Beyond the 5-HT2A Receptor: Classic and Nonclassic Targets in Psychedelic Drug Action.

Serotonergic psychedelics, such as psilocybin and LSD, have garnered significant attention in recent years for their potential therapeutic effects and unique mechanisms of action. These compounds exert their primary effects through activating serotonin 5-HT2A receptors, found predominantly in cortical regions. By interacting with these receptors, serotonergic psychedelics induce alterations in perception, cognition, and emotions, leading to the characteristic psychedelic experience. One of the most crucial aspects of serotonergic psychedelics is their ability to promote neuroplasticity, the formation of new neural connections, and rewire neuronal networks. This neuroplasticity is believed to underlie their therapeutic potential for various mental health conditions, including depression, anxiety, and substance use disorders. In this mini-review, we will discuss how the 5-HT2A receptor activation is just one facet of the complex mechanisms of action of serotonergic psychedelics. They also interact with other serotonin receptor subtypes, such as 5-HT1A and 5-HT2C receptors, and with neurotrophin receptors (e.g., tropomyosin receptor kinase B). These interactions contribute to the complexity of their effects on perception, mood, and cognition. Moreover, as psychedelic research advances, there is an increasing interest in developing nonhallucinogenic derivatives of these drugs to create safer and more targeted medications for psychiatric disorders by removing the hallucinogenic properties while retaining the potential therapeutic benefits. These nonhallucinogenic derivatives would offer patients therapeutic advantages without the intense psychedelic experience, potentially reducing the risks of adverse reactions. Finally, we discuss the potential of psychedelics as substrates for post-translational modification of proteins as part of their mechanism of action.

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.

A pilot study of cerebral metabolism and serotonin 5-HT2A receptor occupancy in rats treated with the psychedelic tryptamine DMT in conjunction with the MAO inhibitor harmine.

Rationale: The psychedelic effects of the traditional Amazonian botanical decoction known as ayahuasca are often attributed to agonism at brain serotonin 5-HT2A receptors by N,N-dimethyltryptamine (DMT). To reduce first pass metabolism of oral DMT, ayahuasca preparations additionally contain reversible monoamine oxidase A (MAO-A) inhibitors, namely ß-carboline alkaloids such as harmine. However, there is lacking biochemical evidence to substantiate this pharmacokinetic potentiation of DMT in brain via systemic MAO-A inhibition. Objectives: We measured the pharmacokinetic profile of harmine and/or DMT in rat brain, and tested for pharmacodynamic effects on brain glucose metabolism and DMT occupancy at brain serotonin 5-HT2A receptors. Methods: We first measured brain concentrations of harmine and DMT after treatment with harmine and/or DMT at low sub-cutaneous doses (1 mg/kg each) or harmine plus DMT at moderate doses (3 mg/kg each). In the same groups of rats, we also measured ex vivo the effects of these treatments on the availability of serotonin 5-HT2A receptors in frontal cortex. Finally, we explored effects of DMT and/or harmine (1 mg/kg each) on brain glucose metabolism with [18F]FDG-PET. Results: Results confirmed that co-administration of harmine inhibited the formation of the DMT metabolite indole-3-acetic acid (3-IAA) in brain, while correspondingly increasing the cerebral availability of DMT. However, we were unable to detect any significant occupancy by DMT at 5-HT2A receptors measured ex vivo, despite brain DMT concentrations as high as 11.3 µM. We did not observe significant effects of low dose DMT and/or harmine on cerebral [18F]FDG-PET uptake. Conclusion: These preliminary results call for further experiments to establish the dose-dependent effects of harmine/DMT on serotonin receptor occupancy and cerebral metabolism.

Pretargeted imaging beyond the blood-brain barrier.

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines and reduce the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain barrier (BBB) is challenging and has not been reported thus far. In this study, we developed Tz imaging agents that are capable of ligating in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. As a non-metal radionuclide, fluorine-18 also allows for development of Tzs with physicochemical properties enabling passive brain diffusion. To develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimentally determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo brain influx and washout as well as on peripheral metabolism profiles. From 18 initially developed structures, five Tzs were selected to be tested for their in vivo click performance. Whereas all selected structures clicked in vivo to TCO-polymer deposited into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted neuroimaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets in the brain that are currently not imageable, such as soluble oligomers of neurodegeneration biomarker proteins. Imaging of such currently non-imageable targets will allow early diagnosis and personalized treatment monitoring. This in turn will accelerate drug development and greatly benefit patient care.

An in vivo Pig Model for Testing Novel Positron Emission Tomography Radioligands Targeting Cerebral Protein Aggregates.

