Neural Data Analysis

Transcription

1 Neural Data Analysis

2 Comprehensive Neural Analysis with AxIS

3 Layers of Neuronal Activity Raw Voltage Data Single electrode analysis provides information on individual neuron firing and bursting Control 100 mv 10 s Single Spike Raster MFR = mean firing rate # of Spikes Burst Duration Inter-burst Interval (IBI) Inter-spike Interval (ISI) Burst Frequency

4 Layers of Neuronal Activity Raw Voltage Data Single electrode analysis provides information on individual neuron firing and bursting Control 100 mv 10 s Single Spike Raster Well Raster Synchrony Multiple electrode analysis provides information on network bursting and synchrony Control Network burst Burst duration Burst frequency Number of spikes in burst Number of electrodes % of spikes in bursts AUCD 20ms HWHHCD Synchrony index

5 Layers of Neuronal Activity Raw Voltage Data Single electrode analysis provides information on individual neuron firing and bursting Control 100 mv 10 s Single Spike Raster Well Raster Multiple electrode analysis provides information on network bursting and synchrony Control Millard et al., SfN 2014 AxIS provides ~25 metrics to characterize the phenotypic behavior of a neuronal culture s firing activity PTX Effect of Compounds

6 Guide to cross-correlation and synchrony Assesses the probability of a spike on 1 channel, relative to another The larger the peak at 0 and area under the curve, the more synchronous two electrodes are AxIS uses frequency domain methods to compute and pool across all pairwise combinations in a well (Halliday, Rosenber, Breeze, & Conway 2006) A B Crosscorrelation for A referenced to B plot density time = 0 Go through data spike-by-spike for electrode B Treat each spike as time = 0 Bin surrounding spike density in electrode A

7 Neural Analysis Workflow Record Raw Data Apply Analysis Configuration (Spike & Burst Detector and Statistics Compiler) Record Advanced Metrics and/or AxIS Spike (From Statistics Compiler and Spike Detector, respectively) Advanced Metrics Output:.csv file, usable in Excel File/Analysis Settings Electrode, Well, and Group Averages for: Mean Firing Bursting Synchrony AxIS Spike Output: Waveform traces for all spikes Import to NeuroExplorer (v4.1+) or MATLAB Used by Neural Metrics Tool (Axion) Creates figures and.csv file

8 Recording Neural Data Hardware Settings Right Click Maestro Stream, choose Settings Specify analog mode (usually Neural Spikes ) Referencing Median (default) For visual monitoring of activity during recording, use Spike Detector and Burst Detector modules Right click Maestro stream -> Configuration -> Neural -> Real-time Spontaneous Statistics Compiler module can only be used when replaying recorded data. If signals look good, record the raw data First play the data and wait for offset correction to finish before pressing record Generally 2-5 min of data is enough to get good average measures Details can be found in the AxIS User Guide section 3.2

9 Neural Data Analysis Before You Begin. Organize the files to be analyzed Add notes, plate maps, and descriptions Ensure they are named properly Tips Segment the files into the regions to be analyzed (see protocol on right) Files analyzed over a network connection will take longer to process Method 1 1. Open the file in AxIS 3b. Play until the desired start time is reached, or click on the green bar 4b. Press Record to begin and stop recording analysis file at the desired time Protocol: Segmenting Files 2. Choose AxIS Raw file type from Experiment Setup Method 2 3a. Double-click the file name under Streams to open the file settings 4a. Select the Segment Type settings and choose the start or end of the file to record Note: Use the Start of File setting and apply a start offset to record a segment in the middle of a file 5a. Select Accelerate Playback, ensure Loop is not selected 6a. Press Record when ready to record analysis file

10 Before You Begin Analysis Protocol: Segmenting Files 1. Open the file in AxIS 2. Choose AxIS Raw file type from Experiment Setup Method 2 3a. Double-click the file name under Streams to open the file settings 4a. Select the Segment Type settings and choose the start or end of the file to record Note: Use the Start of File setting and apply a start offset to record a segment in the middle of a file 5a. Select Accelerate Playback, ensure Loop is not selected 6a. Press Record

11 Neural Analysis Workflow Record Raw Data Apply Analysis Configuration (Spike & Burst Detector and Statistics Compiler) Record Advanced Metrics and/or AxIS Spike (From Statistics Compiler and Spike Detector, respectively) Advanced Metrics Output:.csv file, usable in Excel File/Analysis Settings Electrode, Well, and Group Averages for: Mean Firing Bursting Synchrony AxIS Spike Output: Waveform traces for all spikes Import to NeuroExplorer (v4.1+) or MATLAB Used by Neural Metrics Tool (Axion) Creates figures and.csv file

