Single-sweep spectral analysis of contact heat evoked potentials: a novel approach to identify altered cortical processing after morphine treatment

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1 British Journal of Clinical Pharmacology DOI: /bcp Single-sweep spectral analysis of contact heat evoked potentials: a novel approach to identify altered cortical processing after morphine treatment Tine M. Hansen, 1 Carina Graversen, 2 Jens B. Frøkjær, 1,3 Anne E. Olesen, 2,4 Massimiliano Valeriani 5,6 & Asbjørn M. Drewes 2,3,6 * Correspondence Professor Asbjørn Mohr Drewes MD PhD DMSc, Mech-Sense, Department of Gastroenterology & Hepatology, Aalborg University Hospital, Mølleparkvej 4, 9100 Aalborg, Denmark. Tel.: Fax amd@mech-sense.com Keywords contact heat, electroencephalography, evoked brain potentials, morphine, spectral analysis Received 12 September 2014 Accepted 16 December 2014 Accepted Article Published Online 31 December Mech-Sense, Department of Radiology, Aalborg University Hospital, Aalborg, Denmark, 2 Mech-Sense, Department of Gastroenterology & Hepatology, Aalborg University Hospital, Aalborg, Denmark, 3 Department of Clinical Medicine, Aalborg University, Aalborg, Denmark, 4 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 5 Division of Neurology, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy and 6 Center for Sensory-Motor Interaction (SMI), Department of Health Science and Technology, Aalborg University, Aalborg, Denmark WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Opioids alter spectral indices in evoked brain potentials following electrical stimulation in experimental pain models. Contact heat stimuli activate the nociceptive fibres specifically and thus they can be used to study nociceptive pathways. Advanced analysis is needed to compensate for latency jitter in single-sweep contact heat evoked brain potentials. WHAT THIS STUDY ADDS Spectral analysis of single-sweep contact heat evoked potentials is a reliable method to study nocicpetive pathways. As compared with placebo, morphine administration decreases low frequency oscillations and increases high frequency oscillations. The methodology can be used to identify cortical mechanisms induced by analgesic treatment. AIMS The cortical response to nociceptive thermal stimuli recorded as contact heat evoked potentials (CHEPs) may be altered by morphine. However, previous studies have averaged CHEPs over multiple stimuli, which are confounded by jitter between sweeps. Thus, the aim was to assess single-sweep characteristics to identify alterations induced by morphine. METHODS In a crossover study 15 single-sweep CHEPs were analyzed from 62 electroencephalography electrodes in 26 healthy volunteers before and after administration of morphine or placebo. Each sweep was decomposed by a continuous wavelet transform to obtain normalized spectral indices in the delta (0.5 4Hz), theta(4 8 Hz), alpha (8 12 Hz), beta (12 32 Hz) and gamma (32 80 Hz) bands. The average distribution over all sweeps and channels was calculated for the four recordings for each volunteer, and the two recordings before treatments were assessed for reproducibility. Baseline corrected spectral indices after morphine and placebo treatments were compared to identify alterations induced by morphine. RESULTS Reproducibility between baseline CHEPs was demonstrated. As compared with placebo, morphine decreased the spectral indices in the delta and theta bands by 13% (P =0.04)and9%(P = 0.007), while the beta and gamma bands were increased by 10% (P = 0.006) and 24% (P = 0.04). CONCLUSION The decreases in the delta and theta band are suggested to represent a decrease in the pain specific morphology of the CHEPs, which indicates a diminished pain response after morphine administration. Hence, assessment of spectral indices in single-sweep CHEPs can be used to study cortical mechanisms induced by morphine treatment. 926 / / 79:6 / Br J Clin Pharmacol 2015 The British Pharmacological Society

2 Morphine effect on heat evoked brain potentials Introduction Morphine is a widely used analgesic to treat acute and chronic pain. The drug exerts its main effect on the μ-receptors and acts on different levels of the nervous system, such as the periphery, spinal cord and brain regions [1]. Although morphine is widely used in the clinic, the analgesic mechanisms during acute thermal pain have never been studied [2]. Such knowledge on how morphine modifies central pain processing of the nociceptive input may provide new insight into the multiple complex mechanisms, which may in the future play a key role in mechanism-based pain treatment [3]. To study the altered central pain processing after morphine administration, electroencephalography (EEG) provides an objective method, which has proved useful to study