Fig 1.
Schematic diagrams of the discrimination fear conditioning task and individual trial protocols.
(A) Trials of two different neutral visual stimuli (coloured squares) called the conditioned stimuli (CS) are presented to the subject in pseudo-randomised trials. One is a ‘threat’ cue (CS+), which is paired with the unconditioned stimulus (UCS), an aversive noise, in the Acquisition phase only. A ‘safe’ cue (CS-) is presented alone with no UCS. The ‘threat’ CS+ and ‘safe’ CS- are presented in equal proportions throughout all phases in a pseudo-randomised order. Each block comprises 8 trials, with on average four of each CS per block. In the pre-task calibration phase, no stimuli are presented to allow for baseline skin conductance (SCR) and heart rate (HR) recordings and calibration physiological manoeuvres. The Familiarisation phase (one block) comprises eight CS+/CS- trials presented with no UCS. In the Acquisition phase (three blocks), 10/12 CS+ trials are paired with the aversive UCS (loud white noise) presented binaurally via headphones (~80% reinforcement schedule). After 1–2 hours the Extinction phase is conducted (five blocks), comprising 40 CS+/CS- trials with no paired aversive noise. SCR and HR responses are recorded throughout. (B) Structure of a CS+ trial showing the onset and offset times of conditioned (CS) and unconditioned (UCS) stimuli, and the inter-trial interval (ITI). Each CS+/CS- stimulus is presented for 6s, with onset of UCS after 5s (duration 1s). Both CS and UCS co-terminate 6s into the trial. The ITI begins with a fixation cross for 4s, followed by a neutral picture (black and white drawings of common objects: e.g. house, shoe and car) to maintain attention during the ITI, and ends with a fixation cross prior to commencement of the next trial. The ITI duration is randomised between 13-16s. CS- trials and unreinforced CS+ trials are identical, except for the omission of the UCS. Break periods of 20s occurred between each block of 8 trials.
Fig 2.
Measurement of skin conductance responses.
(A) Schematic diagram of a typical skin conductance response following presentation of the conditioned stimulus, CS. The amplitude of the skin conductance response (SCRamp) is measured from the baseline skin conductance level (SCLbase) to the maximum SCL at the peak of the response (SCLmax). If artefacts in the data affect SCLbase, the minimum SCL before the peak (SCLmin) can be used as a baseline measurement instead. Latency is the time from CS onset to the start of the SCR (defined as when the SCR crosses the 0.01μS detection threshold) and is typically 1-3s. Rise-time is the time from SCR onset to the peak response (SCLmax). The ‘time-to-peak’ (TTP) equals latency + rise-time. For the TTP calculations to determine the sampling window, latency was taken from the CS presentation time rather than the UCS (unconditioned stimulus) presentation time. (B)-(D): SCL data from a single participant analysed with automated event-related data extraction. Upper trace (blue) is the tonic skin conductance level (SCL) from which SCRs are measured. Lower trace (pink) is the phasic SCL, used to determine when threshold of 0.01μS is passed for event-related response identification in the automated analysis. Stimulus onset time (time = 0s) indicated by purple arrow/dashed purple vertical line. Orange dashed horizontal line on phasic SCL indicates the 0.01μS detection threshold. Open parenthesis symbol indicates the start of the SCR when the threshold is crossed. Closed parenthesis symbol indicates the end of the SCR when the phasic SCL drops below 0.01μS. ‘Water drop’ symbol denotes the event-related SCR determined by the automated analysis. Dark blue dotted vertical arrows indicate the automated amplitude measurement; Light blue dotted arrows indicate the actual SCR amplitude measurement. (B) A typical SCR, which has been correctly identified and measured. (C) A non-specific skin conductance response (NS-SCR) starts before CS is presented; a slight hump in the upward NS-SCR indicates where the event-related SCR starts but the automated response measurement (dark blue arrow) overestimates the SCR by measuring the combined amplitude of both responses. This is difficult to accurately re-measure manually so would need to be excluded. (D) A typical response starts but as the phasic response (pink line) does not re-cross the 0.01 μS threshold (orange dashed line) when a second response occurs (e.g. due to a physiological response from a deep breath) the peak of the second response is taken as SCLmax so the automated measurement overestimates the SCR. Responses such as these can be accurately re-measured manually.
