Neuroscience Letters 665 (2018) 246–251

Contents lists available at ScienceDirect

Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Research article

Effects of the antidepressant mirtazapine and zinc on nicotinic acetylcholine T

receptors
Andy Hernández-Abrego, Elizabeth Vázquez-Gómez, Jesús García-Colunga⁎
Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Boulevard Juriquilla
3001, Juriquilla, Querétaro 76230, México

A R T I C L E I N F O A B S T R A C T

Keywords: Nicotinic acetylcholine receptors (nAChRs) and zinc are associated with regulation of mood and related dis-
Nicotinic acetylcholine receptors orders. In addition, several antidepressants inhibit muscle and neuronal nAChRs and zinc potentiates inhibitory
Antidepressants actions of them. Moreover, mirtazapine (a noradrenergic, serotonergic and histaminergic antidepressant) in-
Mirtazapine hibits muscarinic AChRs and its effects on nAChRs are unknown. Therefore, we studied the modulation of muscle
Zinc
α1β1γd nAChRs expressed in oocytes and native α7-containing nAChRs in hippocampal interneurons by mir-
Xenopus oocytes
tazapine and/or zinc, using voltage-clamp techniques. The currents elicited by ACh in oocytes (at −60 mV) were
Hippocampus
similarly inhibited by mirtazapine in the absence and presence of 100 μM zinc (IC50 ∼15 μM); however, the
ACh-induced currents were stronger inhibited with 20 and 50 μM mirtazapine in the presence of zinc.
Furthermore, the potentiation of ACh-induced current by zinc in the presence of 5 μM mirtazapine was
1.48 ± 0.06, and with 50 μM mirtazapine zinc potentiation did not occur. Interestingly, in stratum radiatum
interneurons (at −70 mV), 20 μM mirtazapine showed less inhibition of the current elicited by choline (Ch) than
at 10 μM (0.81 ± 0.02 and 0.74 ± 0.02 of the Ch-induced current, respectively). Finally, the inhibitory effects of
mirtazapine depended on membrane potential: 0.81 ± 0.02 and 0.56 ± 0.05 of the control Ch-induced current
at −70 and −20 mV, respectively. These results indicate that mirtazapine interacts with muscle and neuronal
nAChRs, possibly into the ion channel; that zinc may increase the sensitivity of nAChRs to mirtazapine; and that
mirtazapine decreases the sensitivity of nAChRs to zinc.

1. Introduction and inhibitors of monoamine uptake, is the modulation of cholinergic

Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins
that belong to the superfamily of Cys-loop ligand-gated ion channels.
Muscle nAChRs are formed with α1, β1, γ or ε, and δ subunits; whereas
neuronal nAChRs are composed of the combination of α2-α10 and β2-
β4, which may be assembled as heteromeric nAChRs, or of only one
subunit such as homomeric α7 nAChRs [1–3]. nAChRs are widely dis-
tributed in the organism, in muscle endplate mediating fast neuro-
transmission; whereas in the nervous system they mediate fast neuro-
transmission as well as modulation of transmitter release [1,2]. They
are involved in physiological and pathological roles such as skeletal
muscle contraction, learning, memory, cognition, congenital myas-
thenic syndromes, Alzheimer’s disease, epilepsy, depressive disorders,
and drug addiction [4–7].
The functioning of nAChRs is modulated by a wide variety of sub-
stances interacting in allosteric sites including cations, local anes-
thetics, antidepressants, among others [8–11]. Particularly, a common
feature of different categories of antidepressants, including tricyclics
signaling by inhibiting both nAChRs [8,12–16] and muscarinic AChRs
[17–19]. These facts strengthen the idea that cholinergic signaling is
intimately associated with major depression disorder [20,21].
In the case of mirtazapine – an antidepressant that inhibits nor-
epinephrine, serotonin and histamine receptors [22] – there is one re-
port on cholinergic activity in guinea pig urinary bladder smooth
muscle, in which muscarinic AChRs are competitively inhibited by this
antidepressant [19].
Interestingly, the divalent cation zinc is found throughout the brain,
especially in the cerebral cortex and the hippocampus [23]. This cation
allosterically interacts with nAChRs and can potentiate or inhibit them,
depending on zinc concentration and receptor subunit composition
[24–27]. On other hand, zinc deficiency has been associated with de-
pressive disorders [28–30]. Accordingly, zinc enhances the effect of
several antidepressants both in animal models of depression and in
depressive patients, and it also enhances inhibitory effects of fluoxetine
and bupropion on nAChRs more strongly than the antidepressant alone
[31–36]. In this regard, the aim of this work was to study the

⁎
Corresponding author.
E-mail address: garciacolunga@unam.mx (J. García-Colunga).

https://doi.org/10.1016/j.neulet.2017.12.016
Received 14 September 2017; Received in revised form 29 November 2017; Accepted 6 December 2017
Available online 08 December 2017
0304-3940/ © 2017 Elsevier B.V. All rights reserved.
