Demonstrating Prepulse Inhibition of Acoustic Startle in normals during FMRI: Progress Report Kulkarni, Kirti M; Heidinger, Linda E; Goldman, Morris B Department of Psychiatry, University of Chicago and University of Illinois at Chicago, Chicago, IL, USA. Email: m-goldman@uchicago.edu Accept: initial deflection between 250-450ms (i.e. 70 to 380 ms post Pulse) is downward and has a magnitude >50µvolts. Nonresponse: a net deflection <50µvolts between 200-500ms. Reject: greater than 50µvolts deflection between 200-500ms but (1) there is no downward peak, or (2) first peak is upgoing or (3) downward peak is smaller than 50µvolts. II. METHODS Epoched Channels I. ABSTRACT Introduction: Clarifying the neuroarchitecture of prepulse inhibition of acoustic startle with event related FMRI is appealing, but several barriers exist. Methods: Eleven healthy controls with > 40% PPI in a mock scanner underwent four 10 minute runs during which they each heard a total of 100 pulse alone (40 ms white noise @ 107 db) and 100 prepulse plus pulse (20 ms @ 88 dB, followed 120 ms later by pulse) trials. Continuous assessment of the BOLD response was obtained in a 3T scanner using an EPI. MRI-compatible carbon electrodes (Magnilink, Neuroscan) were attached to each subject: two sub-clavicular, and two above and below each eye. Principle components analysis removed the influence of ballistocardiogram waveform (Neuroscan, 4.3), and the effects of pulse and gradient artifacts were minimized with low pass filters. On epoched waveforms, we then determined the direction, timing and extent of the first peak. This information was used to determine whether a response had occurred (if not scored intensity = 0) or if the trial was to be rejected. Intensity on other non-rejected trials was defined on rectified traces as the area under the curve between 20 and 320 ms post-pulse. Results: Acoustic startle responses can be measured in the fMRI during continuous data acquistion. Out of 11 subjects analyzed in the real scanner, 2 subjects demonstrate PPI >30% and 4 subjects demonstrate PPI >10%. Conclusions: These preliminary data indicate that PPI of acoustic startle can be obtained during continuous fMRI scanning, and is stable across runs. Subjects and Design EKG Epoch ACCEPT RESPONSE NO RESPONSE MOCK SCANNER STUDY Design Two five minute runs Event related: 25 trials/run Two trial types 40 ms Pulse of 107db Pulse preceded 120 ms by 20 ms, 88 db Prepulse Subjects 42, right handed M 25, F 17 Mean Age 23.5 15 out of 42 subjects demonstrated good PPI (>40%) and were studied in the real scanner. REAL SCANNER STUDY Design Four 10 minute runs Structure scans between runs 2 and 3 Event related: 50 trials/run Two trial types 40 ms Pulse of 107db Pulse preceded 120 ms by 20 ms 88 db Prepulse ISI 8-16 sec Left channel Epoch Right channel Epoch 3)The epochs are centered on each trigger marker and then averaged using average transform 4) We perform a spatial Single value Decomposition (SVD). A Spatial SVD is equivalent to a spatial PCA (Principal Components Analysis). This operation produces an LDR (Linear Derivation) file that contains a matrix of linear coefficients or weights used to derive the principal components from existing channels. Removal of the first PCA component created from the spatial SVD results in the EKG ARTIFACT REMOVAL. 1 3 Blink EKG Left COMP1 -0.01872 0.99982 0.00020 The remaining LDR retains the variance explained by the eye leads. 