Pharmacological Reports
https://doi.org/10.1007/s43440-020-00092-4
ARTICLE
Desformylflustrabromine, a positive allosteric modulator
of α4β2‑containing nicotinic acetylcholine receptors, enhances
cognition in rats
Agnieszka Nikiforuk1 · Ewa Litwa1 · Martyna Krawczyk1 · Piotr Popik1 · Hugo Arias2
Received: 29 October 2019 / Revised: 4 February 2020 / Accepted: 5 February 2020
© The Author(s) 2020
Abstract
Rationale The α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) may represent useful targets for cognitive improvement. It has been recently proposed that a strategy based on positive allosteric modulation of α4β2-nAChRs reveals several
advantages over the direct agonist approach. Nevertheless, the procognitive effects of α4β2-nAChR positive allosteric
modulators (PAMs) have not been extensively characterized.
Objectives The aim of the present study was to evaluate the procognitive efficacy of desformylflustrabromine (dFBr), a
selective α4β2-nAChR PAM.
Methods Cognitive effects were investigated in the novel object recognition task (NORT) and the attentional set-shifting
task (ASST) in rats.
Results The results demonstrate that dFBr attenuated the delay-induced impairment in NORT performance and facilitated
cognitive flexibility in the ASST. The beneficial effects of dFBr were inhibited by dihydro-β-erythroidine, a relatively selective α4β2-nAChR antagonist, indicating the involvement of α4β2-nAChRs in cognitive processes. The tested α4β2-PAM was
also effective against ketamine- and scopolamine-induced deficits of object recognition memory. Moreover, procognitive
effects were also observed after combined treatment with inactive doses of dFBr and TC-2403, a selective α4β2-nAChR
agonist.
Conclusions These findings indicate that dFBr presents procognitive activity, supporting the strategy based on α4β2-nAChR
potentiation as a plausible therapy for cognitive impairment.
Keywords Α4β2-nAChRs · Cognition · Desformylflustrabromine · Positive allosteric modulators · Rat
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s43440-020-00092-4) contains
supplementary material, which is available to authorized users.
* Agnieszka Nikiforuk
nikifor@if-pan.krakow.pl
1
Department of Behavioral Neuroscience and Drug
Development, Maj Institute of Pharmacology Polish
Academy of Sciences, 12 Smetna Street, 31-343 Krakow,
Poland
2
Department of Pharmacology and Physiology, Oklahoma
State University College of Osteopathic Medicine,
Tahlequah, OK, USA
Converging lines of evidence indicate that nicotinic acetylcholine receptors (nAChRs) are involved in the regulation
of cognitive processes as well as in the pathophysiology
of disorders that affect cognitive abilities, such as schizophrenia and Alzheimer’s disease (AD) [1–3]. The two most
predominant nAChRs in the brain are heteropentameric
α4β2-nAChRs and homopentameric α7-nAChRs. Recently,
studies on possible therapies for cognitive decline in schizophrenia and AD have focused primarily on α7-nAChRs (e.g.,
[3]). Nevertheless, experimental evidence also supports the
involvement of α4β2-nAChRs in the pathogenesis of schizophrenia and AD [4–9]. For example, post-mortem studies
showed that the density of α4β2-nAChRs was decreased
in the hippocampus [4] and striatum [5] of schizophrenia
patients. Schizophrenia patients also demonstrated lower
13
Vol.:(0123456789)
A. Nikiforuk et al.
cortical β2-nAChR availability associated with executive
dysfunctions [6]. Post-mortem studies also indicated a loss
of α4β2-nAChRs in AD [7]. Moreover, a reduction in α4β2nAChRs in typical AD-affected brain regions, as revealed
by positron emission tomography, occurs at an early stage
of AD and might give prognostic information about a conversion from mild to severe cognitive impairment during
the progression of AD [8]. Abnormalities in α4β2-nAChRs
may be closely linked to histopathological hallmarks of AD,
such as the accumulation of β-amyloid (Aβ) peptides in the
brain. For example, in vivo results showed that the content of α4β2-nAChRs was decreased, whereas Aβ deposits
were increased in the brains of AD patients compared to
the brains of normal elderly subjects [9]. The active peptide
Aβ1–42 may also directly affect nAChR function. More specifically, α4β2-nAChRs can be blocked by Aβ1–42, decreasing its neuronal functions [10]. In line with clinical data, the
cognitive performance of mice lacking the β2 subunit was
deteriorated [11, 12].
Considering these results, it is plausible that a decreased
function or content of α4β2-nAChRs might produce cognitive deficits; consequently, an enhancement of its function
should improve cognition. However, while selective α4β2nAChR agonists enhanced cognition in a variety of animal
models (review in [13]), these preclinical efforts have not
been translated into clinically effective treatments. The clinical lack of efficacy of orthosteric nicotinic agonists might be
due to potential cross activity, overdosing, receptor desensitization and/or upregulation [2, 14]. Thus, drug development has shifted towards the positive allosteric modulation
of α4β2-nAChRs, proposed as an advantageous therapeutic
strategy compared to the direct agonist approach [2, 15, 16].
Since positive allosteric modulators (PAMs) increase the
response of the endogenous neurotransmitter acetylcholine
(ACh), without activating the receptor per se, the temporal
integrity of neurotransmission is preserved, and the risk of
overdosing is limited. In this regard, α4β2-selective PAMs
might produce beneficial activities without generating side
effects, such as those related to receptor desensitization
or receptor upregulation that may occur after the chronic
administration of orthosteric agonists.
Although several α4β2-PAMs have been characterized,
there is only one study thus far that has assessed the potential procognitive efficacy of NS9283 [17], the compound
that was further characterized as an unorthodox α4/α4
site-selective agonist [16]. Another α4β2-selective PAM,
desformylflustrabromine (dFBr) [18], has been previously
shown to suppress nicotine self-administration in rats [19],
ameliorates symptoms of nicotine withdrawal in mice [20]
and attenuates compulsive-like behaviours in a mouse model
of obsessive–compulsive disorder [21]. Nevertheless, to our
knowledge, there is no study demonstrating the procognitive
efficacy of this compound.
13
Therefore, the first objective of our study was to investigate whether dFBr increases cognition in rats by using two
animal tests, i.e., the attentional set-shifting task (ASST)
and the novel object recognition task (NORT), which
determine cognitive flexibility and recognition memory,
respectively. The second goal of this work was to determine
whether α4β2-nAChRs are involved in the procognitive
effects elicited by dFBr. In this regard, the activity of dFBr
was challenged against dihydro-β-erythroidine (DHβE), a
potent competitive antagonist of α4β2-nAChRs with higher
selectivity for this receptor subtype than for α7 and α3β4nAChRs [22]. Moreover, the ability of dFBr to ameliorate
the object recognition deficits elicited by the N-methyl-Daspartate receptor (NMDAR) antagonist ketamine or by
the muscarinic receptor antagonist scopolamine was also
assessed. Finally, the efficacy of combined administration
of the tested PAM with an α4β2-selective agonist, TC-2403
[(E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine; also called
RJR-2403] [23], was tested in the NORT.
