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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.
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published: 06 February 2018
doi: 10.3389/fphar.2018.00017
Frontiers in Pharmacology | 1February 2018 | Volume 9 | Article 17
Edited by:
Andrew Robert Gallimore,
Okinawa Institute of Science and
Technology, Japan
Reviewed by:
A Elizabeth Linder,
Universidade Federal de Santa
Catarina, Brazil
Alfredo Meneses,
Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico
Nacional (CINVESTAV-IPN), Mexico
Thomas Knöpfel
These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
a section of the journal
Frontiers in Pharmacology
Received: 19 October 2017
Accepted: 08 January 2018
Published: 06 February 2018
Buchborn T, Lyons T and Knöpfel T
(2018) Tolerance and Tachyphylaxis to
Head Twitches Induced by the
5-HT2A Agonist 25CN-NBOH in Mice.
Front. Pharmacol. 9:17.
doi: 10.3389/fphar.2018.00017
Tolerance and Tachyphylaxis to Head
Twitches Induced by the 5-HT2A
Agonist 25CN-NBOH in Mice
Tobias Buchborn 1†, Taylor Lyons 1† and Thomas Knöpfel 1,2
1Laboratory for Neuronal Circuit Dynamics, Department of Medicine, Imperial College London, London, United Kingdom,
2Centre for Neurotechnology, Institute of Biomedical Engineering, Imperial College London, London, United Kingdom
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-HT2receptors 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.
Keywords: serotonergic hallucinogen, 25CN-NBOH, 5-HT2A receptor, 5-HT2C receptor, head twitch response,
tachyphylaxis, tolerance, locomotion
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
The serotonin (5-HT) 2A receptor is a member of the 5-
HT2seven-transmembrane receptor family and is expressed
in various cells and tissues across the mammalian body, with
highest expression levels in the brain (see GPCR database;
Regard et al., 2008). 5-HT2A receptors are specifically enriched
in the cerebral cortex, particularly on the apical dendrites of
pyramidal cells in layer V (Weber and Andrade, 2010). By
enhancing cortical glutamate release, 5-HT2A receptors may
modulate cognitive processes, such as working memory and
attention (Williams et al., 2002; Mirjana et al., 2004). The
5-HT2A receptor is considered as an important drug target,
with potential implications of both agonists and antagonists
in the treatment of various psychiatric conditions, including
depression and anxiety (Mascher, 1967; Quesseveur et al., 2012;
Buchborn et al., 2014; Carhart-Harris et al., 2016; Ross et al.,
2016; Carhart-Harris and Goodwin, 2017). Blockade of 5-
HT2A receptors has been shown to counteract alterations of
consciousness induced by serotonergic hallucinogens such as
lysergic acid diethylamide (LSD) (Kraehenmann et al., 2017),
and more specifically, 5-HT2A receptors of layer 5 pyramidal
cells are thought to be a key mediator of psychedelic activity
(Vollenweider and Kometer, 2010; Muthukumaraswamy et al.,
2013; Nichols, 2016). Despite the availability of relatively selective
5-HT2A antagonists (see PDSP Ki database; Roth et al., 2000),
our understanding of the general neurophysiology of (cortical) 5-
HT2A receptors is limited by the lack of highly selective 5-HT2A
agonists. The 4-iodo-2,5-dimethoxy-analog of amphetamine
DOI, for instance, which often has been the drug of choice in
animal studies related to 5-HT2A functions, seems to exhibit
confounded affinity for 5-HT2as well as adrenergic receptors
(Ray, 2010). In search of agonists with higher 5-HT2A selectivity,
a series of 48 compounds was recently developed based on
the phenethylamine backbone shared by DOI-like serotonergic
hallucinogens (Hansen et al., 2014). Amongst these, as indicated
by an extensive follow-up in vitro receptor binding screening,
the newly designed N-(2-hydroxy)benzyl substituted 4-cyano-
2,5-dimethoxyphenethylamine (25CN-NBOH) turned out to
be one of the most promising candidates: 25CN-NBOH is
characterized by a high affinity for 5-HT2A receptors (in vitro
radioligand competition binding, av. Ki: 1.72 nM), by a
100-fold selectivity for binding to this receptor as compared
to a plethora of non-5-HT2targets (including other G-protein
coupled receptors, ion channels, transporters, and enzymes),
and robust 5-HT2A selectivity over 5-HT2B/2C receptors (Ki
in vitro [averaged across studies]: 56 nM for 5-HT2B and
83 nM for 5-HT2C , respectively) (Hansen, 2010; Halberstadt
et al., 2016; Jensen et al., 2017). Therefore, 25CN-NBOH
appears advantageous over other currently used 5-HT2A agonists
subjected to a similarly scrutinous receptor profiling (Ray, 2010;
see also Ki database).
Here, we aimed to determine the suitability of 25CN-NBOH
for prospective brain imaging studies in chronic in vivo mouse
models. To this end, we employed the head twitch response
(HTR) as well as locomotion as a behavioral readout of central
5-HT2A activity. We determined the dose range of 25CN-NBOH
required for parenteral administration to induce central 5-HT2A
activation, the time-course of the central action, sensitivity to
5-HT2A antagonism and experimental familiarity, a possible
involvement of 5-HT2C receptors in 25CN-NBOH induced
HTRs, as well as potential development of tachyphylaxis and
All experimental procedures performed at Imperial College
London were in accordance with the UK Animal Scientific
Procedures Act (1986) under Home Office Personal and Project
licenses (I5B5A6029, IA615553C; PPL 70/7818), following
appropriate ethical review.
Adult mice of both sexes and with mixed genetic background
(stock of mainly C57BL/6JxB6CBAF1 background) were bred
in-house at the Central Biomedical Services (CBS) of Imperial
College London. They were housed in individually ventilated
cages with a 12:12 h light/dark cycle and maintained at an
ambient temperature of 21 ±2C at 55 ±10% humidity. Mice
were provided with standard rodent-chow pellets (Special Diet
Services, #RM1) and water ad libitum.
25CN-NBOH (a kind gift from Jesper L. Kristensen, University
of Copenhagen) and ketanserin (Tocris Biosciences; Avonmouth,
UK) were dissolved in isotonic saline, SB-242084 (Tocris
Biosciences; Avonmouth, UK) in DMSO (10% in saline). Drugs
were applied intraperitoneally or subcutaneously (<10 ml/kg), as
Behavioral Experiments
Animals were allowed to habituate to the (observational) non-
home boxes (25.5 ×12.5 ×12.5 cm [L ×W×H]) for 30 min.
Observational boxes were shielded with red acrylic plates to
minimize the animals’ visual awareness of the experimenter.
Head Twitches
For quantification of HTRs, animals were treated with saline or
the respective dose of 25CN-NBOH (0.5–3.0 mg/kg, i.p. (or 1.5
mg/kg, s.c.]) and obser ved for 20–60 min. The frequency of head
twitches, defined as rapid and brisk rotational movement of the
head around the longitudinal axis of the animals’ body (Handley
and Singh, 1986) (see supplemental Movie), was counted in 2-
or 5-min bins starting directly after injection. The sensitivity
of 25CN-NBOH (1.5 mg/kg [vs. 3.0] mg/kg, i.p.) induced head
twitches to 5-HT2A and/or 5-HT2C antagonism was tested
by pre-treating the animals with ketanserin (0.75 mg/kg, i.p.)
and SB-242084 (0.5 mg/kg, i.p.), respectively, 30 min prior to
agonist administration. The impact of experimental familiarity
was investigated by simulation of aspects of the experimental
procedure (i.e., handling, injection of saline, and exposure to the
observational non-home cages for 30 min before, and for 20 min
after saline injection) 1 day before the actual 25CN-NBOH
Frontiers in Pharmacology | 2February 2018 | Volume 9 | Article 17
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
Tachyphylaxis, defined as in-between-session tolerance
occurring at repeated short-interval application, was tested by
injecting saline or 25CN-NBOH (0.75 or 1.5 mg/kg, i.p.) twice
at a 1.0- or 1.5-h interval, and comparing the average HTR
frequency in each 30-min observation period. For subchronic
tolerance, animals were treated for 4 days with saline or
25CN-NBOH. 25CN-NBOH was injected once (1.5 mg/kg, i.p.
