Abstract by Jannik Nicklas Eliasen

Major depressive disorder (MDD) and substance use disorders (SUDs) represent substantial global health challenges, characterized by high prevalence, marked sex differences, genetic factors, neurobiological alterations, and limited long-term efficacy of conventional pharmacotherapies. Psychedelic compounds such as N,N-dimethyltryptamine (DMT) and ibogaine have re-emerged as promising candidates due to their rapid antidepressant and anti-addictive properties. However, the cellular and circuit-level mechanisms underlying these effects remain incompletely understood. In particular, it is unclear how receptor-level psychedelic signaling translates into functional neuronal outcomes within mesolimbic circuits central to motivation, reward, and attention.

The ventral tegmental area (VTA) is a brain structure that plays a pivotal role in both MDD and SUD through its regulation of dopaminergic and inhibitory signaling that governs goal-directed behavior, motivation and stress responsiveness, and dysfunction within this region is strongly implicated in anhedonia, craving, and relapse. Approximately one-third of the neuronal population in the VTA is GABAergic and not only connects to neighboring dopaminergic neurons, but also projects to several key brain areas implicated in MDD and SUDs. Although the serotonin 2A (5-HT_2A) receptor is classically associated with cortical pyramidal neurons, accumulating evidence indicates the serotonergic modulation of midbrain circuits, including the VTA, can indirectly shape dopaminergic and inhibitory output and plasticity relevant to affective and addictive behaviors.

The present thesis investigates whether transiently transfected human embryonic kidney (HEK) cells constitute a suitable cell culture systems for studying psychedelic activation of the human 5-HT_2A G-protein coupled receptor (GPCR). In addition to receptor activation, study I examined whether transfected HEK cell could be used to evaluate the potential altered effects of single nucleotide polymorphisms (SNPs) in the 5-HT_2A receptor, which have been associated with differential responses to antidepressant treatment. Secondly, the acute effects of DMT and ibogaine were investigated using ex vivo recordings from VTA-containing brain slices.

In study I, the acute membrane effects of DMT and ibogaine were examined in HEK cells transiently transfected with the human serotonin 2A (5-HT_2A) receptor, including the wildtype (WT) receptor and the naturally occurring non-synonymous I197V single nucleotide polymorphism variant. Using whole-cell voltage-clamp recordings, both psychedelics were shown to induce clear membrane current changes and an increase in conductance in the majority of transfected HEK cells, while no effects were observed in non-transfected controls. Additionally, the I197V variant selectively reduced the duration of DMT-induced currents without affecting the response amplitude. These findings demonstrate for the first time that activation of the human 5-HT_2A receptors is sufficient to elicit measurable electrophysiological membrane responses in HEK cells. The results further support the use of this cell line for screening psychedelic effects across the WT and SNP-containing receptors and show that the I197V single-point mutation alters temporal aspects of receptor signaling rather than signaling strength. Current-voltage analyses suggested activation of non-selective cation channels, potentially involving transient receptor potential family channels, although the precise ionic mechanism was not explored. Collectively, study I establishes HEK cells as a rapid and easy system for investigating GPCR responses, particularly psychedelic activation and influence of genetic variation in the 5-HT_2A receptors on signaling kinetics.

Study II extended these findings into a more neurobiologically relevant context by ex vivo examining the acute effects of ibogaine on I_h-negative neurons in mouse VTA-containing brain slices. The absence of I_h current is commonly used as a proxy for identifying γ-aminobutyric acid (GABA)ergic neurons within the VTA. Whole-cell patch clamp recordings and calcium imaging revealed pronounced sex-dependent effects. Specifically, ibogaine significantly reduced the spontaneous firing in male I_h-negative neurons, while firing rates in female neurons were unaffected. In contrast, ibogaine selectively reduced the amplitude and duration of the afterhyperpolarization (AHP) in female neurons without altering the firing rate. Ibogaine did not induce changes in membrane conductance, resting membrane potential, rheobase, or synaptic excitatory transmission in either sex. Notably, baseline sex differences were identified with male neurons exhibiting higher firing rates and larger AHPs. Despite the absence of membrane current changes, ibogaine increased intracellular calcium levels in both sexes. These results indicate that ibogaine modulates VTA inhibitory tone through sex-specific alterations in excitability and repolarization that are also ontologically different, rather than through direct ionotropic mechanisms.

In study III, the acute effects of DMT were examined in the same VTA I_h-negative neuronal population to enable direct comparison with ibogaine. Looking at the I-V curves, DMT did not produce a main effect, as observed in HEK cells, when brain slices were acutely exposed to the same DMT concentration. However, by using a significantly higher concentration than those used in study I, while no main effect was observed, DMT altered the conductance at -40 and -50 mV. DMT, contrary to ibogaine, selectively increased firing rate in female neurons, while male neurons were unaffected. Similar to ibogaine, DMT induced clear intracellular calcium rises without accompanying changes in membrane conductance or synaptic events.

Taken together, these studies demonstrate that DMT and ibogaine can activate 5-HT_2A-linked signaling capable of producing membrane conductance and current changes in transiently transfected HEK cells. However, these effects do not translate directly to native VTA neurons, where the acute relevant actions in VTA neurons observed are expressed primarily through modulation of neuronal excitability, repolarization dynamics, and intracellular calcium signaling. Importantly, the responses of I_h-negative neurons to psychedelics were both dependent on compound and sex. The work reveals clear sex-dependent differences at both baseline and drug-responsive levels, underscoring the importance of incorporating sex as a biological variable in psychedelic research.