Materials and methods
Animals
Male Sprague–Dawley rats (Charles River, Sulzfeld, Germany) weighing 280–350 g on arrival were housed in a
temperature-controlled (21 ± 1 °C) and humidity-controlled
(40–50%) colony room with a 12/12 h light/dark cycle
(lights on at 06:00 h). The rats were group-housed (4–5
rats/cage). For the ASST, rats were subjected to a mild food
restriction (17 g/day food pellets) for at least one week prior
to the testing day. Behavioural testing was performed during the light phase of the light/dark cycle. The experiments
were conducted in accordance with the European Guidelines
for animal welfare (2010/63/EU) and were approved by the
II Local Ethics Committee for Animal Experiments at the
Institute of Pharmacology, Polish Academy of Science, Krakow, Poland.
Attentional set‑shifting task (ASST)
The ASST assesses cognitive flexibility, i.e., the ability to
modify behaviour in response to the altering relevance of
stimuli. In this paradigm, rats must select a bowl containing
a food reward based on the ability to discriminate the odours
or the media covering the bait [24]. The ASST requires rats
to initially learn a rule and form an attentional “set” within
the same stimulus dimensions. At the extra-dimensional
(ED) shift stage, animals must switch their attention to a
previously irrelevant stimulus dimension and, for example,
discriminate between the odours and not between the media
Desformylflustrabromine, a positive allosteric modulator of α4β2‑containing nicotinic…
covering the bait. The animal’s performance at the ED stage
is considered an index of cognitive flexibility.
Apparatus. Testing was conducted in a dimly illuminated (20
lx) Plexiglas apparatus (length x width x height: 38 × 38 × 17
cm) with the grid floor and wall dividing half of the length
of the cage into two sections. During testing, one ceramic
digging pot (internal diameter of 10.5 cm and a depth of 4
cm) was placed in each section. Each pot was defined by a
pair of cues along with two stimulus dimensions. To mark
each pot with a distinct odour, 5 μl of a flavouring essence
(Dr. Oetker®, Poland or The Body Shop, UK) was applied
to a piece of blotting paper fixed to the external rim of the
pot immediately prior to use. A different pot was used for
each combination of digging medium and odour; only one
odour was ever applied to a given pot. The bait (one-half of
a Honey Nut Cheerio, Nestle®) was placed at the bottom
of the “positive” pot and buried in the digging medium. A
small amount of powdered Cheerio was added to the digging
media to prevent the rat from trying to detect the buried
reward by its smell.
Procedure. As described previously (e.g., [25]), the procedure lasted 3 days for each rat.
Day 1, habituation: rats were habituated to the testing area
and trained to dig in the pots filled with sawdust to retrieve
the food reward. The rats were transported from the housing
facility to the testing room where they were presented with
one unscented pot (filled with several pieces of Cheerios)
in their home cages. After the rats had eaten the Cheerio
from the home cage pot, they were placed in the apparatus
and given three trials to retrieve the reward from both of the
sawdust-filled baited pots. With each exposure, the bait was
covered with an increasing amount of sawdust. Animals that
did not dig for a food reward over 3 consecutive daily sessions were excluded from the experiment.
Day 2, training: rats were trained on a series of simple
discriminations (SDs) to a criterion of six consecutive correct trials. For these trials, the rats had to learn to associate
the food reward with an odour cue (e.g., arrack vs. orange,
both pots filled with sawdust) and/or a digging medium (e.g.,
plastic balls vs. pebbles, no odour). All rats were trained
using the same pairs of stimuli. The positive and negative cues for each rat were presented pseudorandomly and
equally. These training stimuli were not used again in later
testing trials.
Day 3, testing: rats performed a series of discriminations
in a single test session. The first four trials at the beginning
of each discrimination phase were discovery trials, during
which the animals were allowed to dig in both bowls. The
first trial of the discovery period was not included in the
six criterion trials. In the subsequent trials, each incorrect
choice was recorded as an error. Digging was defined as any
distinct displacement of the digging media with either the
paw or the nose; the rat could investigate a digging pot by
sniffing or touching without displacing material. Testing was
continued at each phase until the rat reached the criterion of
six consecutive correct trials, after which testing proceeded
to the next phase.
In the simple discrimination involving only one stimulus
dimension, the pots differed along one of two dimensions
(e.g., digging medium). For the compound discrimination
(CD), the second (irrelevant) dimension (i.e., odour) was
introduced, but the correct and incorrect exemplars of the
relevant dimension remained constant. For the reversal of
this discrimination (Rev 1), the exemplars and the relevant
dimension were unchanged, but the previously correct exemplar was now incorrect, and vice versa. The intra-dimensional (ID) shift was then presented, comprising new exemplars of both the relevant and irrelevant dimensions, with
the relevant dimension remaining the same as previously
described. The ID discrimination was then reversed (Rev
2) so that the formerly positive exemplar became the negative one. For the extra-dimensional (ED) shift, a new pair
of exemplars was again introduced; however, this time, the
relevant dimension was also changed. Finally, the last phase
was the reversal (Rev 3) of the ED discrimination.
The following pairs of exemplars were used: Pair 1:
odour: spicy vs. vanilla, medium: cotton wool vs. crumpled
tissue; Pair 2: odour: lemon vs. almond, medium: shredded
pipette tips vs. wooden sticks; and Pair 3: odour: rum vs.
cream, medium: shredded papers vs. silk. The exemplars
were always presented in pairs, and they varied so that only
one animal within each treatment group received the same
combination. The assignment of each exemplar in a pair as
being positive or negative at a given phase and the left–right
positioning of the pots in the test apparatus on each trial
were randomized.
Novel object recognition task (NORT)
The NORT in rodents [26] has been increasingly used as
an ethologically relevant paradigm for the study of visual
recognition memory. This test is based on the spontaneous
exploration of novel and familiar objects. The test consists
of two trials separated by an intertrial interval (ITI). During the first trial, two identical objects are presented. In
the second trial, one of the objects is replaced with a novel
object. Successful object recognition is indicated when an
animal spends more time interacting with the novel object
than with the familiar one in the retention trial. An ITI of
24 h was chosen as a model of natural forgetting based on
our previously published studies [27], which demonstrated
that, at this delay, Sprague–Dawley rats do not discriminate novel objects from familiar ones. Under conditions of
ketamine- or scopolamine-induced deficits, an ITI of 1 h,
13
A. Nikiforuk et al.
at which the animals demonstrate intact object recognition,
was used [28].
