryNames{numel(entryNames) + 1} = entryName;
if dbCount < numel(fnsData)
seriesNameNext = seriesFromEntry(dbStruct.db(dbCount+1).entryName);
end
if dbCount < numel(fnsData) - 1
seriesNameNext2 = seriesFromEntry(dbStruct.db(dbCount+2).entryName);
end
if dbCount == numel(fnsData) || ~strcmpi(seriesName(1:14), seriesNameNext(1:14)) ||...
(dbCount == numel(fnsData)-1 && (area == 5 || area == 10)) || ((area == 5 || area == 10) && ~strcmpi(seriesName(1:14), seriesNameNext2(1:14)))
% LOAD AROUSAL MEASURES
pupilArea = torow(dbStruct.lfpPowerData.areaInterpFilt);
pupilAreaTimes = torow(dbStruct.lfpPowerData.areaInterpTimes);
% Truncate time vectors
[~, iPCs2pupilAreaStart] = min(abs(interpTimes - pupilAreaTimes(1)));
[~, iPCs2pupilAreaEnd] = min(abs(interpTimes - pupilAreaTimes(end)));
PCs2pupilAreaTimes = interpTimes(iPCs2pupilAreaStart:iPCs2pupilAreaEnd);
% Resample arousal measures if necessary
if params.srData ~= dssrLFPinit
pupilArea = torow(interp1(pupilAreaTimes, pupilArea, PCs2pupilAreaTimes));
end
% ADD AROUSAL DATA
neuralActivity = neuralActivity(:,iPCs2pupilAreaStart:iPCs2pupilAreaEnd);
neuralActivity = [neuralActivity; pupilArea];
neuralActivity(isnan(neuralActivity)) = 0;
areas = [areas; 0];
% SUBTRACT THE MEAN AND Z-SCORE THE DATA
neuralActivity = neuralActivity - mean(neuralActivity,2);
neuralActivity = zscore(neuralActivity')';
% RUN PCA
[pcaCoef, explained, PCs, nPCs, prob, individualVarExplained] = pcaGeneric(neuralActivity);
% Calculate a number of variables needed to explain proportions of variance
[explained25, explained50, explained75, explained95, explainedThird, explainedTwoThirds] = explainedProportions(explained);
% CALCULATE EXPLAINED AREA FOR AROUSAL DATA
pcaCoef_arousal = pcaCoef(end);
explained_arousal = 100*mean(individualVarExplained(:,end), 2)';
% Calculate a number of variables needed to explain proportions of variance for arousal data
[explained25_arousal, explained50_arousal, explained75_arousal, explained95_arousal,...
explainedThird_arousal, explainedTwoThirds_arousal] = explainedProportions(explained_arousal);
% CALCULATE EXPLAINED VARIANCE FOR EACH AREA
areasOI = unique(areas);
areasOI(areasOI == 0) = [];
for iArea = 1:numel(areasOI)
dataOI = areas;
dataOI(dataOI ~= areasOI(iArea)) = 0;
dataOI = logical(dataOI);
pcaCoef_area = pcaCoef(dataOI);
explained_area = 100*mean(individualVarExplained(:,dataOI), 2)';
% Calculate a number of variables needed to explain proportions of variance for the area
[explained25_area, explained50_area, explained75_area, explained95_area,...