A. Hernández-Abrego et al.
pharmacological actions of mirtazapine on muscle and α7 nAChRs
hippocampal interneurons, in the absence and presence of zinc.
2. Materials and methods
All experimental procedures were carried out in accordance with
the National Institute of Health Guide for Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care
Committee of the Universidad Nacional Autónoma de México, with an
effort to minimize the number of animals used and their suffering.
2.1. Electrical recordings in Xenopus oocytes
The preparation of cRNA, oocyte injection, and electrical recordings
have been described elsewhere [34,37]. Briefly, cRNAs encoding
muscle α1, β1, γ, and δ nAChRs subunits were transcribed in vitro.
Xenopus laevis oocytes were dissected from pieces of ovary and main-
tained at 16 °C in Barth’s solution containing (in mM): 88 NaCl, 1 KCl,
0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3, 5 HEPES (pH 7.4;
NaOH), and 0.1 mg/mL gentamicin sulfate. Individual oocytes were
injected with 0.5–50 ng of the cRNA mixture, with equal quantities of
muscle subunit RNAs in 50 nL water, and 2 days later the oocytes were
treated with collagenase type I (140 units/mL for 0.5–1 h). ACh-in-
duced currents were recorded 3–9 days after RNA injection using a
voltage-clamp technique with two microelectrodes. Oocytes were
placed in a chamber (0.1 mL) and continuously perfused with Ringer’s
solution containing (in mM): 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 HEPES
(pH 7.0; NaOH) at room temperature (20–23 °C) and at a rate of
7–10 mL/min. Chemicals (acetilcholine, ZnCl2, and mirtazapine) were
obtained from Sigma-RBI and were diluted in Ringer to be applied in
the bath solution. The ZnCl2 stock solution (100 mM) was prepared by
serial dilutions with double-distilled water of a solution containing
100 mM ZnCl2 and 10 mM HCl [38]. Unless otherwise indicated, the
oocyte membrane potential was maintained at −60 mV. Control cur-
rents were elicited by applying 0.5 μM ACh in the superfusing Ringer’s
solution, a low concentration that results in a small extent of desensi-
tization. After the peak of the control ACh-induced current had been
reached, mirtazapine and 100 μM zinc alone or in combination were co-
applied with ACh. The concentration-response relationship for ACh-
induced current in the presence of mirtazapine was obtained by fitting
the data with the Hill equation: Imirt = Ip ∗ IC50nH/([mirtazapi-
ne]nH + IC50nH), where Imirt is the ACh-induced current amplitude in
the presence of mirtazapine, Ip is the control peak current, IC50 is the
half-inhibitory concentration of mirtazapine, and nH is the Hill coeffi-
cient. The fitting was obtained from the pooled averaged data, in which
each point was from five to six independent experiments.
2.2. Electrical recordings in hippocampal slices
The experiments were performed as previously described [39,40].
Briefly, Sprague Dawley rats on postnatal days 13–16 were deeply an-
esthetized with isoflurane and then decapitated. Their brains were re-
moved and placed into an ice-cold (4 °C) solution containing (in mM):
250 sucrose, 2.5 KCl, 1.2 NaH2PO4, 5 MgCl2, 0.5 CaCl2, 26 NaHCO3, 10
glucose, pH 7.4. Coronal slices (350 μm thick) containing the hippo-
campal CA1 area were cut with a Vibratome Leica VT 1000 and sub-
merged in artificial cerebrospinal fluid (ACSF) containing (in mM): 125
NaCl, 2.5 KCl, 1.23 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 10 glu-
cose, pH 7.4. The slices were stabilized in this solution for at least 1 h
before electrical recording. All solutions were continuously bubbled
with 95% O2 and 5% CO2 at room temperature.
One slice was transferred into a chamber and superfused during the
experiment with ACSF at a rate of ∼2 mL/min. Interneurons were vi-
sualized using an infrared video-microscopy system (BX51WI, Olympus
Instruments, Japan) endowed with an 80 x water immersion objective.
Whole-cell voltage-clamp recordings [41] were performed with a PC-
247
Neuroscience Letters 665 (2018) 246–251
ONE Patch/Whole Cell Clamp (Dagan Corporation, MN, USA) using a
Digidata 1440A acquisition system driven with pClamp 10 (Molecular
Devices, CA, USA). Patch-clamp electrodes had a resistance of 3–7 MΩ
when filled with the internal solution (in mM): 140 K-gluconate, 10
HEPES, 2 MgCl2, 0.5 CaCl2, 10 EGTA, and 2 MgATP, (pH 7.4). Data
were acquired in a PC using a Digidata 1440A AD converter at a sam-
pling rate of 10 kHz. Recorded interneurons were located in the striatum
radiatum hippocampal CA1 area and were maintained at a potential of
−70 or −20 mV.