[morning]) or twice (1.5 mg/kg, i.p. [morning] and 3.0 mg/kg,
i.p. [evening of days 1–3]) per day and HTRs were counted
for 20 min after each morning application. The twice-per-
day regimen, with the increased dose (i.e., 3.0 mg/kg) in the
evening, was chosen based on findings for LSD, which indicate
that tolerance development is slightly facilitated by a higher
frequency of application, but more strongly facilitated by a
higher frequency of application with increased dosing every
other application (Buchborn et al., 2016).
Locomotion was quantified for 90 min directly after saline or
25CN-NBOH (1.5 mg/kg, i.p.) injection, averaged across three
30-min bins, and analyzed for distance traveled (in m), mobility
time (in s), and average speed (in m/s) using Any-Maze Video
Tracking Software (Stoelting). Mobility refers to times animals
showed locomotion or rearing (excluding freezing and stationary
grooming); speed was calculated as distance traveled against the
cumulative mobility time periods.
The data from the dose-response, antagonist, and pre-
habituation experiments were analyzed using non-parametric
Kruskal-Wallis test with Dunn’s multiple post-hoc comparisons,
or Mann-Whitney U-testing (as a priori planned). Tolerance
(HTR) and time-course data (HTR and locomotion) were
subjected to a two- and three-factor ANOVA with repeated
measures on one factor, respectively, and followed up on by
Bonferroni-corrected multiple comparisons. For HTRs, time-
course data of interest were fitted by non-linear regression
analysis, and the decay constant provided by the best-fit
exponential equation (HTRt=HTR
0elambdat) subsequently
used for determination of 25CN-NBOH’s half-life (t½ =
ln[2]/lambda). Calculations were carried out by SPSS or
GraphPad Prism software. Statistical significance was assumed if
the null hypothesis (e.g., drug has no action) could be rejected at
0. 05 probability level.
Dose-Response Relationship,
Ketanserin-Sensitivity, and Effect of
Experimental Familiarity on 25CN-NBOH
Induced Head Twitches
Mice received a single injection of either saline (control) or
25CN-NBOH (0.5–3.0 mg/kg, [i.p. or s.c.]) and were monitored
for HTRs during the following 20 min. While control animals
in this observation period rarely showed any behaviors rated
as HTRs [0.83 ±0.31 (mean ±SEM)], the incidence of
HTRs markedly increased for all tested doses of 25CN-NBOH.
Frequencies (mean ±SEM) ranged from 13.67 ±4.26 for the
lowest dose (0.5 mg/kg, i.p.), 37.67 ±6.04 to 40.67 ±5.77 for
the medium dose (1.5 mg/kg, i.p. vs. s.c.), and 27.60 ±4.52 for
the highest dose tested (3.0 mg/kg, i.p.; Figures 1A,B). Inference-
statistically, however, only the 1.5-mg/kg dose significantly
differed from the saline group (X2[10, N=62] =31.49, p
0.001; Dunn’s post-hoc [comparison to control], 1.5 mg/kg i.p. or
s.c., each p0.01). At none of the doses tested, stereotypies other
than HTRs (including any symptoms of the serotonin syndrome)
were noted.
The 5-HT2A antagonist ketanserin (0.75 mg/kg, i.p.), when
applied 30 min before the agonist, near-completely prevented the
25CN-NBOH (1.5-mg/kg, i.p.) induced HTRs (mean ±SEM:
2.0 ±1.29) (X2[4, N=30] =14.47, p0.01; Dunn’s post-
hoc [comparison to 1.5 NBOH w/o antagonist and to control,
respectively], p0.05 and n.s.). The selective 5-HT2C antagonist
SB-242084 (0.5 mg/kg, i.p., 30 min pre-treatment), did not
prevent 25CN-NBOH from inducing significant HTRs (mean ±
SEM: 31.50 ±8.25; Dunn’s post-hoc (comparison to saline), p
0.05]. SB-242084, although not affecting the effect of 1.5-mg/kg
25CN-NBOH, increased the animals’ response to the 3.0-mg/kg
dose of the agonist, leading to HTRs of significant extent (mean
±SEM: 36.20 ±7.02, p0.05; Figure 1B).
Handling the animals and thoroughly habituating them to the
experimental procedures (including exposure to observational
boxes and saline injection 1 day before the 25CN-NBOH
experiment), respectively, facilitated agonist induced HTRs; a
0.75-mg/kg sub-threshold dose was enabled to induce significant
twitching (mean ±SEM: 33.0 ±5.28; p0.01) and the response
to a 1.5-mg/kg dose was significantly increased (mean ±SEM:
61.5 ±6.3; p0.05; Figure 1A).
Time-Course of 25CN-NBOH Induced Head
To investigate the time-course of the behavioral effect of the
different 25CN-NBOH doses, we counted HTRs in 5-min bins
as they occurred over 30 min post-injection (Figure 2). Repeated
measures ANOVA with Bonferroni-corrected post-hoc analysis
revealed significant increases in HTR frequency as a function of
time [F(1.84, 27.66) =57.52, p0.001], treatment [F(2, 15) =30.41,
p0.001], as well as of time ×treatment interaction [F(3.68, 27.66)
=26.52, p0.001]. All but the 0.5- and the 1.0-mg/kg dose
elicited significant HTRs in the first observation window (0–
5 min), where they peaked (1.5 mg/kg: 14.33 ±2.67, p0.001;
2.0 mg/kg: 9.67 ±2.6, p0.05; 2.5 mg/kg: 11.17 ±1.68, p0.01)
and then steadily decreased over time. As depicted in the inset of
Figure 2, the temporal course of 25CN-NBOH (1.5 mg/kg, i.p.)
induced HTRs is well-fitted by an exponential decay function,
with the effect of the drug declining to half after 11 min. The 1.0-,
2.0-, and 2.5-mg/kg doses decayed with similar half-lives between
around 9 and 12 min.
In an additional set of experiments, we studied the time-
course of the effect produced by the 1.5-mg/kg dose over a longer
observation time period. To this end, 25CN-NBOH induced
head twitches (i.p. vs. s.c.) were counted over 60 min at a
Frontiers in Pharmacology | 3February 2018 | Volume 9 | Article 17
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
FIGURE 1 | 25CN-NBOH induced head twitches (HTRs) (as observed for the first 20 min post-application). (A) Dose–response relation (0.5–2.5 mg/kg i.p. vs. 1.5
mg/kg s.c.) and effect of experimental habituation (0.75 vs. 1.5 mg/kg NBOH i.p. +H). (B) Effect of the 5-HT2A antagonist ketanserin [1.5 mg/kg NBOH +0.75
mg/kg K (30 min pre-treatment), i.p.] and the 5-HT2C antagonist SB-242084 [1.5 vs. 3.0 mg/kg NBOH +0.5 mg/kg SB (30 min pre-treatment), i.p.], respectively.
Mean +SEM; n=4–6 per group. Kruskal-Wallis, post-hoc comparison to saline, *p05 and **p01; or to agonist w/o habituation and w/o antagonist
pre-treatment, respectively, #p.05.
FIGURE 2 | Time-course of 25CN-NBOH (0.5–2.5 mg/kg, i.p.) induced head
twitches (HTRs) (as observed for the first 30 min post-application). Mean ±
SEM; n=6 per group. The inset replots the means of the 1.5-mg/kg dose as
fitted by an exponential decay function.