3 3 Blink 0.99965 0.01872 0.00000 EKG 0.01872 0.00035 -0.00020 Left 0.00000 -0.00020 1.00000   What is Prepulse Inhibition(PPI)? Reduced eyeblink when a startling stimulus is preceded by a non-startling stimulus. Subjects 11, right handed M 8, F 7 Mean Age 23.5 REJECT RESPONSE LDR1 Data Acquisition The stimulus is produced by San Diego Instruments Inc., startle box and delivered through custom-designed pneumatic headphones . The response is measured by Neuroscan 4.3 (Neuroscan Inc.) Magnilink electrodes (Neuroscan Inc.) Sampling rate is adjusted based on Nyquist criteria to 10,000/sec for better resolution. IV. RESULTS LDR2 Why measure EMG with fMRI? Independent t-test comparing mean area under the curve for each subject, looking at the difference between Pulse and PreP+P Paired t-test comparing Run1 Vs Run4 (Habituation Effect) Robust phenomenon composed of discrete stimuli Extensively studied in humans Neural circuitry characterized in mammals PPI does not degrade over multiple presentations Real time data acquisition to demonstrate behavioral aspects of PPI during fMRI In data analysis, we incorporate EMG value as a covariate in the deconvolution to explain variability in the fMRI data. EKG Removal Placement of Magnilink electrodes EKG Channel Stimulus Timing 2-way ANOVA looking at trial type effect was p=0.476. (PreP+P) Response Pulse Response 107 107 Spontaneous Eyeblinks dB Subject Response to Pulse & PreP+Pulse dB 88 5)Two successive low pass filters 5Hz (48dB) and 4Hz (48dB) are then applied to remove RF GRADIENT ARTIFACT REMOVAL and the low frequency baseline drift. Steps to facilitate EMG data acquisition %: PPI 8% 20 140 180 140 180 Time (ms) Time (ms) -35% -10% The electrodes (Neuroscan Inc.) are made up of carbon filament. Phantom study supports that they do not cause any susceptibility artifacts WITH LEADS (arrows) WITHOUT LEADS Impedance of the electrodes was decreased by proper scrubbing of the skin in contact with electrodes for better signal to noise Simultaneous ballistocardiogram was acquired for future artifact reduction Stimulus occurrence was marked by triggers to demarcate corresponding responses for data analysis. Pulse Alone PreP+Pulse -50% -80% Filtered Responses 42% 12% 32% 23% 13% 16% III. DATA ANALYSIS Phantom Study The raw data undergoes spatial and frequency filtering resulting in an EMG signal of interest. The signal is then categorized and standardized using a set of specific criteria to determine the extent of PPI. Mock Vs. Real Scanner PPI (PreP+P) Response Pulse Response RAW DATA Spontaneous Eyeblink 6)We epoch the second time for 600ms following the presentation of each trial. 7)Baseline is corrected by taking the average over the first 20ms after the Pulse stimulus. The data next undergoes rectification. V. SUMMARY &DISCUSSION Acoustic startle responses can be measured in the fMRI during continuous data acquisition. Out of 11 subjects analyzed in the real scanner, 2 subjects demonstrate PPI >30% and 4 subjects demonstrate PPI >10%. The response to both trial types was intensified in 5 subjects who did not exhibit PPI. This is possibly due to fear-induced startle in the MRI setting or Variability in the sound presentation due to the in-house designed sound delivery equipment not capable of producing reliable and consistent output. 8)Standardization Criteria: On looking at the analyzed data, there are some spontaneous eyeblinks and movements, some stimulus-induced eyeblinks due to the startle paradigm, and some that are reduced eyeblink responses presumably due to PPI. In order to better delineate these three subtypes, we decided on the following standardized criteria for defining the responses, nonresponses and rejects. 1) We spatially filter the EKG artifact by marking the events using the voltage threshold transform. We select a threshold value based on the EKG wave peak and a refractory period which is between two cyclical consecutive peaks. This transform is applied to the EKG channel and marked as a trigger 9. 2) We epoch the data 200ms before and after the trigger 9 on the EKG channel. Funding provided by NARSAD and BRAIN RESEARCH FOUNDATION, University of Chicago, Chicago, IL Acknowledgements: Dr. Vernon Towle, Dr. Ana Solodkin, Dr. David Zhu, Ana Solomey, Robert Lyons and Dr. Steve Small offered helpful suggestions for the current study.