12 Apply Processors Apply the Spike Detector Recommend to use Adaptive Threshold set to 6 SDs 5.5 is acceptable but may result in some false positives Pre- and post-spike durations should be large enough to capture the entire waveform (~2ms) Apply the Burst Detector Inter-Spike Interval (ISI) A burst is defined as a minimum number of spikes with a maximum time between each spike Poisson Surprise A statistical approach classifying a cluster of spikes that passes a surprise factor threshold Right-click file name to access the Configuration menu and apply processors

13 Apply Processors Apply the Spike Detector Recommend to use Adaptive Threshold set to 6 SDs 5.5 is acceptable but may result in some false positives Pre- and post-spike durations should be large enough to capture the entire waveform (~2ms) Apply the Burst Detector Inter-Spike Interval (ISI) A burst is defined as a minimum number of spikes with a maximum time between each spike Poisson Surprise A statistical approach classifying a cluster of spikes that passes a surprise factor threshold Right-click file name to access the Configuration menu and apply processors Note: Network Burst Detection is not enabled by default. To analyze network bursts, double-click the Burst Detector and click Enable Network Burst Detection.

14 Neural Analysis Workflow Record Raw Data Apply Analysis Configuration (Spike & Burst Detector and Statistics Compiler) Record Advanced Metrics and/or AxIS Spike (From Statistics Compiler and Spike Detector, respectively) Advanced Metrics Output:.csv file, usable in Excel File/Analysis Settings Electrode, Well, and Group Averages for: Mean Firing Bursting Synchrony AxIS Spike Output: Waveform traces for all spikes Import to NeuroExplorer (v4.1+) or MATLAB Used by Neural Metrics Tool (Axion) Creates figures and.csv file

15 Setting up AxIS for Analysis Output 1. Click the Experiment Setup Pane 2. Select Advanced Metrics and AxIS Spike (optional) from the Continuous Streams menu 3. Click Record to Analyze 3. Name file 2. Select the data type to be recorded 1. Experiment Setup Pane

16 Analyzing Multiple Files Use Batch Process when analyzing multiple files Optimized for speed Applies same analysis settings to each file Choose New Batch Process instead of Open Recording Click Add to select files Choose the analysis duration in Segment Type Treat the Batch Process the same as a normal file Add Processors (manually or through a Configuration) Select Start Batch Process Go grab a coffee

17 Neural Analysis Workflow Record Raw Data Apply Analysis Configuration (Spike & Burst Detector and Statistics Compiler) Add Statistics Compiler Stream to Burst Detector Data Stream Record Advanced Metrics and/or AxIS Spike (From Statistics Compiler and Spike Detector, respectively) Advanced Metrics Output:.csv file, usable in Excel File/Analysis Settings Electrode, Well, and Group Averages for: Mean Firing, Weighted Mean Firing # Active Electrodes Bursting Synchrony

18 AxIS Statistics Compiler Output Electrode, Well, and Group Averages for: Mean Firing # of Active Electrodes Weighted Mean Firing Rate (Hz) Bursting Synchrony Details can be found in the AxIS User Guide section 3.3

19 Neural Analysis Workflow Record Raw Data Apply Analysis Configuration (Spike & Burst Detector and Statistics Compiler) Record Advanced Metrics and/or AxIS Spike (From Statistics Compiler and Spike Detector, respectively) Advanced Metrics Output:.csv file, usable in Excel File/Analysis Settings Electrode, Well, and Group Averages for: Mean Firing Bursting Synchrony AxIS Spike Output: Waveform traces for all spikes Import to NeuroExplorer (v4.1+) or MATLAB Used by Neural Metrics Tool (Axion) Creates figures and.csv file

20 Neural Analysis File Types Neural Statistics Compiler CSV A sorted list of neural statistics, including spike, burst, and synchrony metrics Metrics provided for individual electrodes and well-wide averages AxIS.spk file Spike time stamps and voltage waveforms of detected spikes on each electrode Can be used in Axion Neural Metric Tool, and compatible with 3 rd -party analysis tools Spike Count CSV Number of spikes per second Total spikes and firing rate Numbers provided per well and per electrode Spike list, Channel burst list, Network burst list CSV A chronological list of spikes or bursts and their descriptors Time of each spike, channel it occurred on, amplitude, size and duration of bursts Useful for analysis programs that specialize in unsorted data, such as Spotfire

21 Alternate Analysis Tools NMT version 2.0 AxIS Metric Plotting Tool version 1.0

22 Neural Metric Tool- Same Output as AxIS Loaded spike (.spk) file Details can be found in the AxIS User Guide, Appendix B and 2p3 Supplemental

