Distinct 5-HT receptor subtypes regulate claustrum excitability by serotonin and the psychedelic, DOI

Psychedelic drugs have shown clinical promise across a range of psychiatric disorders, with their behavioural effects historically attributed to activation of 5-HT2A receptors in the prefrontal cortex, hippocampus, and thalamus. However, psychedelic drugs can be expected to engage 5-HT2A receptors throughout the brain and are also known to act at non-5-HT2A targets, necessitating detailed investigation of serotonergic signalling within discrete brain circuits. The claustrum (CLA) is a particular focus of interest, as autoradiographic studies consistently identify it as the region with the highest density of serotonin receptor binding in the brain, including for psychedelic hallucinogens. Supporting functional relevance, recent fMRI data show altered claustrum activity and connectivity following psilocybin in humans. Despite this, the specific 5-HT receptor subtypes expressed in the claustrum, their cell-type distribution, and their functional impact on claustrum neuron excitability had not been investigated at the time of this study. The researchers aimed to characterise the expression and functional roles of 5-HT receptor subtypes in regulating excitability of claustrum neurons projecting to the anterior cingulate cortex (CLA-ACC neurons), with particular attention to how endogenous serotonin and the serotonergic psychedelic DOI differentially modulate these cells.

Claustrum neurons projecting to the anterior cingulate cortex (CLA-ACC) were identified by stereotaxic retrograde viral injection in rats. Whole-cell patch-clamp electrophysiology in acute brain slices was used to record spontaneous excitatory post-synaptic currents (sEPSCs), resting membrane potential, membrane resistance, rheobase current, and action potential firing rates in eGFP-labelled CLA-ACC neurons before and during bath application of 5-HT (30 µM) or the psychedelic 5-HT2A/2C agonist DOI (300 nM, 1 µM, and 10 µM). Quantitative RT-PCR was used to compare mRNA expression levels of 13 5-HT receptor subtypes across the CLA, insula, and anterior cingulate cortex. RNAscope in situ hybridisation was then used to examine the cell-type specificity of 5-HT1A, 5-HT2A, and 5-HT2C receptor expression within glutamatergic (Slc17a7-positive) and GABAergic (Gad1-positive) CLA populations. Pharmacological dissection of receptor contributions to 5-HT and DOI effects employed subtype-selective antagonists: WAY 100,635 (5-HT1A), MDL 100907 (5-HT2A), and SB 242,084 (5-HT2C), applied in the presence or absence of the GABAA blocker picrotoxin.

qPCR analysis identified 5-HT2A and 5-HT2C as the most abundantly expressed 5-HT receptor subtypes in the CLA; notably, 5-HT2C expression was approximately five-fold higher in the CLA than in either the ACC or the insula, representing a CLA-selective enrichment. RNAscope confirmed that approximately 40-48% of glutamatergic CLA neurons co-expressed 5-HT1A, 5-HT2A, and 5-HT2C mRNA, whilst GABAergic cells showed markedly lower expression across all three subtypes, particularly 5-HT1A (~5%). Bath application of 5-HT to CLA-ACC neurons significantly decreased sEPSC amplitude (p < 0.0001) and frequency (p = 0.0053), hyperpolarised the resting membrane potential (p = 0.0114), increased rheobase current (p = 0.0003), and suppressed action potential firing (p = 0.0002). Pharmacological dissection showed that the 5-HT1A antagonist WAY 100,635 reversed sEPSC amplitude suppression, whilst the 5-HT2C antagonist SB 242,084 reversed sEPSC frequency suppression; the 5-HT2A antagonist MDL 100907 was without effect on any measured sEPSC parameter — a surprising finding given robust 5-HT2A mRNA expression in the CLA. Both 5-HT1A and 5-HT2C antagonism contributed to reversing 5-HT-induced hyperpolarisation. In contrast to 5-HT, DOI at all concentrations tested (300 nM to 10 µM) significantly increased sEPSC amplitude and frequency. These excitatory effects were abolished by the 5-HT2C antagonist SB 242,084 but not by the 5-HT2A antagonist, indicating that DOI's potentiation of glutamatergic transmission in the CLA is mediated by 5-HT2C rather than 5-HT2A receptors. Picrotoxin experiments confirmed that these effects were independent of local GABAergic inhibition.

The results demonstrate that the claustrum exhibits a distinct serotonergic pharmacological profile compared with other brain regions, characterised by unusually high 5-HT2C expression and a prominent 5-HT2C contribution to the regulation of glutamatergic synaptic transmission. This contrasts with the predominantly 5-HT1A and 5-HT2A-mediated modulation of excitability reported in prefrontal pyramidal neurons, highlighting the CLA as a functionally distinct node in the serotonergic network. The bidirectional regulation of CLA-ACC excitability by 5-HT2C receptors is particularly noteworthy: endogenous 5-HT suppresses glutamatergic transmission, whilst the psychedelic agonist DOI enhances it, both via 5-HT2C. This may reflect differences in receptor occupancy, biased agonism, or concentration-dependent receptor dynamics. The absence of detectable 5-HT2A contributions to synaptic excitability despite robust 5-HT2A mRNA expression remains unexplained, though the researchers propose that 5-HT2A receptors in the CLA may mediate cellular effects not captured by the current experimental approach, including intracellular signalling pathways, dendritic remodelling via BDNF-trkB interactions, or sequestration into intracellular receptor pools. These findings extend recent functional connectivity data implicating the CLA in network responses to psychedelics and position 5-HT2C as a candidate mediator of these circuit-level effects.

These results establish that the serotonergic psychedelic DOI exerts effects on claustrum-ACC neurons that are distinct from those of endogenous 5-HT, and identify 5-HT2C receptors as essential regulators of CLA excitability. Further characterisation of receptor subtype distributions, downstream signalling cascades, and cellular interactions in the claustrum is warranted to advance understanding of the psychoactive and therapeutic effects of psychedelic drugs.