Positron emission tomography (PET) has become an essential clinical tool for diagnosing neurodegenerative diseases with abnormal accumulation of proteins like amyloid-ß or tau. Despite many attempts, it has not been possible to develop an appropriate radioligand for imaging aggregated α-synuclein in the brain for diagnosing, e.g., Parkinson's Disease. Access to a large animal model with α-synuclein pathology would critically enable a more translationally appropriate evaluation of novel radioligands. We here establish a pig model with cerebral injections of α-synuclein preformed fibrils or brain homogenate from postmortem human brain tissue from individuals with Alzheimer's disease (AD) or dementia with Lewy body (DLB) into the pig's brain, using minimally invasive surgery and validated against saline injections. In the absence of a suitable α-synuclein radioligand, we validated the model with the unselective amyloid-ß tracer [11C]PIB, which has a high affinity for ß-sheet structures in aggregates. Gadolinium-enhanced MRI confirmed that the blood-brain barrier was intact. A few hours post-injection, pigs were PET scanned with [11C]PIB. Quantification was done with Logan invasive graphical analysis and simplified reference tissue model 2 using the occipital cortex as a reference region. After the scan, we retrieved the brains to confirm successful injection using autoradiography and immunohistochemistry. We found four times higher [11C]PIB uptake in AD-homogenate-injected regions and two times higher uptake in regions injected with α-synuclein-preformed-fibrils compared to saline. The [11C]PIB uptake was the same in non-injected (occipital cortex, cerebellum) and injected (DLB-homogenate, saline) regions. With its large brain and ability to undergo repeated PET scans as well as neurosurgical procedures, the pig provides a robust, cost-effective, and good translational model for assessment of novel radioligands including, but not limited to, proteinopathies.

Acute sleep deprivation upregulates serotonin 2A receptors in the frontal cortex of mice via the immediate early gene Egr3.

Serotonin 2A receptors (5-HT2ARs) mediate the hallucinogenic effects of psychedelic drugs and are a key target of the leading class of medications used to treat psychotic disorders. These findings suggest that dysfunction of 5-HT2ARs may contribute to the symptoms of schizophrenia, a mental illness characterized by perceptual and cognitive disturbances. Indeed, numerous studies have found that 5-HT2ARs are reduced in the brains of individuals with schizophrenia. However, the mechanisms that regulate 5-HT2AR expression remain poorly understood. Here, we show that a physiologic environmental stimulus, sleep deprivation, significantly upregulates 5-HT2AR levels in the mouse frontal cortex in as little as 6-8 h (for mRNA and protein, respectively). This induction requires the activity-dependent immediate early gene transcription factor early growth response 3 (Egr3) as it does not occur in Egr3 deficient (-/-) mice. Using chromatin immunoprecipitation, we show that EGR3 protein binds to the promoter of Htr2a, the gene that encodes the 5-HT2AR, in the frontal cortex in vivo, and drives expression of in vitro reporter constructs via two EGR3 binding sites in the Htr2a promoter. These results suggest that EGR3 directly regulates Htr2a expression, and 5-HT2AR levels, in the frontal cortex in response to physiologic stimuli. Analysis of publicly available post-mortem gene expression data revealed that both EGR3 and HTR2A mRNA are reduced in the prefrontal cortex of schizophrenia patients compared to controls. Together these findings suggest a mechanism by which environmental stimuli alter levels of a brain receptor that may mediate the symptoms, and treatment, of mental illness.

Dorsal striatal dopamine induces fronto-cortical hypoactivity and attenuates anxiety and compulsive behaviors in rats.

Dorsal striatal dopamine transmission engages the cortico-striato-thalamo-cortical (CSTC) circuit, which is implicated in many neuropsychiatric diseases, including obsessive-compulsive disorder (OCD). Yet it is unknown if dorsal striatal dopamine hyperactivity is the cause or consequence of changes elsewhere in the CSTC circuit. Classical pharmacological and neurotoxic manipulations of the CSTC and other brain circuits suffer from various drawbacks related to off-target effects and adaptive changes. Chemogenetics, on the other hand, enables a highly selective targeting of specific neuronal populations within a given circuit. In this study, we developed a chemogenetic method for selective activation of dopamine neurons in the substantia nigra, which innervates the dorsal striatum in the rat. We used this model to investigate effects of targeted dopamine activation on CSTC circuit function, especially in fronto-cortical regions. We found that chemogenetic activation of these neurons increased movement (as expected with increased dopamine release), rearings and time spent in center, while also lower self-grooming. Furthermore, this activation increased prepulse inhibition of the startle response in females. Remarkably, we observed reduced [18F]FDG metabolism in the frontal cortex, following dopamine activation in the dorsal striatum, while total glutamate levels- in this region were increased. This result is in accord with clinical studies of increased [18F]FDG metabolism and lower glutamate levels in similar regions of the brain of people with OCD. Taken together, the present chemogenetic model adds a mechanistic basis with behavioral and translational relevance to prior clinical neuroimaging studies showing deficits in fronto-cortical glucose metabolism across a variety of clinical populations (e.g. addiction, risky decision-making, compulsivity or obesity).