explainedThird_area, explainedTwoThirds_area] = explainedProportions(explained_area);
% CORRELATE PCs WITH LFP
[rPCs2lfpPearson_area, pvalPCs2lfpPearson_area] = corrMulti(LFPs(iArea,iPCs2pupilAreaStart:iPCs2pupilAreaEnd), PCs, 'Pearson');
[rPCs2lfpSpearman_area, pvalPCs2lfpSpearman_area] = corrMulti(LFPs(iArea,iPCs2pupilAreaStart:iPCs2pupilAreaEnd), PCs, 'Spearman');
% CORRELATE PCs WITH PR
[rPCs2prPearson_area, pvalPCs2prPearson_area] = corrMulti(PRs(iArea,iPCs2pupilAreaStart:iPCs2pupilAreaEnd), PCs, 'Pearson');
[rPCs2prSpearman_area, pvalPCs2prSpearman_area] = corrMulti(PRs(iArea,iPCs2pupilAreaStart:iPCs2pupilAreaEnd), PCs, 'Spearman');
% SAVE DATA
% PCA data for all areas
pcaData.pcaCoef = pcaCoef;
pcaData.explained = explained;
pcaData.nPCs = nPCs;
pcaData.prob = prob;
pcaData.explained25 = explained25;
pcaData.explained50 = explained50;
pcaData.explained75 = explained75;
pcaData.explained95 = explained95;
pcaData.explainedThird = explainedThird;
pcaData.explainedTwoThirds = explainedTwoThirds;
pcaData.explained_arousal = explained_arousal;
pcaData.explained25_arousal = explained25_arousal;
pcaData.explained50_arousal = explained50_arousal;
pcaData.explained75_arousal = explained75_arousal;
pcaData.explained95_arousal = explained95_arousal;
pcaData.explainedThird_arousal = explainedThird_arousal;
pcaData.explainedTwoThirds_arousal = explainedTwoThirds_arousal;
% PCA data for the area of interest
pcaData.pcaCoef_area = pcaCoef_area;
pcaData.explained_area = explained_area;
pcaData.explained25_area = explained25_area;
pcaData.explained50_area = explained50_area;
pcaData.explained75_area = explained75_area;
pcaData.explained95_area = explained95_area;
pcaData.explainedThird_area = explainedThird_area;
pcaData.explainedTwoThirds_area = explainedTwoThirds_area;
pcaData.rPCs2lfpPearson_area = rPCs2lfpPearson_area;
pcaData.pvalPCs2lfpPearson_area = pvalPCs2lfpPearson_area;
pcaData.rPCs2lfpSpearman_area = rPCs2lfpSpearman_area;
pcaData.pvalPCs2lfpSpearman_area = pvalPCs2lfpSpearman_area;
pcaData.rPCs2prPearson_area = rPCs2prPearson_area;
pcaData.pvalPCs2prPearson_area = pvalPCs2prPearson_area;
pcaData.rPCs2prSpearman_area = rPCs2prSpearman_area;
pcaData.pvalPCs2prSpearman_area = pvalPCs2prSpearman_area;
dataString = ['dataStruct.seriesData.' entryNames{iArea} '.pcaData3_pupil = pcaData;'];
eval(dataString);
if intermediateSaving
save(dataFile,'dataStruct','-v7.3'); %#ok<*UNRCH>
end
end
end
end
if ~intermediateSaving
save(dataFile,'dataStruct','-v7.3');
end
clearvars -except dataFile dbStart dbEnd
function [LFPbandPower, wtSpectrogram, fSpectrogram, interpDat] = lfpFileLoad(...
baseFilename, minF, chunkSize, chN, chOI, LFPbands, srRecording, srInterp, medianSubtracted, deleteChans, spectrogram)
% File loading parameters
fid = fopen(baseFilename, 'r');
d = dir(baseFilename);
nSampsTotal = d.bytes/chN/2;
nChunksTotal = ceil(nSampsTotal/chunkSize);
% Load the file
chunkInd = 1;
LFPbandPower = {};
wtSpectrogram = {};
interpDat = {};
while 1
fprintf(1, 'chunk %d/%d\n', chunkInd, nChunksTotal);
dat = fread(fid, [chN chunkSize], '*int16');
% Subtract median and/or delete channels if necessary
if medianSubtracted
if deleteChans
chans2include = ones(1,size(dat,1));
chans2include(deleteChans) = zeros(1,numel(deleteChans));
chm = zeros(size(dat,1),1);
chm(logical(chans2include)) = median(dat(logical(chans2include),:),2);
dat = bsxfun(@minus, dat, int16(chm)); % subtract median of each channel
tm = int16(median(dat(logical(chans2include),:),1));
else
chm = median(dat,2);
dat = bsxfun(@minus, dat, chm); % subtract median of each channel
tm = median(dat,1);
end
dat = bsxfun(@minus, dat, tm); % subtract median of each time point
end
if ~isempty(dat)
% Interpolate data
originalTimes = 1/srRecording:1/srRecording:size(dat,2)/srRecording;
interpTimes = 1/srInterp:1/srInterp:size(dat,2)/srRecording;
interpLFP = interp1(originalTimes, double(dat'), interpTimes)';
for iCh = 1:numel(chOI)
% Calculate wavelet transforms
fb = cwtfilterbank('SignalLength',size(interpLFP,2),'SamplingFrequency',srInterp,...
