Cellular rules underlying psychedelic control of prefrontal pyramidal neurons

The experimental core comprised acute whole‑cell recordings from regular‑spiking layer 5 pyramidal cells in mouse prefrontal slices (prelimbic and anterior cingulate regions). Cells were identified morphologically via biocytin fills and laminar location was determined with NeuroTrace; neurons were included if baseline RMP and series resistance criteria were met, with additional electrophysiological exclusion rules reported. Both current‑clamp and voltage‑clamp protocols were used: evoked spiking was assessed with depolarising square pulses (0.5 s and 1 s steps, up to 400 pA) and firing rate–current (f–I) relations were derived; spontaneous excitatory postsynaptic currents (sEPSCs) were recorded at a holding potential of −70 mV in 30 s sweeps. Key intrinsic measures included rheobase, input resistance, adaptation index and spike waveform metrics; spike detection and feature extraction used automated routines with manual verification. Pharmacology experiments tested multiple serotonergic psychedelics: the 5-HT2AR‑preferring agonist 25CN‑NBOH (referred to as NBOH), DOI (a phenethylamine), and 4‑HO‑DiPT (a tryptamine). Drugs were applied by bath perfusion at a range of concentrations (0.1, 1, 10 and 100 µM) for 10 minutes. Intracellular delivery was performed by including NBOH in the patch pipette at matched concentrations to contrast extracellular versus intracellular effects. Receptor and channel blockers included ketanserin (10 µM) to antagonise 5-HT2Rs and XE‑991 (10 µM) to block M‑current/Kv7 channels. An in vivo component delivered DOI intraperitoneally (2 mg/kg) with ex vivo recordings performed 48–72 hours later in ACC slices. For mechanistic interpretation the team used a machine‑learning–based data assimilation approach to fit conductance‑based Hodgkin–Huxley models to experimental voltage traces. Assimilated models incorporated transient sodium (NaT), delayed rectifier potassium, persistent sodium, M/Kv7 and H currents (with H current pruned where appropriate). Stochastic sEPSC bombardment was modelled as a Poisson process convolved with an exponential kernel; resting depolarisation was simulated as a tonic current. To probe working‑memory–related persistent firing, a slow calcium‑activated non‑specific cation current (ICAN) was added and model perturbations of M/Kv7 conductance and NaT inactivation were simulated. Statistical analyses were performed with GraphPad Prism after testing distributions; specific tests are cited in figure legends. The extracted text does not clearly report the total number of cells or animals used in each experiment.