The method for exploring the effects mirtazapine was previously
described for other substances [39,40]. Choline (Ch, 10 mM) puffs
(2–5 psi, 500 ms) were applied on interneurons through a fine tip glass
micropipette placed ∼10 μm from the recorded cell by using a pneu-
matic picopump (PV830, WPI, FL, USA). Choline was obtained from
Sigma-RBI. Thus, Ch-puffs were applied each 5 min before, during and
after the mirtazapine was added to the bath solution for ∼10 min. The
Ch-induced current amplitude was measured as a function of recording
time.
Hippocampal Ch-induced current in the absence or presence of
mirtazapine was measured with the pClamp 10 software. Origin 7
(MicroCal Software, MA, USA) was used to analyze, fit and graph the
results. Data are presented as mean ± standard error from at least three
independent experiments.
2.3. Statistical analysis
The R project for statistical computing (R V.3.3.2) was used for
statistical analysis. All data are presented as mean ± standard error.
Differences between multiple conditions were evaluated using one- or
two-way ANOVA and post-hoc Tukey’s test; two-tailed Student’s t-tests
were used for comparisons between two conditions; and p < 0.05 were
considered statistically significant different.
3. Results
Initially, when 100 μM ACh or 100 μM zinc were applied to non-
injected oocytes, or when mirtazapine or zinc were applied to injected
Xenopus oocytes expressing α1β1γδ nAChRs, no measurable ion cur-
rents were detected.
3.1. Inhibition of nAChRs by mirtazapine alone and in the presence of zinc
We examined the effects of mirtazapine alone and in the presence of
zinc on the ACh-induced current in oocytes expressing α1β1γδ nAChRs.
The effect of mirtazapine was calculated as the current elicited by ACh
plus mirtazapine divided by the control current value extrapolated to
the time of maximal effect of mirtazapine, giving the fraction of the
remaining control current.
Once ACh-induced current reached the peak, mirtazapine was ap-
plied in the presence of ACh at different concentrations (0.1–50 μM);
the extent of inhibition increased by increasing mirtazapine con-
centration (Fig. 1). In another series of experiments, the current was
elicited by ACh and then 100 μM zinc was added to the bath perfusion,
causing the ACh current to rise (Fig. 1A, bottom record). Thus, mirta-
zapine-concentration/ACh-induced current response relationships were
obtained in the absence and presence of zinc. In the case in which
mirtazapine was tested alone on ACh-induced current, the IC50 was
15.58 ± 2.91 μM (n = 5-6) and the Hill coefficient (nH) was
1.51 ± 0.29 (Fig. 1B, filled circles). When zinc was applied before
mirtazapine, the IC50 was similar (14.78 ± 3.51 μM, n = 5), and nH
was 1.54 ± 0.39 (Fig. 1B, open circles). Note that, at high concentra-
tions of mirtazapine (20 and 50 μM), the currents elicited by ACh were
significantly less inhibited than those in which oocytes were pre-
incubated with zinc (Fig. 1B, p < 0.05). These latter data may suggest
that zinc increases sensitivity of α1β1γδ nAChRs to mirtazapine.
A. Hernández-Abrego et al.
3.2. Potentiation of nAChRs by zinc in the presence of mirtazapine
Here, our interest was to evaluate potentiation exerted by zinc on
the ACh-induced current inhibited by mirtazapine in oocytes expressing
α1β1γδ nAChRs. Hence, after the current was elicited by 0.5 μM ACh,
oocytes were preincubated with different concentrations of mirtazapine
(0.5–50 μM), and then 100 μM zinc was added. Fig. 2A shows two
samples: In the first, zinc potentiated the ACh-induced current in the
presence of 5 μM mirtazapine to 1.48 ± 0.06 of the mirtazapine-in-
hibited ACh-induced current (n = 5); in the second, zinc potentiation
did not occur (n = 5) with a concentration of 50 μM mirtazapine.
248
Neuroscience Letters 665 (2018) 246–251
Fig. 1. Inhibition of the ACh-induced current by
mirtazapine alone and in the presence of zinc. (A)
ACh-induced currents by activation of muscle
α1β1γδ nAChRs: mirtazapine was added alone at
concentrations indicated (upper records), or zinc and
then mirtazapine (bottom record). (B) Mirtazapine-
concentration/current-response relationships for
currents obtained from records as in (A), elicited by
0.5 μM ACh in the absence (filled circles) and in the
presence of 100 μM zinc (open circles). The currents
inhibited by mirtazapine were compared to control
ACh-induced currents. For each concentration of
mirtazapine, the inhibition of currents elicited by
ACh alone (n = 5–6) was compared with those in the
presence of zinc (n = 5). Unless otherwise stated,
oocytes were maintained at a holding potential of
−60 mV. Data were compared with two-way
ANOVA and Tukey’s post test. Asterisks indicate
statistical differences between groups, p < 0.05.