23 Plus. Evoked Activity Analysis for Optical Stimulation Figure Generation Create Crosscorrelograms, raster plots, and heat maps with one click Update detection settings and view the impact on results Remove Coincident Artifacts Can launch the tool more than once to compare different files Details can be found in the AxIS User Guide, Appendix B and 2p3 Supplemental

24 Using the Neural Metric Tool LOAD AN AxIS SPIKE FILE (.SPK) ANALYSIS PARAMETERS Select Analysis Duration and Active Criteria and press Apply BURSTING, SYNCHRONYAND TRIGGERED PARAMETERS CLICK ON PLATE-MAP TO VIEW PLOTS FOR DIFFERENT WELLS SELECT COPY TO GENERATE FIGURES FROM CURRENTLY DISPLAYED WELLS EXPORT RESULTS AS.CSV or.mat Details can be found in the AxIS User Guide, Appendix B and 2p3 Supplemental

25 Plate Map and Analysis Parameters Displayed Metric Active Well Copy/ Refresh Start/ End Analysis The Burst, Synchrony and Triggered plots are automatically uploaded to display the data from the active well Details can be found in the AxIS User Guide, Appendix B

26 Coincident Artifact Blanking This option blanks, a user-specified period of time following each coincident artifact (useful for electrical stimulation). Once enabled, Remove Conicident Artifacts will appear under Analysis Parameters Enabling Remove Conicident Artifacts will algorithmically remove spikes that occur exactly coincident across electrodes in a well, which are often the result of an artifact. Details can be found in the AxIS User Guide 2p3 supplemental.pdf

27 Bursting Options Plot Options Copy Raster Channel Algorithm Network Algorithm Details can be found in the AxIS User Guide, Appendix B

28 Bursting Options (Settings are also in AxIS) ISI Threshold (default) Algorithm relies on defining a burst as a collection of at least N spikes, each separated by an inter-spike (ISI) of no more than T seconds (user can set these parameters (Min # of spikes and Max Inter-Spike Interval) Poisson Surprise Algorithm identifies bursts by assessing the surprise, of observing a collection of spikes, assuming neurons are firing according to Poisson distribution. Adaptive to the mean firing rate of each channel. Min Surprise, will identify bursts more frequently than a high Min Surprise. Details can be found in the AxIS User Guide, Appendix B

29 Synchrony Plot Options Majority of synchrony measures are based on the cross-correlogram: Asseses probability of a spike occurring on channel A at times relative to a spike in channel B to produce a cross-correlogram Additional Synchrony Metrics: None Synchrony Index Kreuz Metric All Window size Details can be found in the AxIS User Guide, Appendix B

30 Guide to cross-correlation and synchrony Assesses the probability of a spike on 1 channel, relative to another The larger the peak at 0 and area under the curve, the more synchronous two electrodes are AxIS uses frequency domain methods to compute and pool across all pairwise combinations in a well (Halliday, Rosenber, Breeze, & Conway 2006) A B Crosscorrelation for A referenced to B plot density time = 0 Go through data spike-by-spike for electrode B Treat each spike as time = 0 Bin surrounding spike density in electrode A

31 Minimum Burst Criterion Minimum burst rate that an electrode must satisfy in order to be included in the electrodeaveraged burst statistics Details can be found in the AxIS User Guide 2p3 supplemental.pdf

32 Evoked Activity Analysis NMT automatically reads the optical stimulation tags for visualization and analysis of evoked data Stimulation event times marked in raster plot Details can be found in the AxIS User Guide 2p3 supplemental.pdf

33 Evoked Activity Analysis Displays the aggregate well-wide response Evoked Data Visualized User can select which stimulus events and time window to include in the analysis Details can be found in the AxIS User Guide 2p3 supplemental.pdf

34 Evoked Activity Endpoints (.csv file EXPORT) For each electrode or well averages # of stimulation events used for analysis Details can be found in the AxIS User Guide 2p3 supplemental.pdf

35 Evoked Activity Endpoints (.csv file EXPORT) For each electrode OR well averages Number of spikes detected on this electrode during the time window specified in the Stimulation Parameters. Avg and Std Dv across trials. Details can be found in the AxIS User Guide 2p3 supplemental.pdf

36 Evoked Activity Endpoints (.csv file EXPORT) For each electrode OR well averages Probability of finding at least one spike during the time window specified in the Stimulation Parameters. Calculated by dividing the # of trials that evoked a response by the # of trials Details can be found in the AxIS User Guide 2p3 supplemental.pdf

37 Evoked Activity Endpoints (.csv file EXPORT) For each electrode OR well averages Amount of time between stimulation event and first poststimulus spike detected on this electrode. Avg across latency across trials Details can be found in the AxIS User Guide 2p3 supplemental.pdf