PET/CT imaging detects intestinal inflammation in a mouse model of doxorubicin-induced mucositis.

Introduction: A severe side effect of cancer chemotherapy is the development of gastrointestinal mucositis, characterised by mucosal inflammation. We investigated if 2-deoxy-2-[18F] fluoro-D-glucose positron emission tomography combined with computed tomography (2-[18F]FDG-PET/CT) could visualise gastrointestinal mucositis in mice treated with the chemotherapeutic agent doxorubicin. Methods: In this study, gastrointestinal inflammation was longitudinally evaluated by 2-[18F]FDG-PET/CT scans before and 1, 3, 6, and 10 days after treatment with doxorubicin. Doxorubicin-treated mice were compared to saline-treated littermates using the abdominal standard uptake value of 2-[18F]FDG corrected for body weight (SUVBW). Results: Abdominal SUVBW was significantly increased on day 1 (p < 0.0001), day 3 (p < 0.0001), and day 6 (p < 0.05) in the doxorubicin-treated group compared to controls. Abdominal SUVBW returned to baseline levels on day 10. In the doxorubicin group, the largest weight loss was observed on day 3 (control vs doxorubicin, mean percent of baseline weight: (98.5 ± 3.2% vs 87.9 ± 4.6%, p < 0.0001). Moreover, in the doxorubicin-treated group, villus lengths were decreased by 23-28% on days 1 and 3 in the small intestine (p < 0.05), and jejunal levels of tumour necrosis factor and interleukin-1ß were significantly increased on day 3 (p < 0.05). Discussion: Together, these findings indicate that sequential 2-[18F]FDG-PET/CT scans can objectively quantify and evaluate the development and resolution of intestinal inflammation over time in a mouse model of doxorubicin-induced mucositis.

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.

Molecular and Functional Imaging Studies of Psychedelic Drug Action in Animals and Humans.

Hallucinogens are a loosely defined group of compounds including LSD, N,N-dimethyltryptamines, mescaline, psilocybin/psilocin, and 2,5-dimethoxy-4-methamphetamine (DOM), which can evoke intense visual and emotional experiences. We are witnessing a renaissance of research interest in hallucinogens, driven by increasing awareness of their psychotherapeutic potential. As such, we now present a narrative review of the literature on hallucinogen binding in vitro and ex vivo, and the various molecular imaging studies with positron emission tomography (PET) or single photon emission computer tomography (SPECT). In general, molecular imaging can depict the uptake and binding distribution of labelled hallucinogenic compounds or their congeners in the brain, as was shown in an early PET study with N1-([11C]-methyl)-2-bromo-LSD ([11C]-MBL); displacement with the non-radioactive competitor ketanserin confirmed that the majority of [11C]-MBL specific binding was to serotonin 5-HT2A receptors. However, interactions at serotonin 5HT1A and other classes of receptors and pleotropic effects on second messenger pathways may contribute to the particular experiential phenomenologies of LSD and other hallucinogenic compounds. Other salient aspects of hallucinogen action include permeability to the blood-brain barrier, the rates of metabolism and elimination, and the formation of active metabolites. Despite the maturation of radiochemistry and molecular imaging in recent years, there has been only a handful of PET or SPECT studies of radiolabeled hallucinogens, most recently using the 5-HT2A/2C agonist N-(2[11CH3O]-methoxybenzyl)-2,5-dimethoxy- 4-bromophenethylamine ([11C]Cimbi-36). In addition to PET studies of target engagement at neuroreceptors and transporters, there is a small number of studies on the effects of hallucinogenic compounds on cerebral perfusion ([15O]-water) or metabolism ([18F]-fluorodeoxyglucose/FDG). There remains considerable scope for basic imaging research on the sites of interaction of hallucinogens and their cerebrometabolic effects; we expect that hybrid imaging with PET in conjunction with functional magnetic resonance imaging (fMRI) should provide especially useful for the next phase of this research.