2-min-observation resolution. After i.p. injection, the frequency
of HTRs peaked within the first 2-min bin (mean ±SEM: 6.8 ±
1.32) and then decayed with a t½ of 8.56 min. For s.c. injection,
the peak occurred with a slight delay in the second 2-min bin
(mean ±SEM: 7.00 ±0.71) and decayed at a of 13.59 min.
Overall, as indicated by repeated measures ANOVA, there were
no route-dependent time-course differences. The average (±
SEM) of the three half-lives calculated for the 1.5-mg/kg dose is
11.05 ±1.45.
25CN-NBOH’s Effect on Locomotor Activity
To investigate how the dose of 25CN-NBOH that is the most
effective in the head-twitch model (i.e., 1.5 mg/kg) would affect
the animals’ overall behavior, we also monitored locomotion.
Splitting the 90-min observational period into three bins of
30 min, we found significant main effects for locomotion [F(1, 14)
=571.22, p0.001], treatment [F(1, 14) =4.62, p0.05], time
[F(2, 28) =140.97, p0.001], as well as significant interactions in
terms of locomotion ×treatment [F(1, 14) =5.44, p0.05] and
locomotion ×time [F(1.71, 23.98) =111.17, p0.001]. Averaged
across the three time-bins, 25CN-NBOH treated animals were
more mobile [mean ±SEM (% of 30 min): 18.23 ±2.75 (saline)
vs. 26.93 ±2.50 (NBOH), post-hoc p 0.05] and traveled a
significantly greater distance [mean ±SEM (m per 30 min): 10.17
±1.45 (saline) vs. 15.07 ±1.75 (NBOH), post-hoc p 0.05]
than saline treated mice. However, both treated and untreated
mice were more often immobile than mobile (see Figures 3A,B).
Also, for the times of mobility, there was no significant difference
in average speed [mean ±SEM (m/s per 30 min): 0.029 ±
0.002 (saline) 0.028 ±0.002 (NBOH), post-hoc, n.s.] (Figure 3C).
Following up on the locomotion ×time interaction, both groups
showed a constant decrease in the locomotion parameter distance
traveled (mean ±SEM [m]: 21.56 ±2.11, 7.48 ±2.31, and
1.48 ±0.51 [saline]; 30.62 ±2.82, 9.67 ±1.94, and 4.91 ±
1.37 [NBOH]) and mobility time (mean ±SEM [% of 30 min]:
37.86 ±3.18, 14.12 ±4.75, and 2.74 ±1.03 [saline]; 50.46
±3.69, 18.04 ±3.43, and 12.29 ±2.98 [NBOH]) from the
first, to the second, to the third interval (Figures 3A,B). For
either parameter, the differences between the three factor levels
of time across groups became significant (post-hoc, each p
0.05). However, no significant time-dependency could be seen for
Frontiers in Pharmacology | 4February 2018 | Volume 9 | Article 17
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
FIGURE 3 | Effect of 25CN-NBOH (1.5 mg/kg, i.p.) on locomotion. Three 30-min intervals for the first 90 min after injection. (A) Fractional mobility (in % of 30 min);
(B) distance traveled (in m per 30 min); (C) average speed (in m/s per 30 min). Mean +SEM; n=8 per group. Repeated measures ANOVA, Bonferroni-corrected
across-time post-hoc comparison to control, *p05.
average speed (mean ±SEM [m/s per 30 min]: 0.031 ±0.001,
0.030 ±0.002, and 0.024 ±0.006 [saline]; 0.033 ±0.001, 0.028 ±
0.002, and 0.023 ±0.003 [NBOH]) (post-hoc across groups, n.s.)
(Figure 3C).
Tachyphylaxis and Tolerance to
25CN-NBOH Induced Head Twitches
In an independent set of experiments, we explored whether
25CN-NBOH induced HTRs showed signs of short-term
tolerance. We therefore repeatedly applied the drug at different
intervals. Similarly, as reported above, 25CN-NBOH (1.5 mg/kg,
s.c.) treated mice exhibited an average of 34.67 ±4.65 HTRs
within 30 min, which was significantly more than observed in
saline treated mice (mean ±SEM: 1.4 ±0.25) (p.01). Re-
applying the same dose of 25CN-NBOH one or 1½ h after the
first application substantially reduced the efficacy of the drug.
Main effects for time [F(1, 24) =64.28, p0.001], group [F(1, 19)
=62.1, p0.001], and time ×group interaction [F(4, 24) =10.47,
p0.001] proved significant. Whereas saline treated animals
at both intervals, did not show significant differences from the
first saline application (not shown), 25CN-NBOH treated mice—
with only 24.98 ±4.54% (1 h) and 19.48 ±4.59% (1.5 h) of the
original responsiveness—twitched significantly less (first vs. resp.
second injection, post-hoc each p0.01; Figure 4). As shown
for the 1.5-h interval, a reduced but still highly significant loss of
drug efficacy could likewise be demonstrated when 25CN-NBOH
was injected twice at a lower dose (0.75 mg/kg, s.c.) to handling-
habituated mice (mean ±SEM [% of the original responsiveness]:
30.66 ±5.86; first vs. second injection, p0.01) (Figure 4).
In addition to tachyphylaxis occurring at repeated short-
interval injection, a substantial loss of responsiveness to 25CN-
NBOH could also be demonstrated when the drug (1.5 mg/kg,
i.p.) was repeatedly applied at 1-day intervals [main effect time:
F(1.84, 27.66) =57.51, p0.001]; main effect group [F(2, 15) =
30.41, p0.001]; time ×group interaction [F(3.68, 27.66) =26.52,
p0.001] (Figure 5). Unlike with the short-interval application,
however, the decrease in HTRs only with the third injection of
25CN-NBOH (i.e. on the 3rd and 4th day) became significant [1x
NBOH per day: Day 1 (mean ±SEM: 37.67 ±5.99) vs. day 3 and
day 4 [56.19 and 54.87% of day 1, respectively], each p0.01].
When a second (higher) dose of the agonist (3.0 mg/kg, i.p.) was
applied in the evening of the first 3 days, the onset of tolerance
proved significant by day 2 already [2x NBOH per day: Day 1
(mean ±SEM: 61.5 ±6.34) vs. day 2, day 3, and day 4 (34.15,
18.69, and 17.88% of day 1, respectively), each p0.001]. Also,
the extent of tolerance was more pronounced. Whereas the HTRs
exhibited by the once-per-day-application mice on day 4 still
were statistically different from the saline-treated control animals
(mean ±SEM: 20.67 ±3.49, p0.001), those in the twice-per-
day mice more closely approached the control level (mean ±
SEM: 11.00 ±1.73, n.s.) (Figure 5).
We characterized the selective 5-HT2A agonist 25CN-NBOH in
terms of HTRs, a behavioral output suggested to engage (Willins
and Meltzer, 1997; González-Maeso et al., 2007), predict and/or
model 5-HT2A receptor function in the cortex (Goodwin et al.,
1984; Zhang and Marek, 2008; e.g., Buchborn et al., 2015).