'FrequencyLimits',[minF 200],'WaveletParameters',[3 16],'VoicesPerOctave',20);
[wt,f] = cwt(interpLFP(chOI(iCh),:),'FilterBank',fb); % Continuous wavelet transform
LFPbandPowerChunk = zeros(numel(LFPbands),size(wt,2));
for iBand = 1:numel(LFPbands)
for iLim = 1:2
[~, LFPbandLimits{iBand}(iLim)] = min(abs(f-LFPbands{iBand}(iLim))); %#ok<*AGROW>
end
LFPbandPowerChunk(iBand,:) = sum(abs(wt(LFPbandLimits{iBand}(2):LFPbandLimits{iBand}(1),:)).^2,1);
end
if chunkInd == 1
LFPbandPower{iCh} = LFPbandPowerChunk;
interpDat{iCh} = interpLFP(chOI(iCh),:);
else
LFPbandPower{iCh} = [LFPbandPower{iCh} LFPbandPowerChunk]; %#ok<*AGROW>
interpDat{iCh} = [interpDat{iCh} interpLFP(chOI(iCh),:)];
end
% Spectrogram
if spectrogram
fb = cwtfilterbank('SignalLength',size(interpLFP,2),'SamplingFrequency',srInterp,...
'FrequencyLimits',[minF 200],'WaveletParameters',[3 16],'VoicesPerOctave',10);
[wtSpectrogramChunk,fSpectrogram] = cwt(interpLFP(chOI(iCh),:),'FilterBank',fb); % Continuous wavelet transform
%helperCWTTimeFreqPlot(wt,interpTimes,f,'surf','Spectrogram for CA1 channel','Seconds','Hz');
%set(gca, 'YScale', 'log')
if chunkInd == 1
wtSpectrogram{iCh} = abs(wtSpectrogramChunk).^2;
else
wtSpectrogram{iCh} = [wtSpectrogram{iCh} abs(wtSpectrogramChunk).^2];
end
else
fSpectrogram = [];
end
end
else
break
end
chunkInd = chunkInd+1;
end
end
function [rippleRate, meanRippleRate] = rippleRateCalculator(rippleBandPower, rippleDuration, sdGaussian, wGaussian, chOI, bandNames, srRippleRate)
for iCh = 1:numel(chOI)
% CALCULATE RIPPLE RATE AS IN MCGINLEY ET AL. (2015)
% Descriptive measures
for iName = 1:numel(bandNames)
if strcmpi(bandNames{iName}, 'ripples/uf')
iRipples = iName;
break
end
end
% figure; plot(timeInit, LFPbandPower{iCh}(iRipples,:)); hold on
medianPower = median(rippleBandPower{iCh},'omitnan');
stdPower = std(rippleBandPower{iCh},'omitnan');
% stdPowerTop = medianPower+1.96*stdPower;
% stdPowerBottom = medianPower-1.96*stdPower;
stdPowerTop = medianPower+5*stdPower;
stdPowerBottom = medianPower-5*stdPower; %#ok<*NASGU>
%plot(medianPower*ones(1,size(LFPbandPower{iCh},2)))
%plot(stdPowerTop*ones(1,size(LFPbandPower{iCh},2)))
%plot(stdPowerBottom*ones(1,size(LFPbandPower{iCh},2)))
% Detect initial ripples
[ripplePowerPeaks, locations] = findpeaks(rippleBandPower{iCh});
locations = locations(ripplePowerPeaks > stdPowerTop);
totalRipplesInit = numel(locations);
% ripplePowerPeaks = ripplePowerPeaks(ripplePowerPeaks > stdPowerTop);
% plot(timeInit(locations),ripplePowerPeaks, 'r.', 'MarkerSize',5)
% Mark ripple initiation and termination
ripplePowerPeaksInit = zeros(1,size(rippleBandPower{iCh},2));