analgesic effects [2, 4]. The EEG response evoked by both radiant laser pulses and contact heat stimuli activate the cutaneous Aδ fibres, which have been used to study the brain response to acute thermal stimulation [5 7].Incomparisonwithlaserevokedpotentials (LEPs), contact heat evoked potentials (CHEPs) have some clinical advantages, as they are easier to be obtained, and unlike laser pulses, contact heat stimuli never produce skin burning. In contrast, CHEPs have smaller amplitudes, longer latencies and higher within-subject and between-subject latency variability, as compared with LEPs [8]. Traditional evoked potential analysis is focused on recording the response to several repeated stimuli, followed by an average procedure in the time domain to improve signal-to-noise ratio [9]. However, this procedure is mostly valid when the main evoked components are phase-locked, as it cancels out non-phase-locked signals. While short latency evoked potentials (within 100 ms of latency) are expected to be strictly phase-locked with the stimuli, this is not true for long latency evoked brain responses, such as LEPs and CHEPs. In particular, CHEP components may present with a large latency variation (jitter) among the different sweeps to be averaged. This is due to two main elements. First, the heating velocity of the commercially available contact heat stimulator is 70 C s 1. This means, that starting from 32 C it takes around 270 ms to reach the target temperature of 51 C, which is known to activate a sufficiently large amount of Aδ nociceptors to evoke brain responses [8]. However, not all Aδ fibres have the same threshold, and thus it is conceivable that the Aδ inputs reach the brain in a widespread time interval among successive sweeps. Second, the main CHEP components are strongly influenced by cognitive activity such as attention that can be different between sweeps [10]. In a previous study from our group we applied spectral analysis on average potentials following morphine treatment [11]. However, to eliminate the influence of jitter and to extract more pertinent information from the evoked potentials in a study of diabetes mellitus patients, we introduced an alternative approach to the time-average procedure where the information in single-sweep evoked potentials was used [12]. The method was subsequently used in a study of opioid treatment, where the single-sweep approach of electrical median nerve evoked potentials was successfully used to assess the analgesic effect of buprenorphine and fentanyl [13]. These two studies were based on average in the frequency domain rather than in the time domain, which has the advantage that it preserves key parameters in the evoked potentials such as inter-trial phase alignment and phase-resetting properties [14]. We hypothesized that single-sweep CHEP analysis is reliable in healthy volunteers and morphine administration reduces the amplitudes of the evoked potentials, which should be reflected in decreased low frequency oscillations. Hence, the aims of this study were: (i) to investigate the reliability of EEG spectral indices extracted from single-sweep CHEPs and (ii) to investigate the effect of morphine administration on the CHEP spectral indices in comparison to placebo treatment. Methods Subjects Forty healthy volunteers participated in this randomized, double-blind, placebo-controlled and two way crossover study. All subjects provided informed consent. The study was carried out in the Research Laboratory at Mech-Sense, Department of Gastroenterology, Aalborg University Hospital, Denmark. The study was conducted in the period from November 2010 April Inclusion criteria were normal blood pressure, no history of drug or alcohol abuse, no intake of opioids or other strong analgesics, no history of allergy to opioids, no planned treatment or surgery during the study period, no history of pain disorders or mental illness and no intake of any medication 24 h prior to the experiment. Female subjects were on safe contraceptive medication and participated in the same phase of their menstrual cycle. All concomitant medications were registered in the case report form. All subjects refrained from eating and drinking for at least 4 h before the start of the experiment. The study was conducted according to the Declaration of Helsinki, approved by the local Ethics Committee (reference no. N ), the Danish Medicines Agency (reference no ) and registered at ClinicalTrials.gov (NCT , EUDRACT no ). The study was conducted according to the rules of Good Clinical Practice and monitored by the Good Clinical Practice unit, Aarhus University Hospital, Denmark. Br J Clin Pharmacol / 79:6 / 927