Fig. 2B depicts zinc potentiation as a function of mirtazapine con-
centration. Notice that zinc-potentiated ACh-induced current decreased
by increasing mirtazapine concentration, except at 5 μM mirtazapine
when zinc potentiation increased compared to 2 μM mirtazapine. These
results suggest that mirtazapine decreased the sensitivity of muscle
α1β1γδ nAChRs to zinc, possibly by allosterically interacting at the
same site.
3.3. Effects of mirtazapine on α7-containing nAChRs
Here we studied the actions of mirtazapine on endogenous rat α7-
Fig. 2. Potentiation of the ACh-induced current by
zinc in the presence of mirtazapine. (A)
Representative currents elicited by 0.5 μM ACh. After
reaching the peak ACh-induced current, the in-
dicated concentrations of mirtazapine were added
followed by 100 μM zinc. (B) Potentiation of the
ACh-induced current by zinc at different concentra-
tions of mirtazapine. The zinc-potentiated ACh-in-
duced currents in the presence of mirtazapine were
compared to currents inhibited by mirtazapine, at
the end of mirtazapine application (n = 5). Data
were compared with one-way ANOVA and Tukey’s
post test. Asterisks indicate significant statistical
difference (p < 0.05) against the lowest concentra-
tion of mirtazapine.
A. Hernández-Abrego et al. Neuroscience Letters 665 (2018) 246–251

Fig. 3. Inhibition of the Ch-induced current by mirtazapine in hippocampal interneurons. The dots in the graph correspond to the amplitude of Ch-induced current as a function of
recording time (representative from n = 5 experiments). Ch-puffs (2–5 psi, 500 ms, arrow above the first record) were applied at 5-min intervals before, during, and after bath application
of mirtazapine. Interneurons were maintained at a holding potential of −70 mV. The timing of mirtazapine application is indicated by the thick black line. Upper inset: Corresponding Ch-
induced currents to data in the plot (representative from n = 5 experiments). Bottom inset: Voltage dependence effects of mirtazapine. Left and central columns are the ratio between the
Ch-induced current in the presence of mirtazapine (ICh+mzp) and the control Ch-induced current, at the holding potential of −70 mV, (10 μM, n = 5; 20 μM, n = 5). Right column
corresponds to the same ratio at 20 μM mirtazapine and at a holding potential of −20 mV (n = 5). Data are the mean ± standard error. Asterisks indicate significant statistical difference
(p < 0.05) performed by Student’s t-test.

containing nAChRs, that is, on Ch-induced current in interneurons from
the stratum radiatum hippocampal CA1 area. Although it was possible to
record postsynaptic currents elicited by electrical stimulation mediated
by nAChRs, which potentially may include all types of nAChRs [42], we
choose Ch-puffs to ensure that responses are mediated only by α7
nAChRs, excluding even other synaptic responses. The electrical ac-
tivity of α7-containing native nAChRs was recorded by applying local
puffs of 10 mM Ch (a selective α7-containing nAChRs agonist, [43])
onto interneurons. The resulting inward Ch-induced current decayed
even in the presence of the agonist due to receptor desensitization
(Fig. 3, inset: records). As control experiments, Ch-puffs were applied at
5-min intervals. The Ch-induced current amplitude remained constant
for up to 60 min (n = 3, data not shown), indicating recovery from
desensitization of α7-containing nAChRs after 5 min.
Additionally, the Ch-induced current in stratum radiatum inter-
neurons was completely inhibited by the selective antagonists methyl-
lycaconitine or α-bungarotoxin, confirming the involvement of α7-
containing nAChRs [40].
To explore the effects of mirtazapine, Ch-puffs were initially applied
at 5-min intervals to obtain control Ch-induced current (Fig. 3). Sub-
sequently, 10 μM mirtazapine was added to the bath solution for
∼10 min. The Ch-induced current amplitude was measured as a func-
tion of recording time before, during and after mirtazapine application.
The Ch-induced current amplitude decreased with respect to the control
current amplitude; this inhibitory effect continued after washing out
mirtazapine and was partially recovered to control level in ∼25 min
(Fig. 3).
Interestingly, mirtazapine at 20 μM inhibited Ch-induced current to
0.81 ± 0.02 control Ch-induced current (n = 5), obtaining a less in-
hibitory effect than at 10 μM (0.74 ± 0.02 control current, n = 5)
(Fig. 3, bottom inset; p < 0.05).
3.4. Voltage dependence of mirtazapine effects
To get more information on the interaction of mirtazapine with α7-
containing nAChRs, we explored the possibility that its effects depend
on membrane potential, which may indicate that mirtazapine interacts
with a site located inside the ion channel of the receptor [44,45]. In this
regard, the Ch-induced current inhibition by 20 μM mirtazapine was
achieved by maintaining membrane potential of hippocampal inter-
neurons at two values (-70 and −20 mV). At −70 mV, the ratio be-
tween the Ch-induced current in the presence mirtazapine and the
control Ch-induced current (ICh+mzp/ICh) was 0.81 ± 0.02 (Fig. 3,
middle column), whereas at −20 mV this ratio was 0.56 ± 0.05 (n = 5,
p < 0.05) for 20 μM mirtazapine (Fig. 3, right column).