Apparatus The rats were tested in a dimly lit (25 lx) open
field made of dull grey plastic (length × width × height:
66 × 56 × 30 cm). After each measurement, the floor was
cleaned and dried.
Procedure The rats were habituated to the arena (without any
objects) for 5 min 24 h prior to testing. The test comprised
two 3-min trials separated by an inter-trial interval (ITI) of
24 h (or 1 h in the ketamine and scopolamine experiments).
During the first trial (familiarization, T1), two identical
objects (A1 and A2) were presented in opposite corners,
approximately 10 cm from the walls of the open field. In the
second trial (retention, T2), one of the objects was replaced
with a novel object (A = familiar and B = novel). The animals
were returned to the home cage after T1. The objects used
included a glass bulb filled with gravel and a plastic bottle
filled with sand. The heights of the objects were comparable
(~ 12 cm), and both objects were heavy enough to not be displaced by the animals. Half of the animals from each group
received the glass bulb as a novel object, and the other half
received the plastic bottle. The location of the novel object
(the left end versus the right end of the open field) in the recognition trial was counterbalanced across the experimental
groups. The exploration of an object was defined by looking,
licking, sniffing or touching the object while sniffing but not
leaning against, standing or sitting on the object. Any rat
spending less than 5 s exploring the two objects within 3
min of T1 or T2 was eliminated from the study. The behaviour of the rats was recorded using a camera placed above
the arena and connected to the Any-maze® tracking system
(Stoelting Co., Illinois, USA). An experimenter blinded to
the treatment conditions manually assessed the exploration
time. Additionally, the distance travelled was automatically
measured using the Any-maze® tracking system. Based on
the exploration time (E) of the two objects, a discrimination
index was calculated as DI = (EB–EA)/(EA + EB).
Drug administration
Desformylflustrabromine hydrochloride (dFBr, an α4β2nAChR PAM; Tocris, Bristol, UK), dihydro-β-erythroidine
hydrobromide (DHβE, an α4β2-nAChR antagonist; Tocris,
Bristol, UK), (E)-N-methyl-4-(3-pyridinyl)-3-butene-1amine (TC-2403, also called RJR-2403, an α4β2-nAChR
partial agonist; Abcam Biochemicals, Cambridge, UK), and
scopolamine (Sigma–Aldrich, Poznan, Poland) were dissolved in distilled water. Ketamine [aqueous solution (115.34
mg/mL), Vetoquinol Biowet, Gorzów Wielkoposki, Poland]
was diluted in distilled water to the appropriate dosage. In
general, the compounds were administered intraperitoneally
13
(IP), except TC-2403, which was given subcutaneously (SC).
The drugs or vehicle (saline) was administered at a volume
of 1 ml/kg of body weight.
ASST: dFBr (0.1, 0.3, and 1.0 mg/kg) or vehicle was
administered 30 min prior to the SD phase of the task. To
determine the ability of 3.0 mg/kg DHβE to block the procognitive effects of 1.0 mg/kg dFBr, the compounds were
administered simultaneously 30 min before testing. The total
number of animals subjected to the ASST experiments was
N = 50 (2 rats were excluded during training). The number
of animals in each experimental group was N = 6. Each rat
was tested only once.
NORT: dFBr (1.0 and 3.0 mg/kg) or vehicle was administered 30 min prior to the acquisition trial (T1). To determine
the ability of 3.0 mg/kg DHβE to block the procognitive
effects of 3.0 mg/kg dFBr, the compounds were administered
simultaneously 30 min before T1.
In the experiments in which amnestic agents were used,
dFBr (1.0 and 3.0 mg/kg) was first administered, followed
by ketamine (20 mg/kg) or scopolamine (1.25 mg/kg) 30
min later; after an additional 45 min (ketamine) or 30 min
(scopolamine), the acquisition trial (T1) was performed. In
the drug interaction studies, an inactive dose of dFBr (1.0
mg/kg) in combination with an inactive dose of TC-2403
(0.01 mg/kg) was administered 30 min prior to T1. The total
number of animals used in the NORT was N = 96. Because
of low (< 5 s) object exploration, 3 rats were excluded from
the analysis. Each rat was tested no more than twice, with
a 7-day washout period between each of the two tests. No
animal received the same treatment twice.
dFBr and TC-2403 doses were based on our preliminary experiments (see Supplement 1) and previous studies
demonstrating drug-evoked procognitive or behavioural
effects [19, 20, 25, 29]. Because the applied dose range was
adjusted to demonstrate the minimal effective dose of the
tested compounds, the dosage schedule differed between
the ASST and NORT. DHβE was administered at a dose
that has been previously demonstrated to block the procognitive effects of TC-2403 [25, 27]. The doses of ketamine
and scopolamine, adopted from our published protocols [28,
30], have been demonstrated to produce reliable impairment
using the NORT.
Statistics
ASST. The number of trials required to achieve the criterion
of six consecutive correct responses (i.e., trials to criterion,
TTC) was recorded for each rat and for each discrimination
phase of the ASST. Data were analysed using a mixed design
ANOVA with dFBr treatment as a between-subject factor
and discrimination phase (SD, CD, Rev 1, etc.) as a repeated
Desformylflustrabromine, a positive allosteric modulator of α4β2‑containing nicotinic…
measure. In the interaction studies, DHβE treatment was a
second between-subject factor in the analysis.
NORT. The data on exploratory preference were analysed
using mixed-design ANOVAs with treatment as a betweensubject factor and object as a repeated measure. The DI data
were analysed using one-way ANOVAs, and the distance
travelled was analysed using mixed-design ANOVAs, with
treatment as a between-subject factor and trials as a repeated
measure. In the interaction studies, DHβE treatment was a
second between-subject factor in the analysis.
Post hoc comparisons were performed using Newman–Keuls tests. The statistical analyses were performed
using Statistica 12.0 for Windows. Statistical significance
was set at p < 0.05.
Results
Desformylflustrabromine (dFBr) enhances rat
cognition in an α4β2‑dependent manner.
Attentional set‑shifting task
The administration of dFBr, an α4β2-nAChR PAM, at doses
of 0.3 and 1.0 mg/kg, but not at a dose of 0.1 mg/kg, reduced
the number of trials to criterion in the ED phase compared to
that in the vehicle-treated group (Fig. 1, a two-way ANOVA
interaction: F[18,120] = 30.18; p < 0.001). There was no significant dFBr effect during any other test phase.
