Cholinergic interneurons (ChIs) provide the main source of acetylcholine in the striatum and have emerged as a critical modulator of behavioral flexibility, motivation, and associative learning. In the dorsal striatum (DS), ChIs display heterogeneous firing patterns. Here, we investigated the spontaneous firing patterns of ChIs in the nucleus accumbens (NAc) shell, a region of the ventral striatum. We identified four distinct ChI firing signatures: regular single-spiking, irregular single-spiking, rhythmic bursting, and a mixed-mode pattern composed of bursting activity and regular single spiking. ChIs from females had lower firing rates compared with males and had both a higher proportion of mixed-mode firing patterns and a lower proportion of regular single-spiking neurons compared with males. We further observed that across the estrous cycle, the diestrus phase was characterized by higher proportions of irregular ChI firing patterns compared with other phases. Using pooled data from males and females, we examined how the stress-associated neuropeptide corticotropin releasing factor (CRF) impacts these firing patterns. ChI firing patterns showed differential sensitivity to CRF. This translated into differential ChI sensitivity to CRF across the estrous cycle. Furthermore, CRF shifted the proportion of ChI firing patterns toward more regular spiking activity over bursting patterns. Finally, we found that repeated stressor exposure altered ChI firing patterns and sensitivity to CRF in the NAc core, but not the NAc shell. These findings highlight the heterogeneous nature of ChI firing patterns, which may have implications for accumbal-dependent motivated behaviors.

Abstract ID 128335Poster Board 337Investigation into the clinical use of the serotonin (5HT-2A) receptor agonist psilocybin in conjunction with psychotherapy has shown promising therapeutic results in the treatment of psychiatric disorders. Correlations between drug-induced cortisol elevation and treatment outcomes have been reported for human studies during psilocybin-assisted psychotherapy; however, the mechanistic relationship between psychedelic-associated alterations in plasma corticosterone (CORT) responses and anxiolytic efficacy remains unclear. A time course for psilocybin to induce anxiolysis in the absence of any intervention was conducted with C57/Bl6 mice and LC-MS/MS. Serum samples tested at doses of 0.3, 1, and 3 mg/kg found peak concentrations at 15 min post-injection. In the open field test 15 minutes post-injection, mice that received 3mg/kg psilocybin demonstrated decreased center time, while the 0.3 mg/kg exhibited a dose-dependent increase. However, at 4 h post-injection, the reverse was found, in that 0.3mg/kg psilocybin decreased center time. This dose-dependent interaction correlates with psilocybin-induced increases in CORT levels that peak at 15 min and return to baseline by 4 h.In addition, the non-hallucinogenic compound lisuride and NDMAR antagonist ketamine were used and demonstrated a transient increase in corticosterone concentrations at 15 min and returned to baseline by 4 h. When tested in the novelty-suppressed feeding test, all three compounds reduced the latency to feed 4 h post-injection. Although following exposure to chronic oral CORT and when administered the glucocorticoid antagonist, mifepristone, both psilocybin and ketamine lost this anxiolytic effect. Chronic exposure to CORT and mifepristone suppressed the psilocybin-induced stress response and increased CORT. At a dose of 3 mg/kg, IP psilocybin was found to have post-acute anxiolytic-like effects that were not altered by pretreatment with the non-hallucinogenic 5-HT2AR antagonist, ketanserin. These results suggest that psilocybin-induced stress response and increased plasma CORT levels are supportive of the observed anxiolytic effects. This study demonstrates that psilocybin-induced corticosterone release is a critical factor driving reductions in anxiety-like behavior in mice in the hours following drug clearance. That ongoing, unresolved glucocorticoid elevations can invert the long-term outcome of psilocybin administration on anxiety-like behavior up to seven days later. These findings point to HPA axis activation and changes in glucocorticoid concentration profiles over time as critical translational factors for mechanistic consideration when investigating classical serotonergic psychedelic-assisted therapy for the treatment of psychiatric disorders.This work was supported through funds from the UW-Madison School of Pharmacy, grant funding to C.J.W. from the National Institute of Mental Health (R01MH122742), a fellowship for N.T.J. from the National Institute of General Medical Sciences (T32GM008688), and fellowships for Z.Z. and J.R. from the National Institute of Neurological Disorders and Stroke (T32NS105602).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

While correlations between drug-induced cortisol elevation,self-reportedanxiety, and treatment outcomes have been reported for human studiesduring psilocybin-assisted psychotherapy, the mechanistic relationshipbetween psychedelic-associated alterations in plasma glucocorticoidresponses and the time course of anxious responsiveness remains unclear.Using rodents, both time-bound manipulation of glucocorticoid concentrationsand assessment of anxiety-like behaviors can be achieved. Here, 3mg/kg IP psilocybin was found to have anxiolytic-like effects in C57BL/6male mice at 4 h after treatment. These effects were not altered bypretreatment with a 5-HT2A antagonist but were bluntedby pretreatment with a glucocorticoid receptor antagonist or suppressionof psilocybin-induced corticosterone elevations. Anxiolytic-like effectswere also observed at 4 h following treatment with the nonpsychedelic5-HT2A agonist lisuride at a dose causing a similar increasein plasma glucocorticoids as that seen with psilocybin, as well asfollowing stress-induced (via repeated injection) glucocorticoid releasealone. Psilocybin's anxiolytic-like effects persisted at 7days following administration. The long-term anxiolytic effects ofpsilocybin were lost when psilocybin was administered to animals withongoing chronic elevations in plasma corticosterone concentrations.Overall, these experiments indicate that acute, resolvable psilocybin-inducedglucocorticoid release drives the postacute anxiolytic-like effectsof psilocybin in mice and that its long-term anxiolytic-like effectscan be abolished in the presence of chronically elevated plasma glucocorticoidelevations.

There are five cloned muscarinic acetylcholine receptors (M1–M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1–M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here, we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the muscarinic agonist oxotremorine, we revealed a unique concentration–response curve and a sensitivity to repeated forced swim stress or restraint stress exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia assayed using the sucrose preference test or on stress-coping strategy assayed using the forced swim test. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in response to environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety. SIGNIFICANCE STATEMENT The basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here, we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without affecting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.