Therefore, inhibition of the Ch-induced current by mirtazapine de-
pended on the membrane potential, being stronger at −20 than at
−70 mV (Fig. 3, bottom inset). These results may indicate that mirta-
zapine interacts at an allosteric site, possibly into the ion channel/re-
ceptor complex.
4. Discussion
Our findings showed that mirtazapine inhibited muscle nAChRs
with an IC5O of ∼15 μM. This value is in the same order as that of other
structurally different antidepressants, such as fluoxetine, imipramine,

249
A. Hernández-Abrego et al.
nefazodone, paroxetine or venlafaxine (with an IC5O in the range of 5.6
− 24 μM), suggesting a common mode of action and affinity on muscle
nAChRs [14,46].
Interestingly, fluoxetine, in addition to treating depression, has been
also used for treating congenital myasthenic syndrome, decreasing the
channel open time that is very long in this disorder [4,47,48]. Similarly,
mirtazapine used for depression, also attenuates cocaine- and nicotine-
induced locomotor sensitization [49,50]. In this regard, it is an open
question whether other antidepressants that inhibit muscle nAChRs,
including mirtazapine, could be used in the treatment of this syndrome
as well as in the treatment of nicotine disorders.
Although both the IC50 for mirtazapine and the Hill coefficient in
the absence and presence of zinc were similar in muscle nAChRs, at
higher concentrations of mirtazapine, it seems to be more efficient for
inhibiting these receptors when zinc first interacts with the receptors,
similar to fluoxetine, imipramine and bupropion that exhibit more
sensitivity for both muscle and neuronal nAChRs when zinc is present
[34,35]. These findings may acquire relevance for the treatment of
depression. In the same direction, zinc makes more efficient the effects
of antidepressants (fluoxetine, imipramine, desipramine, bupropion,
paroxetine) in animal models of depression and in depressive patients
[31–33,36].
It is known that low concentration of zinc potentiates nAChRs
[24,25,27]. However, here we found that the zinc potentiation of ACh-
induced current was slighter or even null in the presence of mirtazapine
(50 μM). The absence of zinc potentiation suggests that these two
substances (zinc and mirtazapine) interact on the same binding site,
with a higher sensitivity to mirtazapine for the receptor. Nevertheless,
the increase in zinc potentiation at 5 μM mirtazapine may suggest an
additional site for zinc on the nAChRs, similar to how α4β2 nAChRs
exhibit at least three interacting sites for zinc [26]. Therefore, the
modulation of muscle nAChRs by zinc and mirtazapine is different
depending which of these two substances first interacts with the re-
ceptor.
On other hand, mirtazapine inhibited α7 nAChRs, similar to keta-
mine metabolites and bupropion [40,51], indicating that α7 nAChRs
are target for structurally different antidepressants and that this inter-
action may contribute to their clinical effect. It is important to note that
the levels of hippocampal α7 nAChRs are involved in depressive-like
behaviors in animal models of depression [52,53]. Although we do not
discard that mirtazapine could affect other nAChRs, the modulation of
α7 nAChRs by this antidepressant may contribute to regulate inter-
neurons of inhibitory circuits in hippocampal stratum radiatum [54,55],
a brain region whose volume is reduced in a genetic animal model of
depression and recovered by ketamine treatment [56].
At 10 μM, mirtazapine inhibited both muscle and α7 nAChRs to the
same extent (∼0.7 of the control response). Surprisingly, by increasing
mirtazapine to 20 μM caused less inhibition in α7 but not in muscle
nAChRs, indicating different actions of mirtazapine on these two ni-
cotinic receptor subtypes.
To account for less inhibition of α7 nAChRs at higher mirtazapine
concentration, one possibility is that mirtazapine exerts a dual effect
(potentiation and inhibition) of α7 nAChRs at higher concentrations,
similar to other compounds [26,39,57].
Additionally, we found that inhibitory effects of mirtazapine on the
Ch-induced current depended on membrane potential; i.e., the inhibi-
tion was stronger at −20 than at −70 mV. In this way, mirtazapine
senses the electric field across the membrane, interacting at an allos-
teric site possibly located in the interface between the membrane and
the ion channel, or into the ion channel, a common action of other
substances on nAChRs [26,39,58–60].
With these results we conclude that the antidepressant mirtazapine
interacts with muscle and neuronal α7 nAChRs; that mirtazapine at
high concentrations inhibits nAChRs more strongly in the presence of
zinc; and that both zinc and mirtazapine possibly interact at the same
site into the ion channel.