The cognitive enhancement elicited by 1.0 mg/kg dFBr
was blocked by 3.0 mg/kg DHβE (an α4β2-nAChR antagonist), demonstrating that the observed effect was α4β2dependent (Fig. 2, a three-way ANOVA interaction: F[6,
120] = 21.27, p < 0.001). The administration of 3.0 mg/kg
DHβE alone did not affect rats’ ASST performance compared to the vehicle-treated group.
Novel object recognition task
No significant differences in the time spent exploring two
identical objects in the acquisition phase in any group were
observed (Supplementary Table 1 S2, a two-way ANOVA
interaction for experiment 1 and a three-way ANOVA for
experiment 2 were F[2, 25] = 0.39, NS and F[1, 28] = 2.22,
NS, respectively).
Vehicle-treated rats did not discriminate the novel object
from the familiar object in the retention trial (Supplementary
Table 1 S2; Fig. 3). This time-induced natural forgetting was
ameliorated by the administration of 3.0 mg/kg dFBr (Supplementary Table 1 S2, two-way ANOVA interaction: F[2,
25] = 30.99, p < 0.001). Moreover, the DI for dFBr (3.0 mg/
kg)-treated rats was significantly higher than that for vehicletreated rats (Fig. 3, a one-way ANOVA: F[2, 25] = 19.68,
p < 0.001). Similar procognitive efficacy was demonstrated
for an α4β2-nAChR agonist, TC-2403, at doses of 0.1 and
0.3 mg/kg (detailed description is provided in Supplement
1). Interestingly, DHβE (3.0 mg/kg) blocked the procognitive
effect elicited by dFBr {a three-way ANOVA interaction for
exploration time: F[1, 28] = 15,47, p < 0.01 (Supplementary
Fig. 1 Dose–response effects
of desformylflustrabromine on
the attentional set-shifting task.
Different doses of dFBr (0.1,
0.3, or 1.0 mg/kg) or vehicle
were administered (IP) to rats
30 min prior to the test. Data
are shown as the mean ± S.E.M.
of the number of trials required
to reach the criterion of six
consecutive correct trials for
each of the discrimination
phases. N = 6 rats per group.
***p < 0.001, significant
improvement in ED performance compared to that of the
vehicle-treated group
13
A. Nikiforuk et al.
Fig. 2 Dihydro-β-erythroidine
inhibits the procognitive effects
of desformylflustrabromine on
the attentional set-shifting task.
DHβE (3.0 mg/kg), dFBr (1.0
mg/kg), and their combinations
were administered (IP) to rats
30 min prior to the test. Data
are shown as the mean ± S.E.M.
of the number of trials required
to reach the criterion of six
consecutive correct trials for
each of the discrimination
phases. N = 6 rats per group.
***p < 0.001, significant
improvement in ED performance compared to the vehicletreated group. ###p < 0.001,
significant reduction in ED
performance compared to the
dFBr (1)-vehicle treated group
Fig. 3 Dose–response effects of desformylflustrabromine on the novel
object recognition task. Different doses of dFBr (1.0 or 3.0 mg/kg)
or vehicle were administered (IP) to rats 30 min prior to the acquisition trial (T1). Data are shown as the mean ± S.E.M. of the discrimination index (DI) during the retention trial (T2) conducted 24 h after
T1. N = 9–10 rats per group; ***p < 0.001 significant increase in DI
compared to the vehicle-treated group
Table 1 S2) and a two-way ANOVA interaction for DI: F[1,
28] = 7.25, p < 0.05 (Fig. 4)}.
No significant treatment effects were observed for the distance travelled by the rats in the familiarization and retention
trials (a two-way ANOVA interaction for experiment 1 and a
three-way ANOVA for experiment 2 were F[2, 25] = 0.82, NS
and F[1,28] = 0.85, NS, respectively, Supplementary Table 2
S2).
13
Fig. 4 Dihydro-β-erythroidine inhibits desformylflustrabromineincreased recognition memory in the novel object recognition task.
DHβE (3.0 mg/kg), dFBr (3.0 mg/kg), and their combinations were
administered (IP) to rats 30 min prior to the acquisition trial (T1).
Data are shown as the mean ± S.E.M. of the discrimination index (DI)
during the retention trial (T2) conducted 24 h after T1. N = 8 rats per
group. ***p < 0.001 significant increase in DI compared to that of the
vehicle + vehicle-treated group; ###p < 0.001 significant reduction in
DI compared to the vehicle + dFBr(3)-treated group
Desformylflustrabromine reverses ketamine‑
and scopolamine‑induced novel object recognition
deficits
There were no significant differences in the time spent
exploring two identical objects in the acquisition phase in
any experimental group (Supplementary Table 1 S2, a twoway ANOVA interactions for ketamine and scopolamine
studies were F[3, 28] = 1.82, NS and F[3, 29] = 0.71, NS,
Desformylflustrabromine, a positive allosteric modulator of α4β2‑containing nicotinic…
respectively). However, the administration of either 20 mg/
kg ketamine (Fig. 5) or 1.25 mg/kg scopolamine (Fig. 6)
abolished the ability of the animal to discriminate novel
and familiar objects in the retention trial. Interestingly,
the ketamine-induced deficit was reversed after treatment
with 3.0 mg/kg dFBr (a two-way ANOVA interaction for
exploration time: F[3, 28] = 22.28, p < 0.001; Supplementary Table 1 S2 and a one-way ANOVA interaction for DI:
F[3, 28] = 26.47, p < 0.001; Fig. 5). Moreover, dFBr (1.0 or
3.0 mg/kg) reversed the impairing effects elicited by scopolamine (a two-way ANOVA interaction for exploration
time: F[3, 29] = 8.99, p < 0.001; Supplementary Table 1 S2,
and a one-way ANOVA interaction for DI: F[3, 29] = 10.76,
p < 0.001; Fig. 6).
No significant treatment effects were observed on the
distance travelled by rats in the familiarization and retention trials (a two-way ANOVA interactions for ketamine and
scopolamine were F[3, 28] = 0.83, NS and F[3, 28] = 0.29,
NS, respectively; Supplementary Table 2 S2).