Contrary to the starting hypothesis, extracellular application of serotonergic psychedelics dose‑dependently suppressed intrinsic excitability of PFC layer 5 pyramidal cells despite producing an RMP depolarisation. Bath‑applied 25CN‑NBOH (NBOH) at 0.1, 1, 10 and 100 µM produced robust, dose‑dependent decreases in maximum firing frequency, f–I gain and total spiking output. Across anatomical position, laminar depth within L5, age, genotype and sex, intrinsic excitability was reduced for all tested doses (with rare exceptions). By contrast, synaptic excitability changed in a dose‑dependent but thresholded manner: sEPSC frequency was increased, with a sustained elevation observed primarily at higher concentrations (10 and 100 µM). The pattern of effects generalised across chemical classes: DOI and 4‑HO‑DiPT produced similar dose‑dependent suppression of intrinsic excitability and enhancement of spontaneous glutamate release. Delivery route mattered: intracellular administration of NBOH via the patch electrode was significantly less effective at suppressing intrinsic excitability and at enhancing sEPSC frequency than equivalent extracellular concentrations, indicating a dominant role for extracellular actions. Mechanistically, spike loss depended on dose. Low to moderate psychedelic doses primarily increased spike frequency adaptation (more spikes early in a depolarising step, fewer later), whereas the highest dose often precipitated depolarisation block, defined as progressive failure of action potential generation associated with reduction in spike amplitude (a state linked to sodium channel inactivation). Low to moderate doses lowered input resistance more than the highest dose, consistent with increased membrane conductance at those concentrations. Pharmacological dissection implicated M‑current/Kv7 channels as a key mediator of intrinsic hypoexcitability that is independent of 5‑HT2AR activation. Pretreatment with ketanserin (10 µM) prevented the psychedelic‑mediated enhancement of sEPSC frequency but did not eliminate the decrease in intrinsic excitability, indicating that 5‑HT2Rs are required for the synaptic effect but not for spike suppression. In contrast, blocking M‑current with XE‑991 (10 µM) abolished the psychedelic‑induced increase in spike frequency adaptation and significantly attenuated firing suppression at low to moderate doses; XE‑991 did not prevent sEPSC frequency enhancement. Among neurons that still showed spike suppression in XE‑991, none exhibited the adaptation signature, implicating M/Kv7 upregulation as responsible for adaptation‑mediated spike loss. Computational data‑assimilation models reproduced key experimental observations. Increasing maximal M/Kv7 conductance or hyperpolarising the half‑inactivation voltage of transient sodium (NaT) individually reduced spike output; combined manipulations produced larger reductions and could produce irregular firing regimes (alternation of spikes and subthreshold oscillations) and depolarisation block at high perturbation levels. Simulations that added stochastic sEPSC bombardment and the observed RMP depolarisation showed that intrinsic conductance changes dominated the cell's input–output relation; stochastic synaptic input minimally altered the f–I relation relative to conductance modulation. Finally, adding a slow calcium‑activated non‑specific cationic current (ICAN) to model persistent firing — a cellular correlate of working memory — revealed dose‑dependent disruption by psychedelic‑like perturbations: moderate M/Kv7 and NaT changes reduced persistent spiking, stronger perturbations caused self‑termination, and the largest perturbations abolished persistent firing, linking the cellular conductance effects to impaired cellular correlates of working memory.

The investigators conclude that, although psychedelics can enhance certain measures of neuronal excitability (for example, sEPSC frequency), the dominant acute effect on PFC layer 5 pyramidal neurons is dose‑dependent suppression of intrinsic excitability. This finding requires revision of the prevailing paradigm that classical psychedelics primarily increase intrinsic excitability via 5‑HT2ARs. Differences from prior studies are attributed to methodological factors: earlier work often focused on RMP changes or near‑threshold current injections, which may miss larger suppressive effects apparent across a wider range of current injections, and many prior studies used serotonin or other 5‑HT2AR agonists without established psychedelic properties. A central mechanistic implication is that serotonergic psychedelics enhance M/Kv7 conductance by a 5‑HT2R‑independent pathway, producing spike frequency adaptation and lowered input resistance even at low doses. Higher doses recruit 5‑HT2AR signalling, producing additional effects on transient sodium currents and thereby increasing susceptibility to depolarisation block. Because Kv7 channels are widely expressed, the authors suggest that M‑current activation could suppress intrinsic excitability broadly across brain regions that express these channels, with particularly strong effects in PFC L5 PCs due to dense 5‑HT2AR expression at higher doses. The authors situate their acute ex vivo findings within in vivo literature showing mixed but often net firing decreases following serotonergic psychedelics, and propose a mechanistic link to longer‑term synaptic remodelling: acute spiking suppression combined with enhanced spontaneous glutamate release could trigger post‑silencing rebound synaptogenesis together with intracellular 5‑HT2AR‑mediated spinogenesis, potentially creating a transient, plastic state that contributes to therapeutic benefit. They acknowledge complexity in extrapolating to in vivo firing rates, given multiple network variables, and note methodological distinctions from prior work as reasons for differing conclusions. Limitations explicitly noted in the extracted text include the lack of clearly reported sample sizes in the methods portion provided and the inherent difficulty of inferring in vivo consequences from acute slice experiments. The authors propose that their cellular rules provide a mechanistic complement to existing models and highlight pathways for future research on how acute ionic mechanisms relate to subjective and therapeutic outcomes.