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

Abstract

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.

Full text

ORIGINAL RESEARCH
published: 06 February 2018
doi: 10.3389/fphar.2018.00017
Frontiers in Pharmacology | www.frontiersin.org 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
*Correspondence:
Thomas Knöpfel
tknopfel@knopfel-lab.net
†These authors have contributed
equally to this work.
Specialty section:
This article was submitted to
Neuropharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 19 October 2017
Accepted: 08 January 2018
Published: 06 February 2018
Citation:
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
INTRODUCTION
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
tolerance.
MATERIALS AND METHODS
Animals
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 ±2◦C at 55 ±10% humidity. Mice
were provided with standard rodent-chow pellets (Special Diet
Services, #RM1) and water ad libitum.
Drugs
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
indicated.
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
experiment.
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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
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.
Statistics
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∗
0e−lambda∗t) 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.
RESULTS
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 p≤0.01). 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, p≤0.01; Dunn’s post-
hoc [comparison to 1.5 NBOH w/o antagonist and to control,
respectively], p≤0.05 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, p≤0.05; 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; p≤0.01) and the response
to a 1.5-mg/kg dose was significantly increased (mean ±SEM:
61.5 ±6.3; p≤0.05; Figure 1A).
Time-Course of 25CN-NBOH Induced Head
Twitches
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, p≤0.001], treatment [F(2, 15) =30.41,
p≤0.001], as well as of time ×treatment interaction [F(3.68, 27.66)
=26.52, p≤0.001]. 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, p≤0.001;
2.0 mg/kg: 9.67 ±2.6, p≤0.05; 2.5 mg/kg: 11.17 ±1.68, p≤0.01)
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
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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, *p≤05 and **p≤01; 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 t½ 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, p≤0.001], treatment [F(1, 14) =4.62, p≤0.05], time
[F(2, 28) =140.97, p≤0.001], as well as significant interactions in
terms of locomotion ×treatment [F(1, 14) =5.44, p≤0.05] and
locomotion ×time [F(1.71, 23.98) =111.17, p≤0.001]. 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
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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, *p≤05.
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, p≤0.001], group [F(1, 19)
=62.1, p≤0.001], and time ×group interaction [F(4, 24) =10.47,
p≤0.001] 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 p≤0.01; 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, p≤0.01) (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, p≤0.001]; main effect group [F(2, 15) =
30.41, p≤0.001]; time ×group interaction [F(3.68, 27.66) =26.52,
p≤0.001] (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 p≤0.01].
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 p≤0.001]. 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, p≤0.001), those in the twice-per-
day mice more closely approached the control level (mean ±
SEM: 11.00 ±1.73, n.s.) (Figure 5).
DISCUSSION
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 | www.frontiersin.org 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), ##p≤01.
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, **p≤01, ***p≤001 (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 | www.frontiersin.org 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
interval.
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.
AUTHOR CONTRIBUTIONS
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.
FUNDING
This study was supported by the European Commission (TB,
TK), funds provided by Imperial College (TK), NIH (TK), and
by MRC (TL).
ACKNOWLEDGMENTS
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
inspiration.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphar.
2018.00017/full#supplementary-material
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
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