3 T. M. Hansen et al. Study protocol The study was conducted on two sessions separated by at least 1 week wash-out period, with subjects treated randomly with morphine on one day and placebo the other day. Prior to the first dosing day, a training session was included to familiarize the subject with the laboratory environment and the protocol procedure. Each session consisted on two CHEP recordings with stimulations applied before and 60 min after drug administration. Hence, in total four conditions were recorded for each subject, before and after morphine administration and before and after placebo administration. The present study was an explorative sub-study of a larger study of morphine effects (main study). The overall aim of the main study was to investigate how morphine modulates pain peripherally, spinally and centrally, and hence additional tests and measurements were performed during the sessions and main findings are reported elsewhere [15]. Heat stimulation Skin heat stimulations were applied using a Pathway Stimulator (Medoc Ltd, Ramat Yishai, Israel) with an activation area of the contact thermode of 573 mm 2.The thermode was placed onto the skin of the right forearm (10 cm from the cubital fossa, fixed position) and 15 successive stimuli of heat were applied (1 s inter-stimulus interval). The start temperature was 32 C and the destination temperature was 51 C. The heating rate was 70 C s 1 and the cooling rate was 40 C s 1. Sensory rating Amodified visual analogue scale (VAS) was used for assessment of rating of the sensory perception. Subjects were asked to rate the sensory level of the last stimulus after the stimulation was applied. This VAS included the following anchor words: 0 = no sensation, 1 = vague perception of mild sensation, 2 = definite perception of mild sensation, 3 = vague perception of moderate sensation, 4=definite perception of moderate sensation, 5 = pain detection threshold, 6 = slight pain, 7 = moderate pain, 8 = medium pain, 9 = intense pain and 10 = unbearable pain [16]. Drug administration Orally administered 30 mg morphine (15 ml morphine oral liquid mixture 2 mg ml 1,TheHospitalPharmacy, Aalborg University Hospital, Denmark) or placebo (15 ml placebo solution, The Hospital Pharmacy, Aalborg University Hospital, Denmark) was mixed together with 5 ml of orange juice concentrate to mask any taste or colour. The placebo solution was similar to the vehicle used for the morphine solution. Thus, flavour and colour matched the characteristics of the morphine solution. EEG recordings EEG signals were recorded using a SynAmp2 system (Neuroscan Compumedics, El Paso, Texas, USA) with standard 62 channel caps (Quick-Cap International, Neuroscan, El Paso, Texas, USA) mounted according to the extended system. The electrode impedances were kept below 5 kohm by applying electro-gel (Electrocap international Inc., Eaton, Ohio, USA). EEG signals were recorded in AC mode with a sampling frequency of 1000 Hz and band-pass filter from 0.05 to 200 Hz. Subjects were instructed to relax with gaze fixed during recordings. Recordings took place in a quiet room with dimmed light. Pre-processing of EEG signals EEG signals were pre-processed offline (Neuroscan 4.3.1, Compumedics,ElPaso,Texas,USA)including:(i)notch filtering (zero phase shift Hz, 24 db/octave), (ii) band-pass filtering (zero-phase shift Hz, 12 db/octave), (iii) epoching ( 50 to 1000 ms) (iv) linear detrending, (v) baseline correction, (vi) re-referencing to ear-electrodes and (vii) interpolation of channels displaying abnormal signals with respect to signal level. Spectral analysis of EEG signals Before spectral analysis, a thorough inspection of the CHEPs was performed manually in the time domain. The inspection was done blinded with respect to treatment in order to validate signal quality and identify peak latencies to narrow the time interval of interest. For the spectral analysis, all 15 sweeps per recording were analyzed in the time interval from 370 ms to 750 ms after stimulus onset. The analysis was performed by a continuous wavelet transform applied with a complex Morlet wavelet function (Matlab 2012a, The Mathworks Inc., Natick, MA, USA). This function was chosen to have optimal time frequency resolution, and the design parameters were set to have bandwidth of 10 Hz and centre frequency of 1 Hz. For each subject and each recording, the continuous wavelet transform was applied to each channel individually to calculate the normalized spectral power distribution in each single-sweep. The spectral power was then averaged over all sweeps to obtain one estimate of the spectral indices for each subject, recording and channel. The indices were calculated in the bands delta ( Hz), theta ( Hz), alpha ( Hz), beta ( Hz) and gamma ( Hz). The normalization was used to present the percentage powerineachbandcomparedwiththetotalpowerin all bands to eliminate inter-sweep and inter-subject power offset. The grand mean topography maps of the spectral indices were assessed. To evaluate the alteration of the spectral indices for each subject, the ratio after administration : before administration was calculated for both the morphine and placebo sessions for each band and 928 / 79:6 / Br J Clin Pharmacol