250
Neuroscience Letters 665 (2018) 246–251
Finally, our data may help to better understand the common actions
of antidepressants on nAChRs, in particular mirtazapine, and how the
combination of zinc and antidepressants might be relevant to treat
depression.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Dirección General de Asuntos del
Personal Académico, UNAM (DGAPA grants IN201313 and IN205016)
to J.G-C; E.V.G. was a postdoctoral fellow from DGAPA, UNAM. We
thank to Patricia Villalobos for technical assistance, Martín García-
Servín for his assistance in taking care of the rats, Leopoldo González-
Santos for helping with the statistical analysis, and Jessica González
Norris for editing the manuscript.
References
[1] E.X. Albuquerque, E.F.R. Pereira, M. Alkondon, S.W. Rogers, Mammalian nicotinic
acetylcholine receptors: from structure to function, Physiol. Rev. 89 (2009) 73–120.
[2] C. Bouzat, S.M. Sine, Nicotinic acetylcholine receptors at the single-channel level,
Br. J. Pharmacol. (2017), http://dx.doi.org/10.1111/bph.13770 (in press),
PubMed: 2017 Mar 5, [Epub ahead of print].
[3] N.S. Millar, C. Gotti, Diversity of vertebrate nicotinic acetylcholine receptors,
Neuropharmacology 56 (2009) 237–246.
[4] C. Deflorio, M. Catalano, S. Fucile, C. Limatola, F. Grassi, Fluoxetine prevents
acetylcholine-induced excitotoxicity blocking human endplate acetylcholine re-
ceptor, Muscle Nerve 49 (2014) 90–97.
[5] K.T. Dineley, A.A. Pandya, J.L. Yakel, Nicotinic ACh receptors as therapeutic targets
in CNS disorders, Trends Pharmacol. Sci. 36 (2015) 96–108.
[6] C. Gotti, F. Clementi, A. Fornari, A. Gaimarri, S. Guiducci, I. Manfredi, M. Moretti,
P. Pedrazzi, L. Pucci, M. Zoli, Structural and functional diversity of native brain
neuronal nicotinic receptors, Biochem. Pharmacol. 78 (2009) 703–711.
[7] H.O. Kalkman, D. Feuerbach, Modulatory effects of α7 nAChRs on the immune
system and its relevance for CNS disorders, Cell. Mol. Life Sci. 73 (2016)
2511–2530.
[8] H.R. Arias, P. Bhumireddy, C. Bouzat, Molecular mechanisms and binding site lo-
cations for noncompetitive antagonists of nicotinic acetylcholine receptors, Int. J.
Biochem. Cell Biol. 38 (2006) 1254–1276.
[9] J. Wang, J. Lindstrom, Orthosteric and allosteric potentiation of heteromeric neu-
ronal nicotinic acetylcholine receptors, Br. J. Pharmacol. (2017) (in press).
[10] H. Wang, Y. Zhang, S.T. Li, The effect of local anesthetics on the inhibition of adult
muscle-type nicotinic acetylcholine receptors by nondepolarizing muscle relaxants,
Eur. J. Pharmacol. 630 (2010) 29–33.
[11] D.K. Williams, J. Wang, R.L. Papke, Positive allosteric modulators as an approach to
nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations,
Biochem. Pharmacol. 82 (2011) 915–930.
[12] J. García-Colunga, J.N. Awad, R. Miledi, Blockage of muscle and neuronal nicotinic
acetylcholine receptors by fluoxetine (Prozac), Proc. Natl. Acad. Sci. U. S. A. 94
(1997) 2041–2044.
[13] J. García-Colunga, K.M. Targowska-Duda, H.R. Arias, Functional and structural
interactions between selective serotonin reuptake inhibitors and nicotinic acet-
ylcholine receptors, Neurotransmitter 3 (2016) e1293.
[14] H. López-Valdés, J. García-Colunga, Antagonism of nicotinic acetylcholine re-
ceptors by inhibitors of monoamine uptake, Mol. Psychiatry 6 (2001) 511–519.
[15] R.D. Shytle, A.A. Silver, R.J. Lukas, M.B. Newman, D.V. Sheehan, P.R. Sanberg,
Nicotinic acetylcholine receptors as targets for antidepressants, Mol. Psychiatry 7
(2002) 525–535.
[16] M.L. Weber, C.M. Hofland, C.L. Shaffer, G. Flik, T. Cremers, R.S. Hurst, H. Rollema,
Therapeutic doses of antidepressants are projected not to inhibit human α2β4 ni-
cotinic acetylcholine receptors, Neuropharmacology 72 (2013) 88–95.
[17] C.B. Brink, S.L. Viljoen, S.E. de Kock, D.J. Stein, B.H. Harvey, Effects of myo-inositol
versus fluoxetine and imipramine pretreatments on serotonin 5HT2A and mus-
carinic acetylcholine receptors in human neuroblastoma cells, Metab. Brain Dis. 19
(2004) 51–70.
[18] M. Johnsson, M. Windera, H. Zawiaa, I. Lödöenb, G. Tobina, B. Götrickb, In vivo
studies of effects of antidepressants on parotid salivary secretion in the rat, Arch.