25CN-NBOH evoked marked HTRs in mice at the full
range of doses tested (i.e., 0.5–3.0 mg/kg). In the context of
the multiple-comparisons analysis, however, only the medium
dose (1.5 mg/kg) turned out significant, which mirrors previous
findings of the drug being most active at around 1.0–3.0 mg/kg
(Fantegrossi et al., 2015; Halberstadt et al., 2016). The inverted
u-shape-like dose-response curve of 25CN-NBOH (with higher
doses entailing a relative loss of responsiveness) might be due
to increasing occupancy of 5-HT2C receptors (whose activity
may counteract the 5-HT2A–mediated HTR induction), and/or
due to 5-HT2receptors requiring a tuned level of occupancy
(compare for DOM, DOB, and DOI: Fantegrossi et al., 2010;
Serafine and France, 2014; Buchborn et al., 2015). Consistent
with its sensitivity to MDL100907 (Fantegrossi et al., 2015),
one of the most selective 5-HT2A antagonists at present (av. Ki
[Ki database]: 1.92 nM for r5-HT2A;112 nM for r5-HT2C),
25CN-NBOH (1.5 mg/kg, i.p.) induced HTRs were blocked
by the 5-HT2A antagonist ketanserin (av. Ki [Ki database]:
1.6 nM for r5-HT2A;52 nM for r5-HT2C ). The selective 5-
HT2C antagonist SB-242084 (av. Ki [Ki database]: 2.85 for h5-
HT2C;505 nM for h5-HT2A), on the other hand, did not affect
HTRs evoked by a 1.5-mg/kg dose of 25CN-NBOH. Interstingly,
it slightly increased the behavioral effect of the higher 3.0-mg/kg
dose of the agonist. Thus, it might be suggested that low to
medium doses of 25CN-NBOH primarily interact with 5-HT2A
Frontiers in Pharmacology | 5February 2018 | Volume 9 | Article 17
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
FIGURE 4 | Tachyphylaxis of 25CN-NBOH (0.75 or 1.5 mg/kg, s.c.) induced
head twitches (HTRs) (as observed for the first 30 min post-application).
Agonist was applied twice at an interval of 60 and 90 min, respectively. Mean
+SEM; n=5–6 per group. Repeated measures ANOVA, Bonferroni-corrected
post-hoc comparison to respective first application (0 min), ##p01.
receptors; but higher doses of 25CN-NBOH may also recruit
5-HT2C receptors, thereby interfering with the actions mediated
by 5-HT2A receptors (compare for DOI: Fantegrossi et al., 2010).
The finding that 3.0 mg/kg 25CN-NBOH given to mice reach
brain concentrations that fall into the range of the agonist’s in
vitro affinity for 5-HT2C receptors (Jensen et al., 2017), would be
in line with such an interpretation.
25CN-NBOH induced HTRs were facilitated when animals
had been thoroughly habituated to the experimental procedures
on the day before the drug application. The latter finding
supports observations made in rats, where the presence of
familiar littermates allowed hallucinogens to evoke substantially
more HTRs than when observed in isolation (Buchborn et al.,
2015). The dependence of 5-HT2A related behavior in rodents
on what may be described as state-environment interaction,
mirrors characteristics of human psychedelia, which is critically
determined by set and setting, i.e., psychological constitution and
environmental circumstances (rev. Hartogsohn, 2016).
25CN-NBOH induced HTRs occurred within <1 min after
i.p. injection, reached maximal frequencies within 2–5 min, and
declined to half-maximal at 11 min (compare for 25I-NBOMe
and 25I-NBMD: Halberstadt and Geyer, 2014). In contrast to the
i.p. route, s.c. administration—as shown for the 1.5-mg/kg dose—
was associated with a slight delay in peak and somewhat longer
duration to half-maximum. 25CN-NBOH, both in plasma and
brain, reaches maximum concentrations within 15 min, where
it remains relatively stable until at least 30 min post-application
(Jensen et al., 2017). The fast decline in the behavioral response
observed here therefore does not seem to be a consequence of
fast drug clearance. One possible explanation for the apparent
disconnect between 25CN-NBOH’s brain concentration and
behavioral output is the development of acute tolerance. Indeed,
DOI which shares the phenethylamine backbone of 25CN-
NBOH, internalizes and desensitizes 5-HT2A receptors within
15–20 min in vitro (Porter et al., 2001; Raote et al., 2013).
To follow up on the possibility of tolerance development,
we applied 25CN-NBOH (1.5 mg/kg) twice at a 1.0- or a 1.5-h
FIGURE 5 | Subchronic tolerance to 25CN-NBOH induced head twitches
(HTRs), with a total of four [1xNBOH: Once per day, 1.5 mg/kg (morning)] and
seven [2xNBOH: Twice per day, 1.5 mg/kg (morning) vs. 3.0 mg/kg (evening)]
i.p. injections, respectively. Head twitches were counted for the first 20 min
after the morning application). Percent values indicate percent of HTRs relative
to respective first day. Mean ±SEM; n=6 per group. Repeated measures
ANOVA, Bonferroni-corrected post-hoc comparison to respective first-day
measurement, **p01, ***p001 (brackets behind asterisks indicate
interval(s) significance applied to).
interval and found a rapid loss of responsiveness to the second
injection (compare for LSD and DOI: Darmani and Gerdes,
1995; Buchborn et al., 2016). To test the hypothesis that the
observed tachyphylaxis was due to substance accumulation (i.e.,
two single 1.5 mg/kg doses accumulating to a less active 3.0-
mg/kg dose), we repeated the experiment injecting a 0.75-
mg/kg dose (which after thorough animal habituation induced
significant HTRs; see Figure 1A) twice at a 1.5-h interval. If the
two 0.75 mg/kg doses had accumulated to form a 1.5-mg/kg
dose, the second dose should have induced unchanged or more
HTRs than the first. However, as the 0.75-mg/kg regimen led
to a similar-degree tachyphylaxis as did the 1.5-mg/kg dose,
it is unlikely that drug accumulation is the underlying cause
for the rapid tolerance development. Serotonergic hallucinogens
have been shown to affect locomotor activity in rodents (e.g.,
Cohen and Wakeley, 1968; Kabes et al., 1972; Wing et al., 1990),
with 5-HT2A activation in mice promoting hyperlocomotion
(Halberstadt et al., 2009). 25CN-NBOH administration (1.5
mg/kg), in line with the latter, during the 1.5-h observation
increased the distance traveled as well as the overall mobility
duration. However, average speed of movement was unchanged.
Also, 25CN-NBOH-treated mice (like the control animals)
overall spent significantly less time mobile than immobile.
Therefore, it appears unlikely that physical exhaustion caused
the observed tachyphylaxis to 25CN-NBOH. Instead processes
of 5-HT2A regulation might play a role; indeed, DOI applied
twice at a 4-h interval in vivo, desensitizes 5-HT2A mediated
glutamate release in the cortex (Scruggs et al., 2003). Finally,
we investigated subchronic tolerance as it would develop with
a once-a-day application of 25CN-NBOH (1.5 mg/kg, i.p.) over
Frontiers in Pharmacology | 6February 2018 | Volume 9 | Article 17
Buchborn et al. Tolerance and Tachyphylaxis to 25CN-NBOH
4 days. HTRs steadily decreased (e.g., compare for DOI and
2C-T-7: Smith et al., 2014), with the most pronounced loss of
responsiveness exhibited in between the first days. The extent of
tolerance could be substantially augmented by applying a second
(higher) daily dose of 25CN-NBOH (3.0 mg/kg, i.p.) at an 8-h
In summary, our data demonstrate that the selective 5-
HT2A agonist 25CN-NBOH induces a dose- and time-dependent,
ketanserin-sensitive increase in HTRs in mice, which is subject
to short-interval and subchronic tolerance. Future research,
correlating HTRs with pharmacokinetic-/dynamic adaptations at
various time points during tolerance development might provide
insight as to a possible mechanism. Furthermore, making use of
new technologies to image cortical activities in awake behaving
mice using genetically encoded, cell-class specifically targeted
voltage and calcium indicators (Carandini et al., 2015; Knöpfel
et al., 2015; Song et al., 2017) might take our mechanistic
understanding of the underlying processes from basic receptor
pharmacology to systemic circuit dynamics.
TB, TL, and TK: have conceptualized the study design; TB and
TL: have collected and plotted the data; TB, TL, and TK: have
interpreted the data and written the manuscript.
This study was supported by the European Commission (TB,
TK), funds provided by Imperial College (TK), NIH (TK), and
by MRC (TL).
