# Cortical Correlates of Psychedelic-Induced Shaking Behavior Revealed by Voltage Imaging
## 1 Introduction  
Serotonergic psychedelics, such as lysergic acid diethylamide (LSD), profoundly affect human psychological functioning. In rodents, psychedelics induce stereotypical motor behaviors, including backward walking, reciprocal forepaw treading, flat body posture, lateral head weaving, and/or head twitches, and wet dog shakes. The last two behavioral components, hereon together referred to as shaking behavior [1, 2], rank among the most widely used animal behavioral correlates of central serotonin activity. As an animal model of neuropsychiatric conditions, shaking behavior is a particularly appealing behavioral readout. In mammals, shaking behavior is innate and has a benign character already infrequently exhibited as a part of the natural repertoire, readily observable by eye, and particularly targets one constituent of serotonin transmission, namely the serotonin (5-HT) 2A receptor. Psychedelic-induced shaking behavior across species has been described from as early as 1956 [3, 4, 5]. Correlation studies showed a close relationship between the potency of diverse antagonists to block shaking behavior and their affinity for 5-HT2A receptors [6, 7, 8]. Further, the importance of 5-HT2A receptors in shaking behavior has recently been reaffirmed using a 5-HT2A receptor knock-out mouse model [9, 10]. Despitehalf a century of research, our understanding of the function and physiology of this behavioral stereotype remains limited. 5-HT2A receptors are most abundantly expressed in the cerebral cortex, and tolerance to shaking behavior has been shown to reflect adaptation in 5-HT2A signaling and/or binding sites in the (frontal) cortex [1; 11]. Further, the inability to display shaking behavior in 5-HT2A receptor knock-out mice is reversed by selective restoration of 5-HT2A receptor expression in cortical pyramidal neurons [9]. Despite these and other findings collectively pointing to a possible role of cortical pyramidal neurons in the generation and/or modulation of shaking behavior under the influence of 5-HT2A receptor signaling [12], this remains controversial due to inconsistencies in the literature [13; 14] and the methodological difficulties in cell type-specific measurement from awake animals.  
To the best of our knowledge, so far there are only two papers that report on event-related electrophysiology of rodent shaking behavior in vivo. Neither of them has provided a cell-type-specific resolution [15; 16]. Here, we address this unknown by taking advantage of recent developments in cell-type-specific voltage imaging approaches using genetically encoded voltage indicators (GEVIs) [17]. Research on the cortical effects of psychedelics is generally focused on pyramidal cells of layer 5 [12; 18]. Layer 2/3 pyramidal cells--despite being major drivers of layer 5 [19]--are largely ignored. We selectively expressed the GEVI VSFP Butterfly 1.2 in cortical layer 2/3 pyramidal neurons [20], a cell population sensitive to psychedelics [21; 22; 23], to investigate the brain activity associated with the shaking behavior induced by the selective 5-HT2A receptor agonist 25 CN-NBOH (N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine) [24]. As 5-HT2A receptor expression is not restricted to neurons but also extends across the vascular system [25; 26], we additionally take advantage of the dual-emission design of VSFP Butterfly 1.2 [27] to delineate both voltage activity for cortical pyramidal neurons as well as blood-volume related hemodynamics associated with shaking behavior.