Non-linear Analysis of Scalp EEG by Using Bispectra: The Effect of the Reference Choice

Bispectral analysis is a signal processing technique that makes it possible to capture the non-linear and non-Gaussian properties of the EEG signals. It has found various applications in EEG research and clinical practice, including the assessment of anesthetic depth, the identification of epileptic seizures, and more recently, the evaluation of non-linear cross-frequency brain functional connectivity. However, the validity and reliability of the indices drawn from bispectral analysis of EEG signals are potentially biased by the use of a non-neutral EEG reference. The present study aims at investigating the effects of the reference choice on the analysis of the non-linear features of EEG signals through bicoherence, as well as on the estimation of cross-frequency EEG connectivity through two different non-linear measures, i.e., the cross-bicoherence and the antisymmetric cross-bicoherence. To this end, four commonly used reference schemes were considered: the vertex electrode (Cz), the digitally linked mastoids, the average reference, and the Reference Electrode Standardization Technique (REST). The reference effects were assessed both in simulations and in a real EEG experiment. The simulations allowed to investigated: (i) the effects of the electrode density on the performance of the above references in the estimation of bispectral measures; and (ii) the effects of the head model accuracy in the performance of the REST. For real data, the EEG signals recorded from 10 subjects during eyes open resting state were examined, and the distortions induced by the reference choice in the patterns of alpha-beta bicoherence, cross-bicoherence, and antisymmetric cross-bicoherence were assessed. The results showed significant differences in the findings depending on the chosen reference, with the REST providing superior performance than all the other references in approximating the ideal neutral reference. In conclusion, this study highlights the importance of considering the effects of the reference choice in the interpretation and comparison of the results of bispectral analysis of scalp EEG.