4 Morphine effect on heat evoked brain potentials each channel. To reduce the number of comparisons and to assess the global effect on brain processing, the ratio was averaged over all channels for each subject. Hence, a ratio above 1 indicated overall increased activity after drug administration, while a ratio below 1 indicated overall decreased activity after drug administration. A schematic illustration of the spectral analysis procedure of theeegsignalsisshowninfigure1. Reliability To validate the approach of extracting spectral indices of CHEPs, the reliability of the two recordings before morphine and placebo administration was assessed, as these two conditions were expected to be reproducible. Reliability was assessed in all bands using three different measurements; coefficient of variation (CV), intra-class correlation coefficient (ICC) and limits of agreement Figure 1 Schematic illustration of the spectral analysis procedure of the EEG signals. For each subject, each condition (before and after morphine and placebo administration), each channel and each single-sweep, the continuous wavelet transform was preformed and the normalized spectral power distribution was calculated. The spectral power distribution was averaged over all sweeps to obtain one estimate of the spectral indices for each subject, condition and channel. An average over all channels was used to calculate the ratio after administration : before administration for both the morphine and placebo sessions. The average sweep distribution was used to calculate grand mean (GM) for all subjects for each condition to create topography maps and statistical analysis of the ratio after administration : before administration was performed for all subjects Br J Clin Pharmacol / 79:6 / 929

5 T. M. Hansen et al. (LOA). As different information regarding variability is provided using the different measurements, results for all measures were considered to decide the reliability of the method. It should be noted that acceptable levels for reliability depend on the application [17]. However, acv< 10% is commonly used [17] and Chinn et al. recommended ICC > 0.6tobethelimittorevealacceptable reliability [18]. For LOA the associated Bland Altman plot shows the measurement error schematically and helps to identify the presence of heteroscedasticity [17]. Statistical analysis SPSS (IBM SPSS Statistics, Version 20.0, Armonk, NY, USA) was used to calculate reliability measurements and for statistical evaluation. CV is reported as the standard deviation of the measurement expressed as a percentage of the mean, ICC was calculated by the one way random model and LOA was calculated as mean of differences ±1.96 SD of differences. Bland Altman plots were used in addition to investigate heteroscedasticity of LOA. Data are presented as mean ± SEM. Differences between pain ratings and ratios of spectral indices for morphine and placebo treatment within all frequency bands were analyzed using a paired t-test. P < 0.05 was considered significant. This study was an explorative sub-analysis of a previously published study including multiple end points [15]. As the main study investigated the effect of morphine on several end points, a calculation of a general sample size was not possible. However, the sample size was estimated based on a previous study assessing opioid effect using heat stimulation [19] and40subjectsshouldbeincluded(α = 0.05, power = 0.90) to detect a difference of 4% in analgesic effect between placebo and morphine [15]. Additionally, previous studies using EEG in conjunction with administration of analgesics have typically enrolled between 10 and 30 subjects [20]. Consequently, we considered the available sample size of 40 enrolled subjects to be sufficient and allow for exclusions based on poor EEG quality. Results Thirty-nine subjects (18 females and 21 males, average age 26.9 ± 6.5 years) completed the study as one subject dropped out due to side effects. Thirty-seven subjects had recordings from all four conditions. However, 11 subjects were excluded from further analysis as at least one out of the four recordings showed bad data quality (e.g. bad signal : noise ratio, artefacts and disturbed sweep morphology caused by external noise). As the single-sweep analysis method was applied to identify alterations induced by morphine, only data sets with valid recordings for all conditions (before and after placebo and morphine) were included. Consequently, 26 subjects had valid data and were used