The co‑administration of inactive doses of dFBr
and TC‑2403 facilitates novel object recognition
memory
There were no significant differences in the time spent
exploring two identical objects in the acquisition phase
in any experimental group (Supplementary Table 1 S2, a
two-way ANOVA interaction: F[1, 38] = 0.01, NS). The coadministration of an inactive dose of TC-2403 (0.01 mg/kg)
with an inactive dose of dFBr (1.0 mg/kg) facilitated object
Fig. 5 Desformylflustrabromine reverses ketamine-induced recognition memory deficits. Ketamine (20 mg/kg) was administered (IP) 45
min prior to the acquisition trial (T1), and dFBr (1.0 or 3.0 mg/kg)
was administered (IP) 30 min prior to the ketamine injection. Data
are shown as the mean ± S.E.M. of the discrimination index (DI)
during the retention trial (T2) conducted 1 h after T1. N = 8 rats per
group. ***p < 0.001 significant reduction in DI compared to that of
the vehicle + vehicle-treated group; ###p < 0.001 significant increase
in DI compared to that of the vehicle + ketamine-treated group
Fig. 6 Desformylflustrabromine reverses scopolamine-induced recognition memory deficits. Scopolamine (1.25 mg/kg) was administered (IP) 30 min prior to the acquisition trial (T1), and dFBr (1.0
or 3.0 mg/kg) was administered (IP) 30 min prior to the scopolamine
injection. Data are shown as the mean ± S.E.M. of the discrimination index (DI) during the retention trial (T2) conducted 1 h after
T1. N = 8–10 rats per group. ***p < 0.001 significant reduction in DI
compared to that of the vehicle + vehicle-treated group; ###p < 0.001
significant increase in DI compared to that of the vehicle + scopolamine-treated group
recognition in the retention trial {a three-way ANOVA interaction for exploration time: F[1, 28] = 19.26, p < 0.001
(Supplementary Table 1 S2), and a two-way ANOVA interaction for DI: F[1, 38] = 16.31, p < 0.001 (Fig. 7)}. No
significant treatment effects were observed for the distance
travelled by rats in the familiarization and retention trials
(a two-way ANOVA interaction: F[3, 38] = 1.58, NS, Supplementary Table 2 S2).
Fig. 7 Effects of co-administration of inactive doses of TC-2403 and
desformylflustrabromine on recognition memory. dFBr (1.0 mg/kg,
IP), TC-2403 (0.01 mg/kg, SC), and their combination were administered to rats 30 min prior to the acquisition trial (T1). Data are
shown as the mean ± S.E.M. of the discrimination index (DI) during
the retention trial (T2) conducted 24 h after T1. N = 10–12 rats per
group. ***p < 0.001 significant increase in DI compared to that of the
vehicle + vehicle-treated group; ###p < 0.001 significant increase in DI
compared to the drug alone-treated groups (i.e., vehicle + TC-2403
and vehicle + dFBr)
13
A. Nikiforuk et al.
Discussion
The present study demonstrated for the first time that dFBr
facilitates cognitive flexibility and recognition memory in
rats. Interestingly, the procognitive activities elicited by
dFBr on the ASST and NORT were blocked by DHβE, a
potent and relatively selective α4β2-nAChR antagonist,
demonstrating that the main targets for the action of the
tested PAM are α4β2-dependent. Moreover, the tested
α4β2-PAM also reversed ketamine- and scopolamineinduced deficits of object recognition memory. Finally,
the procognitive effects were also achieved when dFBr
was combined with TC-2403, an α4β2-nAChR agonist.
There are limited data on the efficacy of α4β2-selective
ligands for enhancing cognitive flexibility in the ASST.
Our previous study demonstrated that TC-2403 facilitated
ED set-shifting in rats and that this effect was blocked by
DHβE [25]. The improvement in rats’ ED performance
was also demonstrated after the administration of 5IA85380, a β2-nAChR-selective agonist [31]. The current
results corroborate these findings by demonstrating the
potential of an α4β2-nAChR PAM to facilitate cognitive
flexibility in an α4β2-nAChR-dependent manner.
Our study also demonstrated that dFBr was effective in
ameliorating delay-induced deficits in object recognition
memory and that this activity was also α4β2-dependent.
However, the effective dose of dFBr (i.e., 3 mg/kg) was
higher than the doses that produced improvement in the
ASST (i.e., 0.3 and 1.0 mg/kg). Likewise, TC-2403 facilitated cognitive flexibility at doses of 0.03–0.1 mg/kg [25],
while a higher dose of 0.3 mg/kg was necessary to enhance
recognition memory (Supplement 1). As a similar trend
was previously noted for other nicotinic acting agents
(e.g., [27]), it may be suggested that delay-dependent forgetting of object memory is a less sensitive task than the
ASST for detecting cognitive enhancement. It should be
noted that the results reported here were obtained from
male rats only and further studies are required to determine potential sex differences.
In line with our data, the enhancement of recognition
memory was observed in rats administered different α4β2selective agonists. For example, the ability to discriminate
between a novel object and a familiar object after a 24-h
delay was improved by TC-1734 (AZD3480, ispronicline)
in mice [32] and by TC-6683 (AZD1446) in rats [33].
The administration of 0.1 mg/kg TC-2403 reversed 6 h
ITI-induced forgetting in rats [29]. Although the efficacy
of TC-2403 was also supported in the current study (Supplement 1), the active dose was higher (i.e., 0.3 mg/kg)
than that used by McLean et al. [29]. Moreover, 0.3 mg/kg
TC-2403 decreased object exploration during the acquisition trial (Supplement 1). On the contrary, dFBr enhanced
13
object recognition in the absence of any deleterious effect
on exploratory or locomotor activity, supporting previous
results in mice where this PAM (up to 6.0 mg/kg) did not
affect open field locomotor activity [21].
The α4β2-PAM not only enhanced the performance of
cognitively unimpaired animals but also reversed object
recognition deficits in a pharmacological model of schizophrenia based on the administration of ketamine, an NMDA
receptor antagonist. The efficacy of α4β2-selective ligands
has not been widely assessed in schizophrenia-like animal
models. For example, ketamine-induced deficits on a tactileto-visual cross modal object recognition task and set-shifting task in rats were reversed by the α4β2-nAChR agonists
ABT-418 [34] and TC-2403 [25], respectively. Additionally,
administration of the β2-nAChR agonist (A-85380) ameliorated object recognition deficits induced by another NMDA
receptor antagonist, phencyclidine [35], while NS9283
had favourable effects on phencyclidine-disrupted sensory
information [17]. Furthermore, dFBr ameliorated recognition memory deficits induced by scopolamine, a muscarinic
receptor antagonist. The full reversal of the deficit was noted
at a dose of 1.0 mg/kg dFBr, while a dose of 3.0 mg/kg was
necessary to block delay- or ketamine-induced forgetting.
According to data in the literature, several α4β2-agonists
are also capable of reversing scopolamine-induced memory
impairments (e.g., [23, 32, 36]). Likewise, the amnestic
effects of scopolamine on the passive avoidance task in rats
were reversed by TC-1734 [32], TC-2559 [36], and TC-2403
[23]. The administration of ABT-089 to scopolamine-treated
[37] and aged [38] rats reversed spatial learning deficits in
the Morris water maze.