Oral Biol. 67 (2016) 54–60.
[19] J. Uno, K. Obara, H. Suzuki, S. Miyatani, D. Chino, T. Yoshio, Y. Tanaka, Inhibitory
effects of antidepressants on acetylcholine-induced contractions in isolated guinea
pig urinary bladder smooth muscle, Pharmacology 99 (2017) 89–98.
[20] W.J. Jeon, B. Dean, E. Scarr, A. Gibbons, The role of muscarinic receptors in the
pathophysiology of mood disorders: a potential novel treatment? Curr.
Neuropharmacol. 13 (2015) 739–749.
[21] Y.S. Mineur, M.R. Picciotto, Nicotine receptors and depression: revisiting and re-
vising the cholinergic hypothesis, Trends Pharmacol. Sci. 31 (2010) 580–586.
A. Hernández-Abrego et al.
[22] D.J. Nutt, Tolerability and safety aspects of mirtazapine, Hum. Psychopharmacol.
17 (2002) S37–S41.
[23] C.J. Frederickson, J.Y. Koh, A.I. Bush, The neurobiology of zinc in health and dis-
ease, Nat. Rev. Neurosci. 6 (2005) 449–462.
[24] J. García-Colunga, M. González-Herrera, R. Miledi, Modulation of α2β4 neuronal
nicotinic acetylcholine receptors by zinc, Neuroreport 22 (2001) 147–150.
[25] B. Hsiao, D. Dweck, C.W. Luetje, Subunit-dependent modulation of neuronal ni-
cotinic receptors by zinc, J. Neurosci. 21 (2001) 1848–1856.
[26] M. Moroni, R. Vijayan, A. Carbone, R. Zwart, P.C. Biggin, I. Bermudez, Non-agonist-
binding subunit interfaces confer distinct functional signatures to the alternate
stoichiometries of the α4β2 nicotinic receptor: an α4α4 interface is required for
Zn2+ potentiation, J. Neurosci. 28 (2008) 6884–6894.
[27] E. Vázquez-Gómez, J. García-Colunga, Neuronal nicotinic acetylcholine receptors
are modulated by zinc, Neuropharmacology 56 (2009) 1035–1040.
[28] M. Siwek, M. Sowa-Kućma, K. Styczeń, B. Szewczyk, W. Reczyński, P. Misztak,
R. Topór-Mądry, G. Nowak, D. Dudek, J. k. Rybakowski, Decreased serum zinc
concentration during depressive episode in patients with bipolar disorder, J. Affect
Disord. 190 (2016) 272–277.
[29] K. Styczeń, M. Sowa-Kućma, M. Siwek, D. Dudek, W. Reczyński, B. Szewczyk,
P. Misztak, R. Topór-Mądry, W. Opoka, G. Nowak, The serum zinc concentration as
a potential biological marker in patients with major depressive disorder, Metab.
Brain Dis. 32 (2017) 97–103.
[30] A. Takeda, H. Tamano, R. Nishio, T. Murakami, Behavioral abnormality induced by
enhanced hypothalamo-pituitary-adrenocortical axis activity under dietary zinc
deficiency and its usefulness as a model, Int. J. Mol. Sci. 17 (2016) e1149.
[31] M.P. Cunha, D.G. Machado, L.E. Bettio, J.C. Capra, A.L. Rodrigues, Interaction of
zinc with antidepressants in the tail suspension test. Prog. Neuropsychopharmacol,
Biol. Psychiatry 32 (2008) 1913–1920.
[32] Q. Ding, H. Li, X. Tian, Z. Shen, X. Wang, F. Mo, J. Huang, H. Shen, Zinc and
imipramine reverse the depression-like behavior in mice induced by chronic re-
straint stress, J. Affect. Disord. 197 (2016) 100–106.
[33] U. Doboszewska, B. Szewczyk, M. Sowa-Kućma, K. Młyniec, A. Rafało, B. Ostacho-
wicz, M. Lankosz, G. Nowak, Antidepressant activity of fluoxetine in the zinc de-
ficiency model in rats involves the NMDA receptor complex, Behav, Brain Res. 287
(2015) 323–330.
[34] J. García-Colunga, E. Vázquez-Gómez, R. Miledi, Combined actions of zinc and
fluoxetine on nicotinic acetylcholine receptors, Pharmacogenomics J. 4 (2004)
388–393.
[35] J. García-Colunga, U. Godoy-García, E. Vázquez-Gómez, Interaction of bupropion
and zinc with neuronal nicotinic acetylcholine receptors, Neuropharmacology 61
(2011) 1202–1209.
[36] E. Ranjbar, M.S. Kasaei, M. Mohammad-Shirazi, J. Nasrollahzadeh, B. Rashidkhani,
J. Shams, S.A. Mostafavi, M.R. Mohammadi, Effects of zinc supplementation in
patients with major depression: a randomized clinical trial, Iran. J. Psychiatry 8
(2013) 73–79.