We would like to thank Jesper L. Kristensen, University of
Copenhagen, for a kind gift of 25CN-NBOH. We would like to
thank Chenchen Song for advice and supervision, Gemma Oliver
for help with animal husbandry, and all members of the Knöpfel
lab for critical comments and encouragement. We would also
like to thank David Nutt and his group members for advice and
The Supplementary Material for this article can be found
online at:
Supplementary Movie 1 | Head twitch response (HTR) induced by a 1.5-mg/kg
i.p. dose of 25CN-NBOH (recorded at 100 Hz). The arrow indicates on- and offset
of the head twitch at 13 s. The twitch is recapitulated by a slow-motion
picture-in-picture replay on the right side of the video. Format: Mp4, H.264+AAC,
720p. Duration: 20 s.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
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Frontiers in Pharmacology | 8February 2018 | Volume 9 | Article 17
... There is scarce data on the effect of chronic administration of NBOMe on neurotransmission and behavior in animal models. However, it has been shown that after repeated administration, other hallucinogens, such as LSD, DOM, and 25CN-NBOH, produce tolerance by downregulation of the 5-HT 2A receptor (Buchborn et al. 2018;Buckholtz et al. 1990;Gresch et al. 2005). Furthermore, it has also been demonstrated that rewarding and reinforcing effects of 25I-NBOMe, 25B-NBOMe, and 25N-NBOMe on mice are related to the dopaminergic system, as changes in expression of dopamine D 1 and D 2 receptors, dopamine transporter DAT, tyrosine hydroxylase, and dopamine (DA) levels were found in the nucleus accumbens and ventral tegmental area (VTA) (Custodio et al. 2019;Jeon et al. 2019;Seo et al. 2019). ...
... Damjanoska et al. (2004) found that a 7-day treatment with DOI decreased 5-HT-stimulated PLC activity in the frontal cortex; it is suggested that desensitization of 5-HT 2A receptors is most likely attributable to post-translational modifications of the receptor due to a change in 5-HT 2A receptor coupling to G proteins. This phenomenon is common to serotonergic hallucinogens, and close congeners in this class shared it: 25CN-NBOH or LSD and DOM (Buchborn et al. 2018;Buckholtz et al. 1990;Gresch et al. 2005). Multiple studies have demonstrated that cortical 5-HT 2A receptors are localized primarily in apical dendrites of pyramidal neurons with a minor localization to GABAergic interneurons (Miner et al. 2003;Willins et al. 1997). ...
Full-text available
Rationale 4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe) is a potent serotonin 5-HT 2A/2C receptor agonist with hallucinogenic activity. There is no data on the 25I-NBOMe effect on brain neurotransmission and animal performance after chronic administration. Objectives We examined the effect of a 7-day treatment with 25I-NBOMe (0.3 mg/kg/day) on neurotransmitters’ release and rats’ behavior in comparison to acute dose. Methods Changes in dopamine (DA), serotonin (5-HT), acetylcholine (ACh), and glutamate release were studied using microdialysis in freely moving rats. The hallucinogenic activity was measured in the wet dog shake (WDS) test. The animal locomotion was examined in the open field (OF) test, short-term memory in the novel object recognition (NOR) test. The anxiogenic/anxiolytic properties of the drug were tested using the light/dark box (LDB) test. Results Repeated administration of 25I-NBOMe decreased the response to a challenge dose of DA, 5-HT, and glutamatergic neurons in the frontal cortex as well as weakened the hallucinogenic activity in comparison to acute dose. In contrast, striatal and accumbal DA and 5-HT release and accumbal but not striatal glutamate release in response to the challenge dose of 25I-NBOMe was increased in comparison to acute treatment. The ACh release was increased in all brain regions. Behavioral tests showed a motor activity reduction and memory deficiency in comparison to a single dose and induction of anxiety after the drug’s chronic and acute administration. Conclusions Our findings suggest that multiple injections of 25I-NBOMe induce tolerance to hallucinogenic activity and produce alterations in neurotransmission. 25I-NBOMe effect on short-term memory, locomotor function, and anxiety seems to be the result of complex interactions between neurotransmitter pathways.
... 20,21,27 This behavioral proxy of human hallucinogenic potential is induced by psychedelics including phenethylamines such as DOI, tryptamines such as psilocybin, and ergolines such as LSD, but not by nonpsychedelic 5-HT 2A R agonists such as lisuride. Using HTR as a behavioral model of psychedelic action, previous studies demonstrate that several psychedelics recapitulate the tolerance 28,29 and cross-tolerance 30 phenomena observed in human subjective effects. ...
... A similar phenotype (i.e., partial reduction of HTR) has been reported after repeated administration of classical psychedelics such as LSD, 1-(2,5-dimethoxy-4bromophenyl)-2-aminopropane (DOB), and N-(2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH). 28,29 This, however, contrasts with previous observations in human subjects suggesting that daily administration of psychoactive doses vanishes the hallucinogenic properties of psychedelics such as LSD and mescaline. 3−5 A potential explanation for these interspecies discrepancies could rely on dosage, length of the treatment, pharmacokinetic properties, or density of the 5-HT 2A R in the frontal cortex. ...
Full-text available
Classical psychedelics represent a subgroup of serotonergic psychoactive substances characterized by their distinct subjective effects on the human psyche. Another unique attribute of this drug class is that such effects become less apparent after repeated exposure within a short time span. The classification of psychedelics as a subgroup within the serotonergic drug family and the tolerance to their effects are replicated by the murine head twitch response (HTR) behavioral paradigm. Here, we aimed to assess tolerance and cross-tolerance to HTR elicited by psychedelic and nonpsychedelic serotonin 2A receptor (5-HT 2A R) agonists in mice. We show that repeated (4 days) administration of the psychedelic 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induced a progressive decrease in HTR behavior. Tolerance to DOI-induced HTR was also observed 24 h after a single administration of this psychedelic. Pretreatment with the 5-HT 2A R antagonist M100907 reduced not only the acute manifestation of DOI-induced HTR, but also the development of tolerance to HTR. Additionally, cross-tolerance became apparent between the psychedelics DOI and lysergic acid diethylamide (LSD), whereas repeated administration of the nonpsychedelic 5-HT 2A R agonist lisuride did not affect the ability of these two psychedelics to induce HTR. At the molecular level, DOI administration led to down-regulation of 5-HT 2A R density in mouse frontal cortex membrane preparations. However, development of tolerance to the effect of DOI on HTR remained unchanged in β-arrestin-2 knockout mice. Together, these data suggest that tolerance to HTR induced by psychedelics involves activation of the 5-HT 2A R, is not observable upon repeated administration of nonpsychedelic 5-HT 2A R agonists, and occurs via a signaling mechanism independent of β-arrestin-2.
... Although we find that mTBI drives hypersensitivity of cortical 5-HT 2A receptors, we find that repetitive agonist-induced activation of 5-HT 2A receptors results in the normalization of aberrant social behaviors in murine subjects. Counterintuitively, the administration of 5-HT 2A receptor agonists results in the pharmacological process known as tachyphylaxis, which is the acute, rapid desensitization of receptors upon repetitive stimulation (Abbas et al., 2009;Buchborn et al., 2018). This includes decreases in detectable 5-HT 2A receptor densities and/or expression in cortical regions known to be involved in the actions of 5-HT 2A receptor agonists (Raval et al., 2021). ...
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Various forms of traumatic brain injury (TBI) are a leading cause of disability in the United States, with the generation of neuropsychiatric complications such as depression, anxiety, social dysfunction, and suicidality being common comorbidities. Serotonin (5-HT) signaling is linked to psychiatric disorders; however, the effects of neurotrauma on normal, homeostatic 5-HT signaling within the central nervous system (CNS) have not been well characterized. We hypothesize that TBI alters specific components of 5-HT signaling within the CNS and that the elucidation of specific TBI-induced alterations in 5-HT signaling may identify novel targets for pharmacotherapies that ameliorate the neuropsychiatric complications of TBI. Herein, we provide evidence that closed-head blast-induced mild TBI (mTBI) results in selective alterations in cortical 5-HT 2A receptor signaling. We find that mTBI increases in vivo cortical 5-HT 2A receptor sensitivity and ex vivo radioligand binding at time points corresponding with mTBI-induced deficits in social behavior. In contrast, in vivo characterizations of 5-HT 1A receptor function revealed no effect of mTBI. Notably, we find that repeated pharmacologic activation of 5-HT 2A receptors post-injury reverses deficits in social dominance resulting from mTBI. Cumulatively, these studies provide evidence that mTBI drives alterations in cortical 5-HT 2A receptor function and that selective targeting of TBI-elicited alterations in 5-HT 2A receptor signaling may represent a promising avenue for the development of pharmacotherapies for TBI-induced generation of neuropsychiatric disorders.