ripplePowerPeaksInit(locations) = 1;
ripplePowerPeaks = ripplePowerPeaksInit;
for dtRipple = 1:round(rippleDuration/2)
ripplePowerPeaks = ripplePowerPeaks + [ripplePowerPeaksInit(1+dtRipple:end) zeros(1,dtRipple)];
ripplePowerPeaks = ripplePowerPeaks + [zeros(1,dtRipple) ripplePowerPeaksInit(1:end-dtRipple)];
end
ripplePowerPeaks(ripplePowerPeaks > 0) = 1;
locations = 1:size(rippleBandPower{iCh},2);
locations = locations(logical(ripplePowerPeaks));
ripplePowerPeaksInit = ripplePowerPeaks;
% ripplePowerPeaks = ripplePowerPeaks(ripplePowerPeaks > 0);
% plot(timeInit(locations),ripplePowerPeaks, 'k.', 'MarkerSize',10); hold on
% Total ripple event count
ripplePowerPeaks2 = findpeaks(ripplePowerPeaksInit);
totalRipples = numel(ripplePowerPeaks2);
% Convolve with Gaussian
w = gausswin(wGaussian*sdGaussian*srRippleRate, (wGaussian*sdGaussian*srRippleRate-1)/(2*sdGaussian*srRippleRate));
w = w/sum(w);
ripplePowerPeaks = filtfilt(w,1,ripplePowerPeaksInit);
ripplePowerPeaks = ripplePowerPeaks/sum(ripplePowerPeaks)*sum(rippleBandPower{iCh});
%plot(ripplePowerPeaks)
%rippleRateInit = ripplePowerPeaks/mean(ripplePowerPeaks)*((totalRipplesInit/size(rippleBandPower{iCh},2))*srRippleRate); % Hz
rippleRate{iCh} = ripplePowerPeaks/mean(ripplePowerPeaks)*((totalRipples/size(rippleBandPower{iCh},2))*srRippleRate); % Hz
meanRippleRate{iCh} = mean(rippleRate{iCh});
%timeInit = 1/srRippleRate:1/srRippleRate:size(rippleBandPower{iCh},2)/srRippleRate;
% plot(timeInit,rippleRate{iCh});
% Down-sample again
%interpTimesFinal = 1/dssrLFPfinal:1/dssrLFPfinal:size(rippleBandPower{iCh},2)/srRippleRate;
%rippleRate{iCh} = torow(interp1(timeInit, rippleRate{iCh}, interpTimesFinal));
%timeFinal = 1/dssrLFPfinal:1/dssrLFPfinal:size(rippleBandPower{iCh},2)/srRippleRate;
%plot(timeFinal,rippleRate{iCh}); hold off
end
end
function [explained25, explained50, explained75, explained95, explainedThird, explainedTwoThirds] = explainedProportions(explained)
explainedCumulitive = cumsum(explained);
explainedCumulitive25 = explainedCumulitive - 25;
explained25 = find(explainedCumulitive25 >= 0);
explainedCumulitive50 = explainedCumulitive - 50;
explained50 = find(explainedCumulitive50 >= 0);
explainedCumulitive75 = explainedCumulitive - 75;
explained75 = find(explainedCumulitive75 >= 0);
explainedCumulitive95 = explainedCumulitive - 95;
explained95 = find(explainedCumulitive95 >= 0);
explainedCumulitiveThird = explainedCumulitive - 100/3;
explainedThird = find(explainedCumulitiveThird >= 0);
explainedCumulitiveTwoThirds = explainedCumulitive - 200/3;
explainedTwoThirds = find(explainedCumulitiveTwoThirds >= 0);
end