for further analysis. Assessment of the evoked potentials in the time domain Latency jitter between single-sweeps in the time domain was present. An example of the single-sweep traces and the corresponding average in a representative subject is shown for the Cz channel in Figure 2. Spectral analysis of EEG signals The continuous wavelet transform coefficients were calculated for all sweeps and in Figure 3 an example of the distribution is shown for a representative sweep and subject for the Cz channel. An example of the topography of the spectral indices for all bands before drug administration is shown in Figure 4. Reliability The reliability of the method was shown in the same subgroup of subjects (n = 26) as was used to identify alterations induced by morphine. Reliability measurements (CV, ICC and LOA) of spectral indices in the different bands are reported in Table 1. CV was below 10% for delta, theta, alpha and beta bands and 14% for the gamma band. ICC values ranged from 0.68 to 0.92 for all bands. The 95% LOA intervals all included zero and the Bland Altman analysis (plots not shown) showed maximum one outlier on each side of the confidence interval and no evidence for heteroscedasticity. Taking all three measurements into account, this indicates that the method is reliable. Morphine effect Sensory ratings of the last stimulus were 5.5 ± 0.3 on the VAS before morphine administration and 5.0 ± 0.4 after morphine administration (P = 0.04) and 5.3 ± 0.3 before placebo administration and 5.1 ± 0.4 after placebo administration (P =0.4). The differences in grand mean topography of the spectral indices over all subjects before and after drug administration are shown in Figure 5. As observed, decreased fronto-central activity was seen after morphine administration in the delta and theta bands, while indication of increased occipital activity was seen in the alpha band. Additionally, increased frontal activity in the beta band and increased central activity in the gamma band was also observed. The ratio of mean spectral indices over all channels was calculated for the morphine and placebo day for each subject. For morphine compared with placebo, a decrease was seen for the delta (0.94 ± vs. placebo 1.07 ± 0.060, P = 0.04) and theta (0.93 ± vs.placebo 1.02 ± 0.025, P = 0.007) bands and an increase was seen for the beta (1.07 ± vs. placebo 0.97 ± 0.028, 930 / 79:6 / Br J Clin Pharmacol

6 Morphine effect on heat evoked brain potentials Figure 2 CHEPs from a recording before drug including 15 stimuli from a representative subject for the Cz channel. The vertical lines show the time interval of interest for the spectral analysis. (A) The 15 single-sweeps (black) and the corresponding average evoked potential (red). The two main peaks (N2 and P2) of the average evoked potential are illustrated and due to the jitter, a large portion of the signal information is lost and the average amplitude is low. (B) Amplitude intensities for all 15 single-sweeps are represented by a colour code to visualize the variability between sweeps. Phase jitter and amplitude variation is present, but not in a dynamic way as would be the case of, for example, habituation P = 0.006) and gamma (1.18 ± vs. placebo: 0.94 ± 0.067, P = 0.04). No significant difference was seen for the alpha band (1.10 ± vs. placebo 1.03 ± 0.043, P = 0.2) (Figure 6). Adverse events During the measurements 67% of the participants experienced side effects to morphine treatment (nine reported nausea, 23 reported dizziness, four reported itching and two reported sweating). Twenty-one % of the participants experienced side effects in the placebo arm (five reported nausea, four dizziness and one itching). [15] Anti-emetics were available on demand. However, none of the participants requested any anti-emetics as the reported nausea was mild to moderate. Discussion To our best knowledge, analysis of EEG single-sweep spectral indices following phasic heat stimuli has never been reported. Due to high latency jitter among single- sweep CHEPs, the approach to assess single-sweep spectral indices was applied. We found this method to be reliable using three different reliability measurements, coefficient of variation, intra-class correlation coefficient and limits of agreement. The main result of the study was identification of decreased low frequency oscillations and increased high frequency oscillations induced by morphine administration compared with placebo. Methodological considerations and limitations By visual inspection of the single-sweeps in the time domain, a clear between-sweeps jitter was observed for the CHEPs. This means that signal information is lost in the average procedure, which calls for more advanced analysis. Kramer et al. suggested to overcome the latency jitter problem by optimizing the design aiming to improve synchronization between single-sweeps and thereby reduce latency jitter [21]. However, other studies also suggested the need for single-sweep analysis of CHEPs [8, 22]. Warbrick et al. suggested an automated single-sweep analysis approach based on multiple linear regression, which was shown to be Br J Clin Pharmacol / 79:6 / 931

