Transient Stimulation with Psychoplastogens Is Sufficient to Initiate Neuronal Growth

Depression is associated with structural degeneration in prefrontal cortical circuits, including dendritic retraction, loss of dendritic spines, and reduced excitatory synapse density. Conventional antidepressants (for example selective serotonin reuptake inhibitors) require chronic dosing and have limited efficacy for many patients, prompting interest in agents that rapidly promote neural plasticity. Psychoplastogens—small molecules such as ketamine, scopolamine and serotonergic psychedelics—can rapidly stimulate regrowth of cortical dendritic arbors and produce relatively long-lasting behavioural effects after single administrations, but the biochemical sequence that allows a short pharmacological exposure to yield sustained structural change is not well defined. Ly and colleagues set out to determine whether very brief exposures to psychoplastogens are sufficient to initiate sustained growth of cortical neurons and to dissect the temporal requirements for key signalling nodes. Using ketamine and lysergic acid diethylamide (LSD) as representative psychoplastogens, the study tests how short stimulation periods (minutes to hours) affect dendritogenesis, spinogenesis and synaptogenesis in primary cortical cultures, and uses pharmacological inhibitors to probe roles for TrkB, AMPA receptors and mTOR during both the initial stimulation and the subsequent drug-free growth phase. The work aims to clarify mechanistic epochs underlying psychoplastogen-driven plasticity and to inform development of neurotherapeutics with rapid onset and reduced side effects.

This was an in vitro study using primary cortical neurons prepared from embryonic day 18 Sprague Dawley rat embryos. Cells were plated on poly-D-lysine-coated plates and maintained in Neurobasal-based media with B27 supplement; cultures were aged to specific days in vitro (DIV) depending on the assay (dendritogenesis assays used DIV3 neurons matured to DIV6; spinogenesis and synaptogenesis assays used older neurons, with drug treatments on DIV19 and fixation on DIV20). Treatments were applied for short intervals (reported durations include 15 min, 0.25 h, 1 h, 6 h, 24 h, and 72 h) after which media were exchanged for drug-free feeding medium and cells were allowed to grow for the remainder of the assay period. Ketamine and LSD were the principal psychoplastogens studied; exogenous brain-derived neurotrophic factor (BDNF, 50 ng/mL) served as a comparator. Concentration–response experiments identified 10 μM psychoplastogen as producing maximal dendritic growth. Pharmacological probes included DNQX (an AMPA receptor antagonist), ANA-12 (a selective TrkB antagonist), rapamycin (mTOR inhibitor) and 8-OH-DPAT (selective 5-HT1A agonist), applied either during the initial stimulation window or during the subsequent growth period to test temporal requirements. A depolarising stimulus (40 mM KCl for 1 h) was also used to test whether increased neuronal activity alone could reproduce psychoplastogen effects. Dendritic arbor complexity was assessed using Sholl-type analyses as in the authors’ prior work. Spinogenesis was measured after transient treatments on DIV19 with fixation on DIV20. Synaptogenesis was quantified in 96-well plates by immunostaining for pre- and postsynaptic markers (VGLUT1 and PSD-95), generating puncta masks and measuring colocalised presynaptic–postsynaptic events normalised to MAP2 mask area (counts per μm2) using high-content imaging and MetaXpress image analysis. Treatments were randomised and analyses were performed blinded. Statistical analysis used GraphPad Prism with data reported as mean ± SEM and conventional p-value thresholds indicated (for example *, p < 0.05). Data sets are stated to be available via Mendeley Data.

