Repeated low doses of psilocybin increase resilience to stress, lower compulsive actions, and strengthen cortical connections to the paraventricular thalamic nucleus in rats.

Psilocybin (a classic serotonergic psychedelic drug) has received appraisal for use in psychedelic-assisted therapy of several psychiatric disorders. A less explored topic concerns the use of repeated low doses of psychedelics, at a dose that is well below the psychedelic dose used in psychedelic-assisted therapy and often referred to as microdosing. Psilocybin microdose users frequently report increases in mental health, yet such reports are often highly biased and vulnerable to placebo effects. Here we establish and validate a psilocybin microdose-like regimen in rats with repeated low doses of psilocybin administration at a dose derived from occupancy at rat brain 5-HT2A receptors in vivo. The rats tolerated the repeated low doses of psilocybin well and did not manifest signs of anhedonia, anxiety, or altered locomotor activity. There were no deficits in pre-pulse inhibition of the startle reflex, nor did the treatment downregulate or desensitize the 5-HT2A receptors. However, the repeated low doses of psilocybin imparted resilience against the stress of multiple subcutaneous injections, and reduced the frequency of self-grooming, a proxy for human compulsive actions, while also increasing 5-HT7 receptor expression and synaptic density in the paraventricular nucleus of the thalamus. These results establish a well-validated regimen for further experiments probing the effects of repeated low doses of psilocybin. Results further substantiate anecdotal reports of the benefits of psilocybin microdosing as a therapeutic intervention, while pointing to a possible physiological mechanism.

Synaptic Density and Neuronal Metabolic Function Measured by Positron Emission Tomography in the Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is caused by progressive neurodegeneration and characterised by motor dysfunction. Neurodegeneration of dopaminergic neurons also causes aberrations within the cortico-striato-thalamo-cortical (CSTC) circuit, which has been hypothesised to lead to non-motor symptoms such as depression. Individuals with PD have both lower synaptic density and changes in neuronal metabolic function in the basal ganglia, as measured using [11C]UCB-J and [18F]FDG positron emission tomography (PET), respectively. However, the two radioligands have not been directly compared in the same PD subject or in neurodegeneration animal models. Here, we investigate [11C]UCB-J binding and [18F]FDG uptake in the CSTC circuit following a unilateral dopaminergic lesion in rats and compare it to sham lesioned rats. Rats received either a unilateral injection of 6-hydroxydopamine (6-OHDA) or saline in the medial forebrain bundle and rostral substantia nigra (n = 4/group). After 3 weeks, all rats underwent two PET scans using [18F]FDG, followed by [11C]UCB-J on a separate day. [18F]FDG uptake and [11C]UCB-J binding were both lower in the ipsilateral striatal regions compared to the contralateral regions. Using [11C]UCB-J, we could detect an 8.7% decrease in the ipsilateral ventral midbrain, compared to a 2.9% decrease in ventral midbrain using [18F]FDG. Differential changes between hemispheres for [11C]UCB-J and [18F]FDG outcomes were also evident in the CSTC circuit's cortical regions, especially in the orbitofrontal cortex and medial prefrontal cortex where higher synaptic density yet lower neuronal metabolic function was observed, following lesioning. In conclusion, [11C]UCB-J and [18F]FDG PET can detect divergent changes following a dopaminergic lesion in rats, especially in cortical regions that are not directly affected by the neurotoxin. These results suggest that combined [11C]UCB-J and [18F]FDG scans could yield a better picture of the heterogeneous cerebral changes in neurodegenerative disorders.

Desorption Electrospray Ionization Mass Spectrometry Imaging of Cimbi-36, a 5-HT2A Receptor Agonist, with Direct Comparison to Autoradiography and Positron Emission Tomography.

PURPOSE: The study demonstrates the use of Desorption Electrospray Ionization mass spectrometry imaging (DESI-MSI) for imaging of the PET tracer compound Cimbi-36 in brain tissue and compares imaging by DESI-MSI to imaging by autoradiography and PET. PROCEDURES: Rats were dosed intraperitoneally with 3 mg/kg of Cimbi-36 and euthanized at t = 5, 10, 15, 30, 60 and 120 min post-injection. The brains were removed, frozen and sectioned, and sagittal sections were imaged by DESI-MSI in positive ion mode. Additionally, brain sections from a non-dosed animal were incubated with 14C-labelled Cimbi-36 and imaged by autoradiography. Finally, PET images were acquired from an animal dosed with 11C-labelled Cimbi-36. RESULTS: DESI-MSI and autoradiography images of a sagittal brain sections showed similar distributions of Cimbi-36, with increased abundance in the frontal cortex and choroid plexus, regions which are high in 5-HT2A and 5-HT2C receptors. The PET image also showed increased abundance in cortex, but the spatial resolution was clearly inferior to DESI-MSI and autoradiography. The DESI-MSI results showed increased abundance of Cimbi-36 in brain tissue until 15 min, after which the abundance was declining. The PET-tracer was still clearly detectable at t = 120 min. Similar imaging of the kidneys showed the abundance of Cimbi-36 peaking at 30 min. Cimbi-36 was quantified in a t = 15 min brain section by quantitative DESI-MSI, resulting in tissue concentrations of 19.8 µg/g in cortex, 15.4 µg/g in cerebellum and 12.5 µg/g in whole brain. CONCLUSIONS: DESI imaging from an in vivo dosing experiment showed distribution of the PET tracer remarkably similar to what was obtained by autoradiography of an in vitro incubation experiment, indicating that the obtained results represent actual binding to certain receptors in the brain. DESI-MSI is suggested as a cost-effective screening tool, which does not rely on labelling of compounds.