Our results also demonstrated that the co-administration
of inactive doses of dFBr and TC-2403 can lead to procognitive effects. This finding corroborates previous reports
in which dFBr enhanced nicotine-induced antinociception in a mouse model of neuropathic pain [39]. Similarly,
NS9283 augmented the antinociceptive properties of the
α4β2-agonist ABT-594 in various pain models [40, 41].
Although we are unaware of any data demonstrating that
α4β2-PAMs may augment the procognitive activities of
selective agonists, this approach was successfully used by
combining α7-nAChR PAMs with direct agonists [42, 43].
For example, the co-administration of inactive doses of an
α7-nAChR PAM, 3-furan-2-yl-N-p-tolyl-acrylamide, with
orthosteric agonists of α7-nAChR (DMXBA or A-582941)
improved object recognition and facilitated attentional setshifting in rats [42]. Moreover, another α7-nAChR PAM,
PNU-120596, enhanced the procognitive efficacy of a subthreshold dose of donepezil, an acetylcholinesterase inhibitor (AChEI) that increases the synaptic concentration of
ACh [43]. This strategy may be particularly beneficial in
conditions with compromised cholinergic function (e.g.,
in AD), in which PAMs alone may be ineffective due to
Desformylflustrabromine, a positive allosteric modulator of α4β2‑containing nicotinic…
scarce ACh levels or when the use of high doses of either
agonists or acetylcholinesterase inhibitors may be limited
due to adverse side effects.
The potential mechanisms underlying the procognitive
effects of α4β2-ligands might be discussed in relation to the
known function of α4β2-nAChRs in the regulation of the
release of neurotransmitters involved in cognitive processes.
For example, α4β2-nAchR activation evoked the release of
ACh in the cortex [44]. Moreover, electrochemical recordings of cholinergic transmission revealed that selective stimulation of α4β2-nAChRs evoked transient increases in prefrontal ACh release that may, in turn, predict enhancement
of attentional functions [45]. There is also a link between
α4β2-induced signalling and glutamate (Glu) release. For
example, a selective α4β2-agonist elicited Glu release from
hippocampal synaptosomes in a DHβE-sensitive fashion
[46]. In line with this finding, in vivo studies demonstrated
NS9283-evoked potentiation of nicotine-evoked Glu release
in the rat medial PFC [47]. It has also been demonstrated
that α4β2-nAchR activation induced dopamine release in
the rat PFC, and this effect was blocked by DHβE [48]. It
cannot be excluded, however, that other brain regions, e.g.,
the nucleus accumbens, ventral tegmental area or substantia nigra, may be also implicated in the observed effects of
α4β2-nAchR stimulation.
Stimulation of α4β2-nAChRs can also be effective
against pathological changes observed in psychiatric disorders, including GABAergic deficits specifically recognized
in schizophrenia or Aβ pathology in AD. For example,
A–85380, a β2-selective agonist, reversed the epigenetically induced transcriptional downregulation of glutamic
acid decarboxylase67 (GAD67) in cortical GABAergic
neurons [49]. Interestingly, dFBr prevented the inhibition
of α4β2-nAChRs by Aβ1–42 peptides [50]. Thus, the potential disease-modifying properties of α4β2-selective ligands
await further studies.
The present study corroborates the concept that α4β2nAChRs are involved in cognitive processes and demonstrates, for the first time, that α4β2 potentiation improves
cognitive flexibility and recognition memory as well as
rescues drug-induced cognitive deficits. The strategy based
on PAM-induced α4β2 enhancement, either alone or in
combination with orthosteric agonists, could offer a useful
approach to treat cognitive deficits associated with schizophrenia or AD.
Acknowledgements This study was supported by the Statutory Activity of the Maj Institute of Pharmacology, Polish Academy of Sciences,
Kraków, Poland and by the Polish National Science Centre (NCN)
Grant No. 2016/23/B/NZ7/01131. Open access publishing of this article was funded by the Ministry of Science and Higher Education under
the agreement No. 879/P-DUN/2019.
Compliance with ethical standards
Conflict of interest None declared.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
1. Lombardo S, Maskos U. Role of the nicotinic acetylcholine
receptor in Alzheimer’s disease pathology and treatment. Neuropharmacology. 2015;96:255–62. https ://doi.org/10.1016/j.
neuropharm.2014.11.018.
2. Grupe M, Grunnet M, Bastlund JF, Jensen AA. Targeting alpha4beta2 nicotinic acetylcholine receptors in central nervous
system disorders: perspectives on positive allosteric modulation as a therapeutic approach. Basic Clin Pharmacol Toxicol.
2015;116(3):187–200. https://doi.org/10.1111/bcpt.12361.
3. Jones C. alpha7 Nicotinic acetylcholine receptor: a potential
target in treating cognitive decline in schizophrenia. J Clin
Psychopharmacol. 2018;38(3):247–9. https://doi.org/10.1097/
JCP.0000000000000859.
4. Freedman R, Hall M, Adler LE, Leonard S. Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry.
1995;38(1):22–33. https://doi.org/10.1016/0006-3223(94)00252
-X.
5. Durany N, Zochling R, Boissl KW, Paulus W, Ransmayr G,
Tatschner T, et al. Human post-mortem striatal alpha4beta2
nicotinic acetylcholine receptor density in schizophrenia and
Parkinson’s syndrome. Neurosci Lett. 2000;287(2):109–12.
6. Esterlis I, Ranganathan M, Bois F, Pittman B, Picciotto MR,
Shearer L, et al. In vivo evidence for beta2 nicotinic acetylcholine receptor subunit upregulation in smokers as compared with
nonsmokers with schizophrenia. Biol Psychiatry. 2014;76(6):495–
502. https://doi.org/10.1016/j.biopsych.2013.11.001.
7. Martin-Ruiz CM, Court JA, Molnar E, Lee M, Gotti C,
Mamalaki A, et al. Alpha4 but not alpha3 and alpha7 nicotinic
acetylcholine receptor subunits are lost from the temporal cortex
in Alzheimer’s disease. J Neurochem. 1999;73(4):1635–40.
8. Kendziorra K, Wolf H, Meyer PM, Barthel H, Hesse S, Becker
GA, et al. Decreased cerebral alpha4beta2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer’s disease assessed with positron emission
tomography. Eur J Nucl Med Mol Imaging. 2011;38(3):515–25.
https://doi.org/10.1007/s00259-010-1644-5.
9. Okada H, Ouchi Y, Ogawa M, Futatsubashi M, Saito Y, Yoshikawa
E, et al. Alterations in alpha4beta2 nicotinic receptors in cognitive decline in Alzheimer’s aetiopathology. Brain. 2013;136(Pt
10):3004–177. https://doi.org/10.1093/brain/awt195.