... Nevertheless, particularly noteworthy is the fact that in a few patients these symptoms occurred despite single administration of the drug. In vivo studies have shown that chronic administration of both agonists and antagonists of the 5-HT2A receptor causes downregulation of the receptor [21]. Thus, it is suggested that the development of tolerance is a result from 5-HT2A downregulation and desensitization [22]. ...
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Background 5-HT2A receptor (e.g. 25I-NBOMe) agonists not only pose risks of acute intoxication but also long-term effects and significant adverse reactions, e.g. hallucinogen persisting perception disorder (HPPD), derealization, and depersonalization. Aims We evaluated the risk associated with single and repeated use of 25I-NBOMe. We aimed to identify factors that may increase the risk of HPPD, increase its severity and determine the time when the first symptoms appear. Herein, we report the first extensive evaluation of 25I-NBOMe-induced HPPD. Method We assessed all reports (58) collected by The Pomeranian Pharmacovigilance Centre (PPC) from 2013 to 2020. Results The study included a total of 58 reports of adverse reactions caused by 25I-NBOMe. In the case of 15 reports (in patients aged 19–26 years), symptoms persisted many months after the discontinuation of 25I-NBOMe. The most common were: pseudohallucinations, bizarre delusions, derealizations and in some cases development or worsening of depression has been diagnosed. HPPD-like symptoms were most common in patients who took the drug regularly (i.e., several times a month). The risk of HPPD-like symptoms is higher in patients who have severe visual pseudohallucinations, severe bizarre delusions, derealization and/or depersonalization onset immediately after taking the drug. Recurrence of HPPD symptoms may be provoked by many factors, however, there is some cases there is no apparent reason. HPPD after 25I-NBOMe use can last from 2 months up to 2 years. In some patients, pharmacological treatment was necessary due to 25I-NBOMe-induced HPPD and depression. Conclusions The study showed long-lasting effects after 25I-NBOMe administration and allowed for the determination of HPPD risk factors.
... (25CN-NBOH) (structure shown in Fig. 2) has emerged as a selective 5-HT 2A agonist [5,6] and several in vivo investigations with this compound have been published [7][8][9][10]. ...
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 slowlier 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.
Disruption of neuronal chloride ion (Cl- ) homeostasis has been linked to several pathological conditions, including substance use disorder, yet targeted pharmacotherapies are lacking. In this study, we explored the potential of serotonin 2A receptor (5-HT2A R) agonism to reduce alcohol consumption in male wild-type C57Bl/6J mice and to ameliorate alcohol-induced inhibitory plasticity in the midbrain. We found that administration of the putative 5-HT2A R agonist TCB-2 attenuated alcohol consumption and preference but did not alter water or saccharin consumption. We hypothesized that the selective behavioural effects of TCB-2 on alcohol drinking were due, at least in part, to effects of the agonist on ventral tegmental area (VTA) neurocircuitry. Alcohol consumption impairs Cl- transport in VTA GABA neurons, which acts as a molecular adaptation leading to increased alcohol self-administration. Using ex vivo electrophysiological recordings, we found that exposure to either intermittent volitional alcohol drinking or an acute alcohol injection diminished homeostatic Cl- transport in VTA GABA neurons. Critically, in vivo TCB-2 administration normalized Cl- transport in the VTA after alcohol exposure. Thus, we show a potent effect of alcohol consumption on VTA inhibitory circuitry, in the form of dysregulated Cl- homeostasis that is reversible with agonism of 5-HT2A Rs. Our results provide insight into the potential therapeutic action of 5-HT2A R agonists for alcohol abuse.
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Cartilage diseases currently affect a high percentage of the world’s population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples’ physicochemical properties were studied with SEM, micro-CT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-α and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-α and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage.
Alcohol use is a prominent contributor to global disease burden and a leading cause of preventable mortality. Long-term severe problem drinking is clinically diagnosed as alcohol use disorder (AUD), one of the most prevalent neuropsychiatric diseases worldwide. Despite the persistent public health concern posed by alcohol abuse, treatments targeting specific brain mechanisms impacted by and driving pathological alcohol use are lacking. Behavioral animal models have revealed that the mesolimbic dopamine reward pathway is a critical mediator of alcohol’s reinforcing effects. The ventral tegmental area (VTA) is a central hub of this reward circuitry and subversion of alcohol-induced neuronal activity in the VTA has been linked to increased alcohol consumption. Among its many effects in the brain, alcohol enhances release of the inhibitory neurotransmitter GABA in the VTA. This acute effect of alcohol is exacerbated by prior alcohol, drug, or stress exposure, which are all risk factors for subsequent alcohol abuse. For these reasons, the central hypothesis of this dissertation was that disruptions in alcohol-induced inhibitory GABA signaling in the VTA contribute to escalated alcohol consumption. To delineate the mechanisms of disturbed VTA inhibitory transmission that may lead to increased alcohol consumption, electrophysiological recordings, in vivo pharmacological manipulations, and alcohol self-administration paradigms were performed in rodents. The second and third chapters provide evidence that an acute stressor in adulthood or chronic nicotine exposure in adolescence promote subsequent alcohol self-administration behavior via chloride ion (Cl-) dysregulation in VTA GABA neurons. Normalizing Cl- homeostasis by intra-VTA pharmacological upregulation of the potassium-Cl- cotransporter KCC2 prevented the increased alcohol drinking phenotype observed after stress or nicotine. In the fourth and fifth chapters, rescue of disrupted Cl- homeostasis via serotonin 2A receptor (5-HT2AR) activation is demonstrated ex vivo and correlated with 5-HT2AR agonist-mediated reduction in heavy alcohol consumption. Collectively, this body of work suggests that dysregulation of VTA Cl- transport, dictated largely by KCC2 function, increases risk for alcohol abuse. Targeting this form of inhibitory plasticity represents a novel interventional approach for AUD. Therefore, future work is needed to identify clinically safe and efficacious pharmacotherapies to reverse disturbances in midbrain Cl- homeostasis in alcohol-dependent individuals.
4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH) was first reported as a potent and selective serotonin 2A receptor (5-HT 2A R) agonist in 2014, and it has since found extensive use as a pharmacological tool in a variety of in vitro, ex vivo and in vivo studies. 25CN-NBOH is readily available from a synthetic perspective using standard chemical transformations, and displays favorable physiochemical properties in terms of stability and solubility. Due to its superior selectivity for 5-HT 2A R, 25CN-NBOH has been used to investigate the effects of selective 5-HT 2A R activation in vivo, and has thus become an important pharmacological tool for the exploration of 5-HT 2A R signaling in a range of animal models. In the present review, we outline the discovery of 25CN-NBOH, its pharmacological profile and major findings from studies where it has been used.
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4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH) was first reported as a potent and highly selective serotonin 2A receptor (5-HT2AR) agonist in 2014. The compound has since found extensive use as a pharmacological tool in a variety of in vivo and in vitro studies. In the present study, we present an improved and scalable synthesis of 25CN-NBOH making this compound readily available to the scientific community.