A quantitative method for the selective 5-HT2A agonist 25CN-NBOH in rat plasma and brain.

In recent years, agonists of the 5-HT2A receptor have gained increasing attention for their potential therapeutic use to treat psychological disorders such as anxiety and depression. Here, we report the development and validation of an LC-MSMS based analytical method for the quantification of the novel selective 5-HT2A agonist 25CN-NBOH in rat plasma and brain. As simple and efficient sample clean-up we applied the Phree Phospholipid Removal approach from Phenomenex, which is particularly novel for brain samples. In order to investigate the metabolic stability of 25CN-NBOH in vitro biotransformation studies with recombinant enzymes and human liver microsomes were conducted. Several biotransformation products and pathways could be identified. Based on the in vitro study one of the putative metabolites (2C-CN) was included in the analytical method development. To test the methods applicability 25CN-NBOH was quantified in plasma and brain samples from a pharmacokinetic in vivo study with Wildtype Long Evans rats. Both the in vitro metabolism data as well as the in vivo PK data suggest that 25CN-NBOH is susceptible to metabolism, but is degraded slower and is more stable compared to other NBOMe's investigated to date. The developed analytical method might serve as basis to include further 25CN-NBOH metabolites. It is expected to facilitate further preclinical and clinical investigations of 25CN-NBOH in biological matrices.

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.

Development and Evaluation of Two Potential 5-HT7 Receptor PET Tracers: [18F]ENL09 and [18F]ENL10.

The latest addition to the serotonin (5-HT) receptor family is the 5-HT7 receptor (5-HT7R). This receptor has gained interest as a drug target due to its involvement in various disorders such as depression or schizophrenia. There is currently no clinically validated positron emission tomography (PET) tracer for the 5-HT7R available. But, the (arylpiperazinyl-butyl)oxindole scaffold provides a promising lead structure for this purpose. Here, we synthesized 12 (arylpiperazinyl-butyl)oxindole derivatives and in vitro affinity screening identified two structures with suitable affinity and selectivity to be radiolabeled and tested as 5-HT7R selective PET tracers. Next, the radiolabeled products [18F]ENL09 and [18F]ENL10 were evaluated as PET tracers in rats. Both tracers were found to be P-gp substrates, but after P-gp inhibition the brain uptake showed a regional distribution in line with the known 5-HT7R distribution.  The [18F]ENL10 brain binding was displaceable with a 5-HT7R selective ligand, whereas [18F]ENL09 was not. We find that [18F]ENL10 is a promising 5-HT7R selective PET tracer candidate that should be investigated in higher species.

The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels.

Chemogenetic studies with the ligand clozapine N-oxide (CNO) are predicated upon the assumption that CNO is devoid of actions at natural neuroreceptors. However, recent evidence shows that CNO may be converted back to clozapine (CLZ) in vivo, which could yield plasma concentrations that may be sufficient to occupy inter alia dopamine D2/3 and serotonin 5HT2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D2/3 receptor occupancy with [18F]fallypride PET and serotonin 5HT2A occupancy ex vivo using [18F]MH.MZ. We found a CNO dose-dependent effect on the availability of both neuroreceptor sites. In parallel MR spectroscopy experiments, we found that CNO reduced creatine + phosphcreatine (Cr+PCr) and increased N-acetylaspartate + N-acetylaspartylglutamate (NAA+NAAG) signals in the prefrontal cortex, and also reduced the glutamate signal in dorsal striatum, with peak effect at 2 mg/kg. Thus, our findings suggest that conversion of CNO to CLZ in living rats imparts significant occupancy at endogenous neuroreceptors and significant changes to neurometabolite levels.

Effects of common anesthetic agents on [18F]flumazenil binding to the GABAA receptor.

BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [18F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [18F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood radioactivity concentrations were measured ex vivo by scintillation counting or in vivo by PET in four groups of mice following administration of [18F]flumazenil: awake mice and mice anesthetized with isoflurane, dexmedetomidine, or ketamine/dexmedetomidine. Dynamic PET recordings were obtained for 60 min in mice anesthetized by either isoflurane or ketamine/dexmedetomidine. Static PET recordings were obtained at 25 or 55 min after [18F]flumazenil injection in awake or dexmedetomidine-treated mice acutely anesthetized with isoflurane. The apparent distribution volume (VT*) was calculated for the hippocampus and frontal cortex from either the full dynamic PET scans using an image-derived input function or from a series of ex vivo experiments using whole blood as the input function. RESULTS: PET images showed persistence of high [18F]flumazenil uptake (up to 20 % ID/g) in the brains of mice scanned under isoflurane or ketamine/dexmedetomidine anesthesia, whereas uptake was almost indiscernible in late samples or static scans from awake or dexmedetomidine-treated animals. The steady-state VT* was twofold higher in hippocampus of isoflurane-treated mice and dexmedetomidine-treated mice than in awake mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [18F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.