Fig 3.
Comparison of skin conductance response amplitude data derived by Event-related and Epoch analysis methods.
(A) Mean skin conductance responses (SCR) in μS from second stage of task development study (DMD and Control participants) in all phases of the fear conditioning paradigm (n = 17). SCRs are shown both for analysis using the epoch analysis extraction method (‘Epoch’; circular markers) and the event-related analysis extraction method (‘Event’; triangular markers), for both CS+ and CS- trials. Each data point represents the mean of four CS+ or CS- trials in each block, and error bars indicate 95% confidence intervals. ‘Raw’ data includes all data as extracted using these methods. ‘Clean’ data has excluded data points affected by artefacts, or data points that have been remeasured after manual inspection. FAM = Familiarisation phase; ACQ = Acquisition phase (1–3 blocks); EXT = Extinction (1–5 blocks). (B) Bar chart indicating percentages of trials included with and without adjustment, and those excluded following manual inspection. ‘Max-min switch’ refers to the use of the minimum skin conductance level (SCLmin) value within the trial window where it was more appropriate to use this than the SCL at the trial onset (SCLbase). ‘Remeasured’ refers to manual remeasurement, where artefacts prevented automated SCR measurement and a max-min switch was not appropriate or available.
Fig 4.
Comparison of physiological startle response metrics.
(A) & (B) show comparison of raw and cleaned SCR data for all phases. (A) Mean skin conductance response amplitude (SCRamp) for raw (AmplRaw) and cleaned (AmplClean) data by block. Unconditioned SCR startle response taken as the SCRamp on the first CS+ trial (termed ‘SCRuc’). (B) Mean skin conductance response area (SCRarea) for raw (AmplRaw) and cleaned (AmplClean) data by block. Baseline = baseline recordings taken from mean of Familiarisation phase SCR; ACQ = Acquisition phase (1–3 blocks); EXT = Extinction (1–5 blocks). (C) Change in heart rate from baseline (ΔHR) in Acquisition phase. Baseline heart rate (HR) taken from mean HR in Familiarisation phase. Data points are mean ΔHR for each numbered trial, where ‘+’ denotes a CS+ trial, ‘-’ denotes a CS- trial and and ‘o’ indicates a non-reinforced CS+ trial, i.e. no unconditioned stimulus (UCS) presented. The unconditioned HR startle response metric was taken from the change in HR after first CS+ trial, i.e. from trial ‘3+’ to trial ‘4-’ (termed ΔHRuc). All data were obtained by Epoch analysis extraction. Error bars show 95% confidence intervals.
Table 1.
Baseline demographics of DMD and Control participants.
Table 2.
Comparison of skin conductance responses in paired CS+ and CS- trials.
Fig 5.
Comparison of SCR and HR responses in DMD participants at initial and follow-up visits.
Data for n = 11 DMD participants who performed the fear-conditioning task at the initial visit (Visit 1)and at a second follow-up visit after 3 months (Visit 2). Paired HR data available for n = 11 and paired SCR data available for n = 6. (A) Mean SCR by block for CS+ and CS- trials over all task phases. Error bars show 95% confidence intervals. (B) Paired data for individual participants showing the SCR for the first unconditioned response (SCRuc) in Visit 1 and Visit 2. (C) Mean absolute HR by trial in the first Acquisition block. Error bars show 95% confidence intervals. (D) Paired data for individual participants showing the Change in HR (ΔHR) from Acquisition trial 3+ to trial 4-, i.e. the unconditioned ΔHR response (ΔHRuc). (E) & (F). Bland-Altman Limits of Agreement plots for: (E) Unconditioned skin conductance response (SCR; n = 6) and (F) Unconditioned change in heart rate (ΔHR; n = 11). Plots show the mean difference (solid pink line) and upper and lower limits of agreement, LOA, in dashed purple lines. Data labels indicate the values for upper LOA, mean difference and lower LOA (from top to bottom). All data points for both SCRuc and ΔHRuc lie within these LOA boundaries, indicating there is agreement between the test-retest outcomes.