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Serotonin (5‐HT) is an important neurotransmitter that is responsible for the regulation of a number of behavioral effects such as mood, appetite and sleep. Abnormalities in the serotonin system are associated with a broad range of disorders in the central nervous system (CNS) such as schizophrenia, depression, anxiety and migraine. The 5‐HT2A receptor is the primary excitatory 5‐HT receptor in the human brain and mediates the hallucinogenic effects of drugs such as lysergic acid diethylamide (LSD) and is the target of atypical antipsychotics. Positron emission tomography (PET) is a powerful technique to study receptors in the living brain and is widely used for investigating 5‐HT receptors in both human and animal studies. Currently, only antagonist PET tracers are in use for the 5‐HT2A receptor. Agonist PET tracers could selectively label 5‐HT2A receptors in the high‐affinity state and thereby serve as a better functional measure of 5‐HT2A receptor function. Furthermore, agonist PET tracers are potentially more sensitive to changes in endogenous neurotransmitter levels than antagonist tracers. The aims of this project were: 1) To design and synthesize new 5‐HT2A agonists with the aim to increase affinity and selectivity for 5‐HT2A receptor. 2) To synthesize, radiolabel and evaluate a number of 5‐HT2A agonists for use as PET tracers. These were based on some known 5‐HT2A agonists from the literature as well as newly designed compounds. We synthesized four groups of compounds derived from the N‐benzylphenethylamine scaffold. Group 1, 2 and 4 compounds were synthesized by a general strategy comprising reductive amination of the appropriate phenethylamine and benzaldehyde building blocks. The more complicated Group 3 compounds were synthesized by a variety of methods. Group 1 compounds focused on the 4‐position of the phenethylamine‐moiety with minor variations in the N‐benzyl moiety. We found that most compounds had high affinity for the 5‐HT2A, 5‐HT2B and 5‐HT2C receptors. Compounds containing a cyano‐group in the 4‐position showed high selectivity towards the 5‐HT2A receptor, a property that has previously been elusive. Group 2 compounds were designed with the aim to further investigate the 2’‐ and 3´‐ position of the N‐benzyl moiety and many of these were designed as benzo‐fused heterocycles. When necessary, the required aldehydes were synthesized de novo. The preliminary biological screening showed a mix of good and passable compounds. Some of these compounds have the highest affinity for the 5‐HT2A receptor when compared to known compounds from the literature. Group 3‐compounds were designed as conformationally restricted analogues of the known agonists 25B‐NBOMe and 25B‐NB. Most of the compounds had significantly lower binding affinity at the 5‐HT2A when compared to group 2 or 3 compounds, but the study gave valuable information on the binding conformation of N‐benzylphenethylamines. One compound (4.7) showed good selectivity for the 5‐HT2A receptor as well as high affinity. IV Group 4 compounds were designed using a homology model of the 5‐HT2A receptor made using a template from an in silico‐activated model of the human β2‐adrenergic receptor. The predicted compounds were synthesized and submitted for biological evaluation. The results obtained so far, however, show that the predicted affinity does not correlate well with the experimental results, necessitating further refinement of the model. A total of nine compounds were selected for in vivo evaluation as PET‐tracers in pigs. 6.1 ([11C]‐CIMBI‐5) was able to label 5‐HT2A receptors in the brain and the cortical binding of 6.1 was blocked by treatment with the antagonist ketanserin. 6.1 had a non‐displaceable binding potential (BPND) in the pig brain comparable to [18F]‐altanserin. Using 6.1 as the lead compound, eight other compounds as well as the 6.1 isotopomer 6.2, were synthesized and tested. Compound 6.7 ([11C]‐CIMBI‐36) showed both better brain uptake and higher targetto‐ background ratio than 6.1. The cortical BPND of 6.7 was decreased by ketanserin, indicating high selectivity for 5‐HT2A receptors.
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The pioneering work of Amiram Grinvald established voltage-sensitive dye imaging (VSDI) in the mammalian cortex in the 1980s and inspired decades of cortical voltage imaging and the associated technological developments. The recent conception and development of genetically encoded voltage indicators (GEVIs) overcome many of the limitations of classical VSDI, and open experimental approaches that provide accruing support for orchestrated neuronal circuit dynamics of spatially distributed neuronal circuit underlying behaviors. We will review recent achievements using GEVIs to optically monitor the cortical activity in mammalian brains in vivo and provide a perspective for potential future directions. © 2017 The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
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Plant-based psychedelics such as psilocybin have an ancient history of medicinal use. After the first English-language report on LSD in 1950, psychedelics enjoyed a short-lived relationship with psychology and psychiatry. Used most notably as aides to psychotherapy for the treatment of mood disorders and alcohol dependence, drugs such as LSD showed initial therapeutic promise before prohibitive legislature in the mid-1960s effectively ended all major psychedelic research programmes. Since the early 1990s, there has been a steady revival of human psychedelic research: last year saw reports on the first modern brain imaging study with LSD and 3 separate clinical trials of psilocybin for depressive symptoms. In this Circumspective piece, Robin Carhart-Harris and Guy Goodwin share their opinions on the promises and pitfalls of renewed psychedelic research, with a focus on the development of psilocybin as a treatment for depression.Neuropsychopharmacology accepted article preview online, 26 April 2017. doi:10.1038/npp.2017.84.
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RationaleAccumulating evidence indicates that the mixed serotonin and dopamine receptor agonist lysergic acid diethylamide (LSD) induces an altered state of consciousness that resembles dreaming. Objectives This study aimed to test the hypotheses that LSD produces dreamlike waking imagery and that this imagery depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods Twenty-five healthy subjects performed an audiorecorded guided mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). Cognitive bizarreness of guided mental imagery reports was quantified as a standardised formal measure of dream mentation. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) questionnaire. ResultsLSD, compared with placebo, significantly increased cognitive bizarreness (p < 0.001). The LSD-induced increase in cognitive bizarreness was positively correlated with the LSD-induced loss of self-boundaries and cognitive control (p < 0.05). Both LSD-induced increases in cognitive bizarreness and changes in state of consciousness were fully blocked by ketanserin. ConclusionsLSD produced mental imagery similar to dreaming, primarily via activation of the 5-HT2A receptor and in relation to loss of self-boundaries and cognitive control. Future psychopharmacological studies should assess the differential contribution of the D2/D1 and 5-HT1A receptors to cognitive bizarreness.
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Background: Clinically significant anxiety and depression are common in patients with cancer, and are associated with poor psychiatric and medical outcomes. Historical and recent research suggests a role for psilocybin to treat cancer-related anxiety and depression. Methods: In this double-blind, placebo-controlled, crossover trial, 29 patients with cancer-related anxiety and depression were randomly assigned and received treatment with single-dose psilocybin (0.3 mg/kg) or niacin, both in conjunction with psychotherapy. The primary outcomes were anxiety and depression assessed between groups prior to the crossover at 7 weeks. Results: Prior to the crossover, psilocybin produced immediate, substantial, and sustained improvements in anxiety and depression and led to decreases in cancer-related demoralization and hopelessness, improved spiritual wellbeing, and increased quality of life. At the 6.5-month follow-up, psilocybin was associated with enduring anxiolytic and anti-depressant effects (approximately 60-80% of participants continued with clinically significant reductions in depression or anxiety), sustained benefits in existential distress and quality of life, as well as improved attitudes towards death. The psilocybin-induced mystical experience mediated the therapeutic effect of psilocybin on anxiety and depression. Conclusions: In conjunction with psychotherapy, single moderate-dose psilocybin produced rapid, robust and enduring anxiolytic and anti-depressant effects in patients with cancer-related psychological distress. Trial registration: Identifier: NCT00957359.