10. Lamb PW, Melton MA, Yakel JL. Inhibition of neuronal
nicotinic acetylcholine receptor channels expressed in
13
A. Nikiforuk et al.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Xenopus oocytes by beta-amyloid1-42 peptide. J Mol Neurosci.
2005;27(1):13–211. https://doi.org/10.1385/JMN:27:1:013.
Guillem K, Bloem B, Poorthuis RB, Loos M, Smit AB, Maskos U,
et al. Nicotinic acetylcholine receptor beta2 subunits in the medial
prefrontal cortex control attention. Science. 2011;333(6044):888–
91. https://doi.org/10.1126/science.1207079.
Granon S, Faure P, Changeux JP. Executive and social behaviors
under nicotinic receptor regulation. Proc Natl Acad Sci USA.
2003;100(16):9596–601. https ://doi.org/10.1073/pnas.15334
98100.
Radek RJ, Kohlhaas KL, Rueter LE, Mohler EG. Treating the
cognitive deficits of schizophrenia with alpha4beta2 neuronal
nicotinic receptor agonists. Curr Pharm Des. 2010;16(3):309–22.
Bertrand D, Terry AV Jr. The wonderland of neuronal nicotinic
acetylcholine receptors. Biochem Pharmacol. 2018;151:214–25.
https://doi.org/10.1016/j.bcp.2017.12.008.
Pandya A, Yakel JL. Allosteric modulators of the alpha4beta2
subtype of neuronal nicotinic acetylcholine receptors. Biochem Pharmacol. 2011;82(8):952–8. https://doi.org/10.1016/j.
bcp.2011.04.020.
Wang J, Lindstrom J. Orthosteric and allosteric potentiation of
heteromeric neuronal nicotinic acetylcholine receptors. Br J Pharmacol. 2018;175(11):1805–21. https://doi.org/10.1111/bph.13745
.
Timmermann DB, Sandager-Nielsen K, Dyhring T, Smith M,
Jacobsen AM, Nielsen EO, et al. Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric
modulator of alpha2- and alpha4-containing nicotinic acetylcholine receptors. Br J Pharmacol. 2012;167(1):164–82. https://doi.
org/10.1111/j.1476-5381.2012.01989.x.
Weltzin MM, Schulte MK. Pharmacological characterization of
the allosteric modulator desformylflustrabromine and its interaction with alpha4beta2 neuronal nicotinic acetylcholine receptor
orthosteric ligands. J Pharmacol Exp Ther. 2010;334(3):917–26.
https://doi.org/10.1124/jpet.110.167684.
Liu X. Positive allosteric modulation of alpha4beta2 nicotinic
acetylcholine receptors as a new approach to smoking reduction:
evidence from a rat model of nicotine self-administration. Psychopharmacology. 2013;230(2):203–13. https://doi.org/10.1007/
s00213-013-3145-2.
Hamouda AK, Jackson A, Bagdas D, Imad DM. Reversal of nicotine withdrawal signs through positive allosteric modulation of
alpha4beta2 nicotinic acetylcholine receptors in male mice. Nicotine Tob Res. 2018;20(7):903–7. https://doi.org/10.1093/ntr/ntx18
3.
Mitra S, Mucha M, Khatri SN, Glenon R, Schulte MK, Bult-Ito A.
Attenuation of compulsive-like behavior through positive allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors
in non-induced compulsive-like mice. Front Behav Neurosci.
2016;10:244. https://doi.org/10.3389/fnbeh.2016.00244.
Chavez-Noriega LE, Crona JH, Washburn MS, Urrutia A, Elliott
KJ, Johnson EC. Pharmacological characterization of recombinant
human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2,
h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta
2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes. J
Pharmacol Exp Ther. 1997;280(1):346–56.
Lippiello PM, Bencherif M, Gray JA, Peters S, Grigoryan
G, Hodges H, et al. RJR-2403: a nicotinic agonist with CNS
selectivity II. In vivo characterization. J Pharmacol Exp Ther.
1996;279(3):1422–9.
Birrell JM, Brown VJ. Medial frontal cortex mediates perceptual
attentional set shifting in the rat. J Neurosci. 2000;20(11):4320–4.
Potasiewicz A, Golebiowska J, Popik P, Nikiforuk A. Procognitive
effects of varenicline in the animal model of schizophrenia depend
on alpha4beta2- and alpha 7-nicotinic acetylcholine receptors. J
13
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Psychopharmacol. 2018;2018:269881118812097. https ://doi.
org/10.1177/0269881118812097.
Ennaceur A, Delacour J. A new one-trial test for neurobiological
studies of memory in rats. 1: Behavioral data. Behav Brain Res.
1988;31(1):47–59.
Nikiforuk A, Kos T, Potasiewicz A, Popik P. Positive allosteric modulation of alpha 7 nicotinic acetylcholine receptors
enhances recognition memory and cognitive flexibility in rats.
Eur Neuropsychopharmacol. 2015;25(8):1300–13. https ://doi.
org/10.1016/j.euroneuro.2015.04.018.
Nikiforuk A, Kos T, Holuj M, Potasiewicz A, Popik P. Positive
allosteric modulators of alpha 7 nicotinic acetylcholine receptors
reverse ketamine-induced schizophrenia-like deficits in rats. Neuropharmacology. 2016;101:389–400. https://doi.org/10.1016/j.
neuropharm.2015.07.034.
McLean SL, Grayson B, Marsh S, Zarroug SH, Harte MK, Neill
JC. Nicotinic alpha7 and alpha4beta2 agonists enhance the formation and retrieval of recognition memory: Potential mechanisms
for cognitive performance enhancement in neurological and psychiatric disorders. Behav Brain Res. 2016;302:73–80. https://doi.
org/10.1016/j.bbr.2015.08.037.
Vanda D, Soural M, Canale V, Chaumont-Dubel S, Satala G,
Kos T, et al. Novel non-sulfonamide 5-HT6 receptor partial
inverse agonist in a group of imidazo[4,5-b]pyridines with cognition enhancing properties. Eur J Med Chem. 2018;144:716–
29. https://doi.org/10.1016/j.ejmech.2017.12.053.
Wood C, Kohli S, Malcolm E, Allison C, Shoaib M. Subtypeselective nicotinic acetylcholine receptor agonists can improve
cognitive flexibility in an attentional set shifting task. Neuropharmacology. 2016;105:106–13. https://doi.org/10.1016/j.neuro
pharm.2016.01.006.