38 Evoked Activity Endpoints (.csv file EXPORT) For each electrode OR well averages Std Dv of the Evoked First Spike Latency, across trials Details can be found in the AxIS User Guide 2p3 supplemental.pdf

39 Compatible with 3 rd -party tools AxIS raw and spike files are compatible with NeuroExplorer and Matlab AxIS spike files are compatible with Plexon Offline Sorter AxIS spike and bursts list files are compatible with Spotfire Details can be found at

40 Alternate Analysis Tools NMT version 2.0 AxIS Metric Plotting Tool version 1.0

41 AxIS Metric Plotting Tool Import baseline, file from statistics compiler (.csv) as a reference point for comparison files. Comparison files will be loaded alphabetically If there is no plate-map in your statistics compiler (.csv) file, import separately (.csv or.platemap files) Tool allows rapid visualization of experiment results and organization of endpoints according to treatment condition Save.csv file from AxIS (use batch-processing for multiple files) Can also use.csv files generated from NMT; however with no treatment information Details can be found in the AxIS User Guide 2p3 supplemental.pdf

42 AxIS Metric Plotting Tool Details can be found in the AxIS User Guide 2p3 supplemental.pdf

43 One Treatment Per Plot Each treatment is a separate plot, with files on the x-axis Details can be found in the AxIS User Guide 2p3 supplemental.pdf

44 One File Per Plot Each file will be plotted on a separate plot, with treatments on x-axis Details can be found in the AxIS User Guide 2p3 supplemental.pdf

45 Additional Data Options Error Bars (STD or SEM) Plot Display (% change or raw values) Well Exclusion (exclude from averages) Clear (removes all files and data) Details can be found in the AxIS User Guide 2p3 supplemental.pdf

46 Additional Data Options Details can be found in the AxIS User Guide 2p3 supplemental.pdf

47 Figure and Data Export COPY to export figure Export Recommended Metrics to.csv ümean Firing Rate (Hz) ünumber of Active Electrodes ünumber of Bursting Electrodes üburst Frequency Avg (Hz) üburst Duration Avg (s) ünormalized Duration IQR Avg üibi Coefficient of Variation Avg üburst Percentage Avg ünetwork Burst Frequency (Hz) ünetwork Burst Duration Avg (sec) ünetwork Burst Percentage ünetwork IBI Coefficient of Variation ünetwork Normalized Duration IQR üarea Under Normalized Cross-Correlation Details can be found in the AxIS User Guide 2p3 supplemental.pdf

48 Data Export Export Metrics to.csv Details can be found in the AxIS User Guide 2p3 supplemental.pdf

49 Support for Standalone Tools NMT and AMP Tools The AxIS User Guide is located in Help User Guide Software and AxIS 2.3 tutorial videos can be downloaded from For additional questions, contact

50 References for Neural Metrics Bakkum, D. J., Radivojevic, M., Frey, U., Franke, F., Hierlemann, A., & Takahashi, H. (2013). Parameters for burst detection. Frontiers in Computational Neuroscience, 7, 193. doi: /fncom Chiappalone, M., Novellino, a., Vajda, I., Vato, a., Martinoia, S., & van Pelt, J. (2005). Burst detection algorithms for the analysis of spatio-temporal patterns in cortical networks of neurons. Neurocomputing, 65-66, doi: /j.neucom Defranchi, E., Novellino, A., Whelan, M., Vogel, S., Ramirez, T., van Ravenzwaay, B., & Landsiedel, R. (2011). Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro. Frontiers in Neuroengineering, 4(April), 6. doi: /fneng Halliday, D. M., Rosenberg, J. R., Breeze, P., & Conway, B. a. (2006). Neural spike train synchronization indices: definitions, interpretations, and applications. IEEE Transactions on Bio- Medical Engineering, 53(6), doi: /tbme Kreuz, T., Chicharro, D., Houghton, C., Andrzejak, R. G., & Mormann, F. (2013). Monitoring spike train synchrony. Journal of Neurophysiology, 109(5), doi: /jn Legéndy, C. R., & Salcman, M. (1985). Bursts and recurrences of bursts in the spike trains of spontaneously active striate cortex neurons. Journal of Neurophysiology, 53(4), Retrieved from Paiva, A. R. C., Park, I., & Príncipe, J. C. (2010). A comparison of binless spike train measures. Neural Computing and Applications, 19(3), doi: /s Wagenaar, D. a, Pine, J., & Potter, S. M. (2006). An extremely rich repertoire of bursting patterns during the development of cortical cultures. BMC Neuroscience, 7, 11. doi: /