The Chronic Treatment With 5-HT2A Receptor Agonists Affects the Behavior and the BDNF System in Mice.

Serotonin 5-HT2A receptors and the brain-derived neurotrophic factor (BDNF) are involved in the pathophysiology and treatment of many psychiatric diseases. However, the interaction between 5-HT2A and BDNF is still poorly understood. In the present paper, the effects of chronic treatment with mixed 5-HT2A/2C receptor agonist DOI, highly selective 5-HT2A agonists TCB-2 and 25CN-NBOH on behavior and the BDNF system have been investigated. Chronic treatment of males of C57Bl/6 mice with DOI, TCB-2 and 25CN-NBOH (1 mg/kg, i.p., 14 days) resulted in desensitization of 5-HT2A receptors. Treatment with 25CN-NBOH significantly increased startle amplitude. At the same time all used drugs failed to affect anxiety, exploratory and stereotyped behavior as well as spatial memory and learning. TCB-2 and 25CN-NBOH increased the BDNF mRNA level. All 5-HT2A agonists increased the proBDNF level but failed to alter the mature BDNF protein level. TrkB and p75NTR mRNA levels were affected by all utilized agonists. All drugs decreased the total level as well as membrane TrkB protein one indicating downregulation of TrkB receptors. All agonists decreased the membrane p75NTR protein level. Thus, we have shown for the first time that the chronic activation of the 5-HT2A receptor with agonists has affected the BDNF system almost on all levels-transcription, proBDNF production, TrkB and p75NTR receptors' level. The obtained data suggested possible suppression in BDNF-TrkB signaling under chronic treatment with 5-HT2A agonists.

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.

Acute Lysergic Acid Diethylamide Does Not Influence Reward-Driven Decision Making of C57BL/6 Mice in the Iowa Gambling Task.

While interest in psychedelic drugs in the fields of psychiatry and neuroscience has re-emerged in recent last decades, the general understanding of the effects of these drugs remains deficient. In particular, there are gaps in knowledge on executive functions and goal-directed behaviors both in humans and in commonly used animal models. The effects of acute doses of psychedelic lysergic acid diethylamide (LSD) on reward-driven decision making were explored using the mouse version of the Iowa Gambling Task. A total of 15 mice were trained to perform in a touch-screen adaptation of the rodent version of the Iowa Gambling Task, after which single acute doses of LSD (0.025, 0.1, 0.2, 0.4 mg/kg), serotonin 2A receptor-selective agonist 25CN-NBOH (1.5 mg/kg), d-amphetamine (2.0 mg/kg), and saline were administered before the trial. 25CN-NBOH and the three lowest doses of LSD showed no statistically significant changes in option selection or in general functioning during the gambling task trials. The highest dose of LSD (0.4 mg/kg) significantly decreased premature responding and increased the omission rate, but had no effect on option selection in comparison with the saline control. Amphetamine significantly decreased the correct responses and premature responding while increasing the omission rate. In conclusion, mice can perform previously learned, reward-driven decision-making tasks while under the acute influence of LSD at a commonly used dose range.

The serotonin 2A receptor agonist 25CN-NBOH increases murine heart rate and neck-arterial blood flow in a temperature-dependent manner.

BACKGROUND: Serotonin 2A receptors, the molecular target of psychedelics, are expressed by neuronal and vascular cells, both of which might contribute to brain haemodynamic characteristics for the psychedelic state. AIM: Aiming for a systemic understanding of psychedelic vasoactivity, here we investigated the effect of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine - a new-generation agonist with superior serotonin 2A receptor selectivity - on brain-supplying neck-arterial blood flow. METHODS: We recorded core body temperature and employed non-invasive, collar-sensor based pulse oximetry in anesthetised mice to extract parameters of local blood perfusion, oxygen saturation, heart and respiration rate. Hypothesising an overlap between serotonergic pulse- and thermoregulation, recordings were done under physiological and elevated pad temperatures. RESULTS: N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (1.5 mg/kg, subcutaneous) significantly increased the frequency of heart beats accompanied by a slight elevation of neck-arterial blood flow. Increasing the animal-supporting heat-pad temperature from 37°C to 41°C enhanced the drug's effect on blood flow while counteracting tachycardia. Additionally, N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine promoted bradypnea, which, like tachycardia, quickly reversed at the elevated pad temperature. The interrelatedness of N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine's respiro-cardiovascular effects and thermoregulation was further corroborated by the drug selectively increasing the core body temperature at the elevated pad temperature. Arterial oxygen saturation was not affected by N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine at either temperature. CONCLUSIONS: Our findings imply that selective serotonin 2A receptor activation modulates systemic cardiovascular functioning in orchestration with thermoregulation and with immediate relevance to brain-imminent neck (most likely carotid) arteries. As carotid branching is a critical last hub to channel cardiovascular output to or away from the brain, our results might have implications for the brain haemodynamics associated with psychedelia.

