# Cortical Correlates of Psychedelic-Induced Shaking Behavior Revealed by Voltage Imaging
## 3 Discussion  
5-HT2A receptor activation is the primary molecular substrate of psychedelic action in humans and underlies psychedelic-induced shaking behavior in rodents. Further, nonsychedelic 5-HT2A receptor agonists fail to induce shaking behavior, indicating that shaking behavior may be a readout of a psychedelic-specific active state of the 5-HT2A receptor [9]. Given the relevance of 5-HT2A receptors for both neuronal and cardiovascular functioning, here we sought to investigate the psychedelic-induced shaking behavior, through optical monitoring of both voltage activity patterns of pyramidal neurons in the motor cortex and blood-volume-related corticodynamics. First, we demonstrated that the selective 5-HT2A receptor agonist 25 CN-NBOH reduced the amplitude and frequency of synchronous pulse blood-volume-related signal oscillations (BvSO) in the motor cortex. These reductions were accompanied by a change in the shape of the BvSO frequency spectrum, with low-amplitude frequency components emerging. Unlike its effect on locomotion in freely moving mice [28], 25 CN-NBOH did not affect motor activity under head fixation, hence the observed BvSO effect does not appear to be a consequence of the behavioral recruitment of the cardiovascular system. BvSOs reflect blood flow mainly within large-amplitude arteries of the superficial brain tissue rather than parenchymal microcirculation. Psychedelics increase blood pressure [29; 30; 31], possibly resulting from 5-HT2A receptor-related increase in total peripheral resistance [32]. Consequently, the baroreceptor reflex may counteract the sympathetically  
Figure 7: Time-course of cortical events associated with shaking behavior. Oscillatory components detected in the motor cortex are depicted (**a**) in the time domain and (**b**) as peak-power contours in the time-frequency domain (mean \(\pm\) SEM: Y-scale is arbitrarily offset for visualization). Peak-power contours were extracted from the spectrograms in Figures 4–6; upper inset in (**b**) depicts the associated motor output. (**c**) Distribution of oscillatory peaks within the motor cortex relative to the shaking event. Median and quartiles. Green = primarily voltage-related; red = primarily blood-related. (**d**) Fourier transform of a representative shaking event (ear ROI) with the window size fixed in frequency domain. Note: LFO set the motoric platform for HFO. BvSO = Blood-volume-related signal oscillations; HFO = High-frequency oscillations; LFO = Low-frequency oscillations. driven tachycardia [33; 34] thereby potentially affecting brain surface BvSOs. Additionally, thermoregulatory changes [35] and/or interaction with 5-HT2A receptors within the Circle of Willis might play a role [36]. Next, we investigated the brain voltage and hemodynamic activity associated with the shaking behavior induced by 25 CN-NBOH. Despite head fixation, shaking events could be readily detected from various body parts. Wavelet and spectral characteristics of shaking events, measured from ROI-specific pixel variations analysis of the behavioral monitoring videos comparable to those quantified by magnetometer detection of head-mounted or ear-clipped magnets [37; 38] or video-based tracking of a fur-glued fluorescent marker [39]. We then used synchronized fluorescence activity imaging to examine the voltage and hemodynamic activity components in the motor cortex associated with shaking behavior. We detected a bimodal low-frequency oscillation (LFO) peak, which could be attributed to whole-body movements setting the \"motoric platform\" for the execution of shaking behavior. Shaking behavior has been previously linked to fronto-cortical LFO detected via electroencephalography and local field potential recordings [15; 16] which is much more prominent in the striatum than in the motor cortex [16]. The LFO summit in the motor cortex detected here reflects neuro- and hemodynamics and might be a component of cortico-striatal coupling.