Blocking of efflux transporters in rats improves translational validation of brain radioligands.

BACKGROUND: Positron emission tomography (PET) is a molecular imaging technique that can be used to investigate the in vivo pharmacology of drugs. Initial preclinical evaluation of PET tracers is often conducted in rodents due to the accessibility of disease models as well as economic considerations. Compared to larger species, rodents display a higher expression and/or activity of efflux transporters such as the P-glycoprotein (P-gp). Low brain uptake could, therefore, be species-specific and uptake in rodents not be predictive for that in humans. We hypothesized that a better prediction from rodent data could be achieved when a tracer is evaluated under P-gp inhibition. Consequently, we compared the performance of eight neuroreceptor tracers in rats with and without P-gp inhibition including a specific binding blockade. This data set was then used to predict the binding of these eight tracers in pigs. METHODS: PET tracers targeting serotonin 5-HT2A receptors ([18F]MH.MZ, [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin), serotonin 5-HT7 receptors ([11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10) and dopamine D2/3 receptors ([18F]Fallypride) were used in the study. The brain uptake and target-specific binding of these PET radiotracers were evaluated in rats with and without inhibition of P-gp. Rat data were subsequently compared to the results obtained in pigs. RESULTS: Without P-gp inhibition, the amount of target-specific binding in the rat brain was sufficient to justify further translation for three out of eight evaluated tracers. With P-gp inhibition, results for five out of eight tracers justified further translation. The performance in pigs could correctly be predicted for six out of eight tracers when rat data obtained under P-gp inhibition were used, compared to four out of eight tracers without P-gp inhibition. CONCLUSIONS: P-gp strongly affects the uptake of PET tracers in rodents, but false prediction outcomes can be reduced by evaluating a tracer under P-gp inhibition.

The selective 5-HT2A receptor agonist 25CN-NBOH: Structure-activity relationship, in vivo pharmacology, and in vitro and ex vivo binding characteristics of [3H]25CN-NBOH.

The remarkable effects exhibited by classical psychedelics in recent clinical trials have spawned considerable interest in 5-HT2A receptor (5-HT2AR) activation as a treatment strategy for several psychiatric/cognitive disorders. In this study we have continued our development of 25CN-NBOH, one of the most 5-HT2AR-selective agonists reported to date, as a pharmacological tool for exploration of 5-HT2AR expression and functions. The importance of the 2' and 3' positions in 25CN-NBOH as structural hotspots for its 5-HT2AR activity was investigated by synthesis and pharmacological characterization of six novel analogs at 5-HT2AR and 5-HT2CR in binding and functional assays. While the 5-HT2AR activity of 25CN-NBOH was retained in 3'-methyl, 2',3'-chroman, 2',3'-dihydrofuran and 2',3'-furan analogs, the 3'-methoxy and 3'-ethyl analogs displayed substantially lower binding affinities and agonist potencies than 25CN-NBOH. Interestingly, the 2',3'-substitution pattern was also a key determinant of agonist efficacy, as all six analogs exhibited low-efficacy partial agonism or de facto antagonism at the 5-HT2AR in the functional assays. Systemic administration of 25CN-NBOH and its close structural analog 25CN-NBMD induced robust head-twitch response in mice, a well-established behavioural effect of 5-HT2AR activation in vivo, and 25CN-NBOH mediated robust reductions in the activity of mice in an anxiety-related marble burying assay, which supports the proposed beneficial effects of 5-HT2AR activation on disorders characterized by cognitive rigidity. Finally, tritiated 25CN-NBOH exhibited high 5-HT2AR binding affinity (KD ~1 nM) and selectivity against 5-HT2BR and 5-HT2CR in equilibrium and kinetic binding studies of the recombinant receptors, and in concordance [3H]25CN-NBOH displayed substantial specific, ketanserin-sensitive binding to cortex and small levels of binding to choroid plexus in rat brain slices in autoradiography studies. In conclusion, this work delineates the subtle molecular determinants of the 5-HT2AR activity in 25CN-NBOH, substantiates the potential in this compound and its analogs as tools for in vivo studies of the 5-HT2AR, and introduces a novel selective agonist radioligand as another potentially valuable tool for future explorations of this receptor.