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.

Tolerance and Tachyphylaxis to Head Twitches Induced by the 5-HT2A Agonist 25CN-NBOH in Mice.

The serotonin (5-HT) 2A receptor is the primary molecular target of serotonergic hallucinogens, which trigger large-scale perturbations of the cortex. Our understanding of how 5-HT2A activation may cause the effects of hallucinogens has been hampered by the receptor unselectivity of most of the drugs of this class. Here we used 25CN-NBOH (N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine), a newly developed selective 5-HT2A agonist, and tested it with regard to the head-twitch-response (HTR) model of 5-HT2A activity and effects on locomotion. 25CN-NBOH evoked HTRs with an inverted u-shape-like dose-response curve and highest efficacy at 1.5 mg/kg, i.p. HTR occurrence peaked within 5 min after agonist injection, and exponentially decreased to half-maximal frequency at ~11 min. Thorough habituation to the experimental procedures (including handling, saline injection, and exposure to the observational boxes 1 day before the experiment) facilitated the animals' response to 25CN-NBOH. 25CN-NBOH (1.5 mg/kg, i.p.) induced HTRs were blocked by the 5-HT2A antagonist ketanserin (0.75 mg/kg, 30 min pre), but not by the 5-HT2C antagonist SB-242084 (0.5 mg/kg, i.p., 30 min pre). SB-242084 instead slightly increased the number of HTRs occurring at a 3.0-mg/kg dose of the agonist. Apart from HTR induction, 25CN-NBOH also modestly increased locomotor activity of the mice. Repeated once-per-day injections (1.5 mg/kg, i.p.) led to reduced occurrence of 25CN-NBOH induced HTRs. This intermediate tolerance was augmented when a second (higher) dose of the drug (3.0 mg/kg) was interspersed. Short-interval tolerance (i.e., tachyphylaxis) was observed when the drug was injected twice at intervals of 1.0 and 1.5 h at either dose tested (1.5 mg/kg and 0.75 mg/kg, respectively). Inducing ketanserin-sensitive HTRs, which are dependent on environmental valences and which show signs of tachyphylaxis and tolerance, 25CN-NBOH shares striking features common to serotonergic hallucinogens. Given its distinct in vitro selectivity for 5-HT2A over non5-HT2 receptors and its behavioral dynamics, 25CN-NBOH appears to be a powerful tool for dissection of receptor-specific cortical circuit dynamics, including 5-HT2A related psychoactivity.

Effect of Hallucinogens on Unconditioned Behavior.

Because of the ethical and regulatory hurdles associated with human studies, much of what is known about the psychopharmacology of hallucinogens has been derived from animal models. However, developing reliable animal models has proven to be a challenging task due to the complexity and variability of hallucinogen effects in humans. This chapter focuses on three animal models that are frequently used to test the effects of hallucinogens on unconditioned behavior: head twitch response (HTR), prepulse inhibition of startle (PPI), and exploratory behavior. The HTR has demonstrated considerable utility in the neurochemical actions of hallucinogens. However, the latter two models have clearer conceptual bridges to human phenomenology. Consistent with the known mechanism of action of hallucinogens in humans, the behavioral effects of hallucinogens in rodents are mediated primarily by activation of 5-HT2A receptors. There is evidence, however, that other receptors may play secondary roles. The structure-activity relationships (SAR) of hallucinogens are reviewed in relation to each model, with a focus on the HTR in rats and mice.