7 T. M. Hansen et al. Figure 3 (A) A representative sweep for the Cz channel in the time domain and (B) a selected part of the time frequency coefficients squared to obtain the power distribution. The vertical lines show the time interval of interest for the spectral analysis. The maximum power is found in the theta band (red colour) within the interval of interest. The frequency range is limited to 20 Hz in the figure as the pain specific morphology of the N2-P2 complex of the evoked potential is also located in the theta band ( Hz) superior to standard averaging of CHEPs [22]. Other studies have also applied automated single-sweep analysis approaches of LEPs [23, 24], although they used automated correction for eye blinks and movements and rejection of artefacts by visual inspection. We included all 15 sweeps for all recordings knowing that some sweepsmightcontainartefactssuchaseyeblinksand loss of signal quality. However, as artefact rejection is highly subjective, inter-observer dependent and time consuming, we decided to apply a method robust enough to overcome those issues. Additionally, our method proved to be reliable, easy to apply and totally objective which allowed a certain level of noise. Importantly, our method also has future clinical aspects, as the methodology only requires a person to detect bad data quality caused by external noise, which is easier to recognize and does not require a trained EEG expert to first reject sweeps before data can be analyzed. A short inter-stimulus interval was used in order to investigate a design which could be comparable with an fmri block design. A short inter-stimulus interval reduces the amplitude and habituation may occur and a longer inter-stimulus interval could be preferable. However, we showed this method to be robust and reliable even with a short inter-stimulus interval. As manual readings of amplitude characteristics of the two main peaks, N2 and P2, are not feasible in single-sweeps, we chose to perform spectral analysis in the time interval of interest. This decision was further supported by previous studies, where spectral analysis of single-sweep evoked potentials has proved to be superior to manual inspection [13]. By visual inspection of all recorded signals, the main morphology of the evoked potentials was identified to be in the time interval 370 to 750 ms after stimulus onset. Hence, we chose to use this narrow window although it cannot be excluded that brain oscillations prior to this time point might play an important role in pain processing. Additionally, the Pathway Stimulator makes auditory noise simultaneously with the heat stimuli onset, which also motivates for this late time point to discard the auditory P300 response from the analysis [25]. 932 / 79:6 / Br J Clin Pharmacol

8 Morphine effect on heat evoked brain potentials To obtain a measurement of the overall spectral indices in all 62 channels, we calculated an average over all channels. This is an improvement compared with Gram et al., who only used recordings from one single electrode in the analysis of EPs [13]. This is further emphasized in the topography plots showing morphine-induced changes in frontal, central and occipital parts of the scalp. The explorative end points in the present study were not viewed as being directly related to the primary objectives of the main study but were thought to provide potentially worthwhile information about morphine central effects. Exploratory end points do not require any Figure 5 Topography plots of the altered spectral indices after treatment compared with before treatment in all analyzed bands correction for multiplicity [26]. However, in this exploratory study, significant changes in spectral indices should be interpreted with caution. Different opioids exert different mechanisms in both experimental and clinical settings [2, 27]. Other administrations and dosing intervals may also affect the outcome. Hence, it cannot be excluded that other Figure 4 Grand mean topography plot of spectral indices in all analyzed bands before drug administration Table 1 Between day reliability of the two recordings before drug administration for the spectral indices delta ( Hz), theta ( Hz), alpha ( Hz), beta ( Hz) and gamma ( Hz) CV ICC Delta Theta ± 4.18 Alpha ± 5.87 Beta Gamma LOA 0.43 ± ± ± 1.35 CV, coefficient of variation (%); ICC, intra-class correlation coefficient; LOA, limits of agreement (%). Figure 6 Mean ratios of spectral indices over all channels for all analyzed bands (mean ± SEM). *significant difference between morphine and placebo treatment, P < 0.05., morphine;, placebo Br J Clin Pharmacol / 79:6 / 933