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Background: Psilocybin is a serotonin receptor agonist that occurs naturally in some mushroom species. Recent studies have assessed the therapeutic potential of psilocybin for various conditions, including end-of-life anxiety, obsessive-compulsive disorder, and smoking and alcohol dependence, with promising preliminary results. Here, we aimed to investigate the feasibility, safety, and efficacy of psilocybin in patients with unipolar treatment-resistant depression. Methods: In this open-label feasibility trial, 12 patients (six men, six women) with moderate-to-severe, unipolar, treatment-resistant major depression received two oral doses of psilocybin (10 mg and 25 mg, 7 days apart) in a supportive setting. There was no control group. Psychological support was provided before, during, and after each session. The primary outcome measure for feasibility was patient-reported intensity of psilocybin's effects. Patients were monitored for adverse reactions during the dosing sessions and subsequent clinic and remote follow-up. Depressive symptoms were assessed with standard assessments from 1 week to 3 months after treatment, with the 16-item Quick Inventory of Depressive Symptoms (QIDS) serving as the primary efficacy outcome. This trial is registered with ISRCTN, number ISRCTN14426797. Findings: Psilocybin's acute psychedelic effects typically became detectable 30-60 min after dosing, peaked 2-3 h after dosing, and subsided to negligible levels at least 6 h after dosing. Mean self-rated intensity (on a 0-1 scale) was 0·51 (SD 0·36) for the low-dose session and 0·75 (SD 0·27) for the high-dose session. Psilocybin was well tolerated by all of the patients, and no serious or unexpected adverse events occurred. The adverse reactions we noted were transient anxiety during drug onset (all patients), transient confusion or thought disorder (nine patients), mild and transient nausea (four patients), and transient headache (four patients). Relative to baseline, depressive symptoms were markedly reduced 1 week (mean QIDS difference -11·8, 95% CI -9·15 to -14·35, p=0·002, Hedges' g=3·1) and 3 months (-9·2, 95% CI -5·69 to -12·71, p=0·003, Hedges' g=2) after high-dose treatment. Marked and sustained improvements in anxiety and anhedonia were also noted. Interpretation: This study provides preliminary support for the safety and efficacy of psilocybin for treatment-resistant depression and motivates further trials, with more rigorous designs, to better examine the therapeutic potential of this approach. Funding: Medical Research Council.
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The first reports on tolerance to the serotonergic hallucinogen lysergic acid diethylamide (LSD) were published half a century ago, yet hitherto, a systematic review on this topic is not available. In this chapter, we discuss tolerance to LSD with regard to its psychedelic and somatic effects in humans, as well as selected behaviors in animals. In humans, mental tolerance to LSD substantially manifests 24 h after its first administration and reaches a maximum by around the fourth day. Once established, tolerance cannot be overcome even if the initial dose is quadrupled. Mental tolerance to LSD generalizes to psilocybin and mescaline but not to tetrahydrocannabinol or amphetamine. As to LSD’s somatic effects, mental tolerance most reliably is accompanied by tolerance to mydriasis. Five days of abstinence is sufficient for tolerance to be reversed; symptoms of withdrawal are not encountered. In animals, LSD-induced shaking behavior, limb flicking, and hallucinogenic pausing are undermined by tolerance, too; the first-mentioned behaviors, for instance, are subject to tachyphylaxis. Mechanistically, pharmacodynamic adaptations of serotonin 5-HT2A and/or (downstream) glutamate receptors are likely to account for tolerance; a learning-related precipitation, however, has also been described. The rapid onset of mental tolerance probably is a main reason LSD generally is not taken on an everyday basis by humans. Given its rapid reversal, on the other hand, a once-per-week abuse cannot be excluded.
Therapeutic interest in augmentation of 5-hydroxytryptamine2A (5-HT2A) receptor signaling has been renewed by the effectiveness of psychedelic drugs in the treatment of various psychiatric conditions. In this study, we have further characterized the pharmacological properties of the recently developed 5-HT2 receptor agonist 2-hydroxybenzyl)-2,5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH) and three structural analogs at recombinant 5-HT2A, 5- HT2B and 5-HT2C receptors and investigated the pharmacokinetic properties of the compound. 25CN-NBOH displayed robust 5-HT2A selectivity in [3H]ketanserin/[3H]mesulergine, [3H]LSD and [3H]Cimbi-36 binding assays (Ki 2C/Ki 2A ratio range 52-81, Ki 2B/Ki 2A ratio 37). Moreover, in inositol phosphate and intracellular Ca2+ mobilization assays 25CN-NBOH exhibited 30- to 180-fold 5-HT2A/5-HT2C selectivities and 54-fold 5-HT2A/5-HT2B selectivity as measured by ?log(Rmax/EC50) values. In an off-target screening 25CN-NBOH (10 ?M) displayed either substantially weaker activity or inactivity at a plethora of other receptors, transporters and kinases. In a toxicological screening, 25CN-NBOH (100 ?M) displayed a benign acute cellular toxicological profile. 25CNNBOH displayed high in vitro permeability (Papp = 29 x 10-6 cm/s) and low P-glycoprotein-mediated efflux in a conventional model of cellular transport barrier. In vivo, administration of 25CN-NBOH (3 mg/kg, s.c.) in C57 mice produced plasma and brain concentrations of the free (unbound) compound of ?200 nM within 15 min, further supporting that 25CN-NBOH rapidly penetrates the blood-brain barrier and is not subjected to significant efflux. In conclusion, 25CNNBOH appears to be a superior selective and brain-penetrant 5-HT2A receptor agonist compared to DOI, and thus we propose that the compound could be a valuable tool for future investigations of physiological functions mediated by this receptor.
Placebo response theory and set and setting theory are two fields which examine how non-biological factors shape the response to therapy. Both consider factors such as expectancy, preparation and beliefs to be crucial for understanding the extra-pharmacological processes which shape the response to drugs. Yet there are also fundamental differences between the two theories. Set and setting concerns itself with response to psychoactive drugs only; placebo theory relates to all therapeutic interventions. Placebo theory is aimed at medical professionals; set and setting theory is aimed at professionals and drug users alike. Placebo theory is primarily descriptive, describing how placebo acts; set and setting theory is primarily prescriptive, educating therapists and users on how to control and optimize the effects of drugs. This paper examines how placebo theory and set and setting theory can complement and benefit each other, broadening our understanding of how non-biological factors shape response to drugs and other treatment interventions.
Timing deficits are observed in patients with schizophrenia. Serotonergic hallucinogens can also alter the subjective experience of time. Characterizing the mechanism through which the serotonergic system regulates timing will increase our understanding of the linkage between serotonin (5-HT) and schizophrenia, and will provide insight into the mechanism of action of hallucinogens. We investigated whether interval timing in mice is altered by hallucinogens and other 5-HT2 receptor ligands. C57BL/6J mice were trained to perform a discrete-trials temporal discrimination task. In the discrete-trials task, mice were presented with two levers after a variable interval. Responding on lever A was reinforced if the interval was < 6.5 s, and responding on lever B was reinforced if the interval was > 6.5 s. A 2-parameter logistic function was fitted to the proportional choice for lever B (%B responding), yielding estimates of the indifference point (T50) and the Weber fraction (a measure of timing precision). The 5-HT2A antagonist M100907 increased T50, whereas the 5-HT2C antagonist SB-242,084 reduced T50. The results indicate that 5-HT2A and 5-HT2C receptors have countervailing effects on the speed of the internal pacemaker. The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI; 3 mg/kg IP), a 5-HT2 agonist, flattened the response curve at long stimulus intervals and shifted it to the right, causing both T50 and the Weber fraction to increase. The effect of DOI was antagonized by M100907 (0.03 mg/kg SC) but was unaffected by SB-242,084 (0.1 mg/kg SC). Similar to DOI, the selective 5-HT2A agonist 25CN-NBOH (6 mg/kg SC) reduced %B responding at long stimulus intervals, and increased T50 and the Weber fraction. These results demonstrate that hallucinogens alter temporal perception in mice, effects that are mediated by the 5-HT2A. It appears that 5-HT regulates temporal perception, suggesting that altered serotonergic signaling may contribute to the timing deficits observed in schizophrenia and other psychiatric disorders.