Very short exposures to psychoplastogens were sufficient to initiate sustained structural growth in cortical neurons. Treating DIV3 cortical cultures with ketamine or LSD for as little as 15 min, followed by growth in drug-free medium until DIV6, produced robust increases in dendritic arbor complexity measured by Sholl-type analyses. By comparison, exogenous BDNF (50 ng/mL) required about 1 h of exposure to elicit comparable effects. A 1 h psychoplastogen exposure followed by a 71 h growth period produced maximal dendritic complexity (Nmax) comparable with a continuous 72 h treatment. Concentration–response data indicated 10 μM as the concentration giving maximal dendritic growth. Fixing cultures immediately after 1 h of treatment showed no change in arborisation, supporting a model with distinct stimulation and subsequent growth phases. Transient psychoplastogen stimulation also increased dendritic spine density when applied to mature cultures (DIV19), with LSD producing spine increases similar in magnitude to BDNF and ketamine producing more modest effects. A 6 h stimulation period was generally optimal for spine density increases; longer stimulations tended to produce smaller changes, which the investigators attribute to homeostatic responses to excessive excitation. Synaptogenesis, assessed by colocalisation of VGLUT1 and PSD-95, paralleled spine results: a 6 h stimulation followed by an 18 h growth period produced the largest psychoplastogen-induced increases in synapse density (~2-fold). By contrast, BDNF produced a larger and faster synaptogenic effect (approximately a 6-fold increase after 15 min stimulation), indicating that synaptogenesis and structural growth can be dissociated and that psychoplastogens at tested concentrations are less synaptogenic than BDNF. Pharmacological dissection revealed distinct temporal requirements for signalling nodes. Blocking AMPA receptors with DNQX during either the initial stimulation or the growth phase prevented psychoplastogen-induced dendritic growth, indicating that sustained AMPA receptor activation is required both to initiate and to maintain the growth response. In contrast, blockade of TrkB with ANA-12 inhibited growth only when applied during the initial stimulation period and was ineffective when applied during the drug-free growth phase, suggesting TrkB activation is required early but not continuously. Inhibition of mTOR (rapamycin) during either the stimulation or growth periods abrogated neuronal growth, consistent with mTOR-dependent protein synthesis being necessary for both initiation and maintenance. Increasing neuronal activity with 40 mM KCl for 1 h did not reproduce the psychoplastogen-induced growth phenotype, nor did combining KCl with ketamine or LSD augment dendritogenesis, indicating that simple depolarisation/overexcitation is insufficient. Finally, activation of 5-HT1A receptors with 8-OH-DPAT failed to increase dendritic growth, suggesting 5-HT1A receptors are unlikely to mediate the shared psychoplastogenic effects of ketamine and LSD.

The investigators interpret their findings to show that transient exposure to chemically distinct psychoplastogens can catalyse a sustained growth programme in cortical neurons, with mechanistic separation into an initial stimulation epoch that requires TrkB activation and a subsequent growth epoch that depends on sustained AMPA receptor and mTOR signalling. They propose a positive autoregulatory feedback loop in which initial AMPA receptor activation leads to BDNF secretion and TrkB engagement, activating mTOR and protein synthesis, which then supports further BDNF production and continued glutamate release to maintain AMPA activity. Although ketamine and LSD have different primary targets (NMDA receptor antagonism and 5-HT2A receptor agonism, respectively), the authors suggest both may converge on glutamate bursts that trigger this downstream cascade; however, the data show that depolarisation alone does not reproduce the effect, leaving open the possibility of an additional shared target or mechanism. The discussion emphasises translational implications: behaviourally relevant brain concentrations of ketamine and DMT (>10 μM in rodents) are within the range that produced growth in culture, and short-acting psychoplastogens that achieve high brain concentrations but are rapidly cleared could retain therapeutic plasticity-promoting effects while limiting prolonged hallucinogenic or dissociative adverse effects. The authors note variability among psychoplastogens in durability of effects—for example, psilocybin can produce longer-lasting antidepressant-like responses than ketamine—and acknowledge that the precise biochemical details and duration of induced plasticity remain unresolved. They highlight the potential for non-hallucinogenic psychoplastogens and infrequent dosing paradigms to reduce healthcare burden and side effects, while calling for further work to clarify the signalling logic and to identify compounds with enhanced synaptogenic efficacy.