9 T. M. Hansen et al. findings would be seen if different opioids or doses were administered and a dose-dependent relationship or more measurements would be very interesting in a future study. Furthermore, evoked potentials following opioid treatment can also be assessed with other methods such as electrical and mechanical stimuli, which may be less susceptible to jitter. Reliability Single-sweep analysis using the continuous wavelet transform with a complex Morlet function was proven to be a reliable method using three different reliability measurements, CV, ICC and LOA. The delta band showed the lowest ICC value and highest 95% confidence interval for LOA, which might be explained by sweeps containing eye blinks and movements. The gamma band showed the highest CV value, which may be explained by noise from muscle contractions in this band [28]. Morphine effect Morphine revealed significant decreased delta and theta and increased beta and gamma band spectral indices. The main peaks, N2 and P2, of the CHEPs are expected to appear at approximately 460 ms and 550 ms, respectively, when stimulating the volar side of the forearm [29]. Hence, the peak-to-peak latency interval, presenting the pain specific morphology, is approximately 90 ms, which corresponds to an oscillation in the theta band. A decrease in the theta band spectral indices after morphine administration therefore reflects decreased N2-P2 peak-to-peak amplitude. This is in line with previous studies, where analgesics typically decrease the N2-P2 peak-to-peak amplitude of evoked potentials [2, 30]. This decrease in amplitude following morphine treatment has even previously been associated with a decrease in subjective pain ratings [31 33]. Consistent with our study, increased oscillations in the higher frequencies of spontaneous EEG during morphine treatment have previously been reported [34]. The decrease in delta and theta bands likely reflect a lowering of the amplitude of the evoked potentials as the main complexes are localized in these frequency bands. The physiological implications are likely related to a change in the processing of pain by the brain as we have shown in previous studies that morphine changed the dominant electrical activity and networks in the limbic system, where the density of opioid receptors are richer. Hence, such neuroplastic changes will invariably be reflected in alterations in the corresponding surface potentials [35 37]. Only a minimal morphine effect was found on subjective pain ratings during skin heat stimulation. The effects of opioids on heat pain have been tested through different stimulation paradigms and conflicting results exist [38, 39]. It could be speculated that A-δ fibres were mainly activated as heat stimulation in the present study was phasic. Hence, the fact that limited subjective morphine analgesia was demonstrated may reflect minimal effectiveness to attenuate the A-δ mediated nociceptive component [15]. However, the method for skin heat stimulation was chosen to detect changes in evoked brain potentials and not to explore the subjective analgesic effects of morphine. In conclusion, in this study we proved the reliability of EEG spectral indices in single-sweep evoked potentials following phasic heat stimuli using CHEPs. Morphine administration induced decreased low frequency oscillations and increased high frequency oscillations. This proposed methodology is valid in experimental settings and can be used to assess and understand cortical analgesic mechanisms induced by opioid treatment. Competing Interests All authors have completed the Unified Competing Interest form at (available on request from the corresponding author) and declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work. This study was funded by The Danish Agency for Science, Technology and Innovation(Det Strategiske Forskningsråd), grant no Contributors T.M.H.,C.G.andA.E.O.:conceptionanddesignofthe study, acquisition of data, analysis and interpretation of data, drafting the manuscript. J.B.F., M.V. and A.M.D.: conception and design of the study, analysis and interpretation of data. All authors discussed the results and commented on the manuscript and revised it critically for important intellectual content and did final approval of the version to be published. REFERENCES 1 Inturrisi CE. Clinical pharmacology of opioids for pain. Clin J Pain 2002; 18: Malver LP, Brokjaer A, Staahl C, Graversen C, Andresen T, Drewes AM. Electroencephalography and analgesics. Br J Clin Pharmacol 2014; 77: / 79:6 / Br J Clin Pharmacol

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