In vivo assessment of behavioral recovery and circulatory exchange in the peritoneal parabiosis model.

The sharing of circulation between two animals using a surgical procedure known as parabiosis has created a wealth of information towards our understanding of physiology, most recently in the neuroscience arena. The systemic milieu is a complex reservoir of tissues, immune cells, and circulating molecules that is surprisingly not well understood in terms of its communication across organ systems. While the model has been used to probe complex physiological questions for many years, critical parameters of recovery and exchange kinetics remain incompletely characterized, limiting the ability to design experiments and interpret results for complex questions. Here we provide evidence that mice joined by parabiosis gradually recover much physiology relevant to the study of brain function. Specifically, we describe the timecourse for a variety of recovery parameters, including those for general health and metabolism, motor coordination, activity, and sleep behavior. Finally, we describe the kinetics of chimerism for several lymphocyte populations as well as the uptake of small molecules into the brains of mice following parabiosis. Our characterization provides an important resource to those attempting to understand the complex interplay between the immune system and the brain as well as other organ systems.

Further validation to support clinical translation of [(18)F]FTC-146 for imaging sigma-1 receptors.

BACKGROUND: This study aims to further evaluate the specificity and selectivity of [(18)F]FTC-146 and obtain additional data to support its clinical translation. METHODS: The binding of [(19)F]FTC-146 to vesicular acetylcholine transporter (VAChT) was evaluated using [(3)H]vesamicol and PC12(A123.7) cells in an in vitro binding assay. The uptake and kinetics of [(18)F]FTC-146 in S1R-knockout mice (S1R-KO) compared to wild-type (WT) littermates was assessed using dynamic positron emission tomography (PET) imaging. Ex vivo autoradiography and histology were conducted using a separate cohort of S1R-KO/WT mice, and radiation dosimetry was calculated from WT mouse data (extrapolated for human dosing). Toxicity studies in Sprague-Dawley rats were performed with a dose equivalent to 250× the anticipated clinical dose of [(19)F]FTC-146 mass. RESULTS AND DISCUSSION: VAChT binding assay results verified that [(19)F]FTC-146 displays negligible affinity for VAChT (K i = 450 ± 80 nM) compared to S1R. PET images demonstrated significantly higher tracer uptake in WT vs. S1R-KO brain (4.57 ± 1.07 vs. 1.34 ± 0.4 %ID/g at 20-25 min, n = 4, p < 0.05). In S1R-KO mice, it was shown that rapid brain uptake and clearance 10 min post-injection, which are consistent with previous S1R-blocking studies in mice. Three- to fourfold higher tracer uptake was observed in WT relative to S1R-KO mouse brains by ex vivo autoradiography. S1R staining coincided well with the autoradiographic data in all examined brain regions (r (2) = 0.85-0.95). Biodistribution results further demonstrated high [(18)F]FTC-146 accumulation in WT relative to KO mouse brain and provided quantitative information concerning tracer uptake in S1R-rich organs (e.g., heart, lung, pancreas) for WT mice vs. age-matched S1R-KO mice. The maximum allowed dose per scan in humans as extrapolated from mouse dosimetry was 33.19 mCi (1228.03 MBq). No significant toxicity was observed even at a 250X dose of the maximum carrier mass [(19)F]FTC-146 expected to be injected for human studies. CONCLUSIONS: Together, these data indicate that [(18)F]FTC-146 binds specifically to S1Rs and is a highly promising radiotracer ready for clinical translation to investigate S1R-related diseases.

Semiconducting Polymer Nanoparticles with Persistent Near-Infrared Luminescence for In Vivo Optical Imaging.

Materials with persistent luminescence are attractive for in vivo optical imaging since they have a long lifetime that allows the separation of excitation of fluorophores and image acquisition for time-delay imaging, thus eliminating tissue autofluorescence associated with fluorescence imaging. Persistently luminescent nanoparticles have previously been fabricated from toxic rare-earth metals. This work reports that nanoparticles made of the conjugated polymer MEH-PPV can generate luminescence persisting for an hour upon single excitation. A near-infrared dye was encapsulated in the conjugated polymer nanoparticle to successfully generate persistent near-infrared luminescence through resonance energy transfer. This new persistent luminescence nanoparticles have been demonstrated for optical imaging applications in living mice.