Gatto GJ, Bohme GA, Caldwell WS, Letchworth SR, Traina
VM, Obinu MC, et al. TC-1734: an orally active neuronal nicotinic acetylcholine receptor modulator with antidepressant, neuroprotective and long-lasting cognitive effects. CNS Drug Rev.
2004;10(2):147–66.
Mazurov AA, Miao L, Bhatti BS, Strachan JP, Akireddy S,
Murthy S, et al. Discovery of 3-(5-chloro-2-furoyl)-3,7-diazabicyclo[3.3.0]octane (TC-6683, AZD1446), a novel highly
selective alpha4beta2 nicotinic acetylcholine receptor agonist for the treatment of cognitive disorders. J Med Chem.
2012;55(21):9181–94. https://doi.org/10.1021/jm3006542.
Cloke JM, Winters BD. alpha(4)beta(2) Nicotinic receptor stimulation of the GABAergic system within the orbitofrontal cortex
ameliorates the severe crossmodal object recognition impairment in ketamine-treated rats: implications for cognitive dysfunction in schizophrenia. Neuropharmacology. 2015;90:42–52.
https://doi.org/10.1016/j.neuropharm.2014.11.004.
Miyauchi M, Neugebauer NM, Oyamada Y, Meltzer HY.
Nicotinic receptors and lurasidone-mediated reversal of
phencyclidine-induced deficit in novel object recognition.
Behav Brain Res. 2016;301:204–12. https://doi.org/10.1016/j.
bbr.2015.10.044.
Bencherif M, Bane AJ, Miller CH, Dull GM, Gatto GJ. TC-2559:
a novel orally active ligand selective at neuronal acetylcholine
receptors. Eur J Pharmacol. 2000;409(1):45–55.
Decker MW, Bannon AW, Curzon P, Gunther KL, Brioni JD,
Holladay MW, et al. ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride]: II. A novel cholinergic channel modulator with effects on cognitive performance in rats and
monkeys. J Pharmacol Exp Ther. 1997;283(1):247–58.
Rueter LE, Anderson DJ, Briggs CA, Donnelly-Roberts DL, Gintant GA, Gopalakrishnan M, et al. ABT-089: pharmacological
properties of a neuronal nicotinic acetylcholine receptor agonist
for the potential treatment of cognitive disorders. CNS Drug Rev.
2004;10(2):167–82.
Desformylflustrabromine, a positive allosteric modulator of α4β2‑containing nicotinic…
39. Bagdas D, Ergun D, Jackson A, Toma W, Schulte MK, Damaj
MI. Allosteric modulation of alpha4beta2* nicotinic acetylcholine receptors: Desformylflustrabromine potentiates antiallodynic
response of nicotine in a mouse model of neuropathic pain. Eur J
Pain. 2018;22(1):84–93. https://doi.org/10.1002/ejp.1092.
40. Zhu CZ, Chin CL, Rustay NR, Zhong C, Mikusa J, Chandran
P, et al. Potentiation of analgesic efficacy but not side effects:
co-administration of an alpha4beta2 neuronal nicotinic acetylcholine receptor agonist and its positive allosteric modulator
in experimental models of pain in rats. Biochem Pharmacol.
2011;82(8):967–76. https://doi.org/10.1016/j.bcp.2011.05.007.
41. Lee CH, Zhu C, Malysz J, Campbell T, Shaughnessy T, Honore
P, et al. alpha4beta2 neuronal nicotinic receptor positive allosteric modulation: an approach for improving the therapeutic index
of alpha4beta2 nAChR agonists in pain. Biochem Pharmacol.
2011;82(8):959–66. https://doi.org/10.1016/j.bcp.2011.06.044.
42. Potasiewicz A, Kos T, Ravazzini F, Puia G, Arias HR, Popik
P, et al. Pro-cognitive activity in rats of 3-furan-2-yl-N-p-tolylacrylamide, a positive allosteric modulator of the alpha7 nicotinic
acetylcholine receptor. Br J Pharmacol. 2015;172(21):5123–35.
https://doi.org/10.1111/bph.13277.
43. Callahan PM, Hutchings EJ, Kille NJ, Chapman JM, Terry AV
Jr. Positive allosteric modulator of alpha7 nicotinic-acetylcholine
receptors, PNU-120596 augments the effects of donepezil on
learning and memory in aged rodents and non-human primates.
Neuropharmacology. 2013;67:201–12. https://doi.org/10.1016/j.
neuropharm.2012.10.019.
44. Obinu MC, Reibaud M, Miquet JM, Pasquet M, Rooney T.
Brain-selective stimulation of nicotinic receptors by TC-1734
enhances ACh transmission from frontoparietal cortex and memory in rodents. Prog Neuropsychopharmacol Biol Psychiatry.
2002;26(5):913–8.
45. Howe WM, Ji J, Parikh V, Williams S, Mocaer E, TrocmeThibierge C, et al. Enhancement of attentional performance by selective stimulation of alpha4beta2(*) nAChRs:
46.
47.
48.
49.
50.
underlying cholinergic mechanisms. Neuropsychopharmacology.
2010;35(6):1391–401. https://doi.org/10.1038/npp.2010.9.
Zappettini S, Grilli M, Salamone A, Fedele E, Marchi M. Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct
coupling mechanisms. Br J Pharmacol. 2010;161(5):1161–71.
https://doi.org/10.1111/j.1476-5381.2010.00958.x.
Grupe M, Paolone G, Jensen AA, Sandager-Nielsen K, Sarter M,
Grunnet M. Selective potentiation of (alpha4)3(beta2)2 nicotinic
acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats. Biochem Pharmacol. 2013;86(10):1487–96. https://doi.org/10.1016/j.
bcp.2013.09.005.
Livingstone PD, Srinivasan J, Kew JN, Dawson LA, Gotti C,
Moretti M, et al. alpha7 and non-alpha7 nicotinic acetylcholine
receptors modulate dopamine release in vitro and in vivo in the
rat prefrontal cortex. Eur J Neurosci. 2009;29(3):539–50. https://
doi.org/10.1111/j.1460-9568.2009.06613.x.
Maloku E, Kadriu B, Zhubi A, Dong E, Pibiri F, Satta R, et al.
Selective alpha4beta2 nicotinic acetylcholine receptor agonists
target epigenetic mechanisms in cortical GABAergic neurons.
Neuropsychopharmacology. 2011;36(7):1366–74. https ://doi.
org/10.1038/npp.2011.21.
Pandya A, Yakel JL. Allosteric modulator Desformylflustrabromine relieves the inhibition of alpha2beta2 and alpha4beta2
nicotinic acetylcholine receptors by beta-amyloid(1–42) peptide.
J Mol Neurosci. 2011;45(1):42–7. https://doi.org/10.1007/s1203
1-011-9509-3.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
13