[37] J. García-Colunga, R. Miledi, Effects of serotonergic agents on neuronal nicotinic
acetylcholine receptors, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 2919–2923.
[38] P. Paoletti, P. Ascher, J. Neyton, High-affinity zinc inhibition of NMDA NR1-NR2A
receptors, J. Neurosci. 17 (1997) 5711–5725.
[39] J.J. López, E.G. Pérez, J. García-Colunga, Dual effects of a 2- benzylquinuclidinium
derivative on α7-containing nicotinic acetylcholine receptors in rat hippocampal
interneurons, Neurosci. Lett. 607 (2015) 35–39.
[40] E. Vázquez-Gómez, H.R. Arias, D. Feuerbach, M. Miranda-Morales, S. Mihailescu,
K.M. Targowska-Duda, K. Jozwiak, J. García-Colunga, Bupropion induced inhibi-
tion of α7 nicotinic acetylcholine receptors expressed in heterologous cells and
neurons from dorsal raphe nucleus and hippocampus, Eur. J. Pharmacol. 740
(2014) 103–111.
[41] O.P. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth, Improved patch-clamp
251
Neuroscience Letters 665 (2018) 246–251
techniques for high-resolution current recording from cells and cell-free membrane
patches, Pflugers Arch. 391 (1981) 85–100.
[42] C.J. Frazier, A.V. Buhler, J.L. Weiner, T.V. Dunwiddie, Synaptic potentials mediated
via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippo-
campal interneurons, J. Neurosci. 18 (1998) 8228–8235.
[43] Q. Liu, Y. Huang, J. Shen, S. Steffensen, J. Wu, Functional α7β2 nicotinic acet-
ylcholine receptors expressed in hippocampal interneurons exhibit high sensitivity
to pathological level of amyloid β peptides, BMC Neurosci. 13 (2012) 155.
[44] J. García-Colunga, R. Miledi, Serotonergic modulation of muscle acetylcholine re-
ceptors of different subunit composition, Proc. Natl. Acad. Sci. U. S. A. 93 (1996)
3990–3994.
[45] A.M. Woodhull, Ionic blockage of sodium channels in nerve, J. Gen. Physiol. 61
(1973) 687–708.
[46] J.D. Fryer, R.J. Lukas, Antidepressants noncompetitively inhibit nicotinic acet-
ylcholine receptor function, J. Neurochem. 72 (1999) 1117–1124.
[47] A. Chaouch, J.S. Müller, V. Guergueltcheva, M. Dusl, U. Schara, V. Rakocević-
Stojanović, C. Lindberg, R.H. Scola, L.C. Werneck, J. Colomer, A. Nascimento,
J.J. Vilchez, N. Muelas, Z. Argov, A. Abicht, H. Lochmüller, A retrospective clinical
study of the treatment of slow-channel congenital myasthenic syndrome, J. Neurol.
259 (2012) 474–481.
[48] H. Zhu, G.E. Grajales-Reyes, V. Alicea-Vázquez, J.G. Grajales-Reyes, K. Robinson,
P. Pytel, C.A. Báez-Pagán, J.A. Lasalde-Dominicci, C.M. Gomez, Fluoxetine is neu-
roprotective in slow-channel congenital myasthenic syndrome, Exp. Neurol. 270
(2015) 88–94.
[49] S. Barbosa-Méndez, N. Jurado, M. Matus-Ortega, S. Martiñon, G. Heinze, A. Salazar-
Juárez, Mirtazapine attenuates the expression of nicotine-induced locomotor sen-
sitization in rats, Eur. J. Pharmacol. (2017) (in press).
[50] S. Barbosa-Méndez, M. Matus-Ortega, A. Flores-Zamora, N. Jurado, A. Salazar-
Juárez, Dose- and time-dependent effects of mirtazapine on the expression of co-
caine-induced behavioral sensitization in rats, Psychiatry Res. 254 (2017) 301–310.
[51] R. Moaddel, G. Abdrakhmanova, J. Kozak, K. Jozwiak, L. Toll, L. Jimenez,
A. Rosenberg, T. Tran, Y. Xiao, C.A. Zarate, I.W. Wainer, Sub-anesthetic con-
centrations of (R, S)-ketamine metabolites inhibit acetylcholine-evoked currents in
α7 nicotinic acetylcholine receptors, Eur. J. Pharmacol. 698 (2013) 228–234.
[52] R.G. Hunter, E.B. Bloss, K.J. McCarthy, B.S. McEwen, Regulation of the nicotinic
receptor α7 subunit by chronic stress and corticosteroids, Brain Res. 1325 (2010)
141–146.
[53] Y.S. Mineur, T.N. Mose, S. Blakeman, M.R. Picciotto, Hippocampal α7 nicotinic ACh
receptors contribute to modulation of depression-like behaviour in C57BL/6J mice,
Br. J. Pharmacol. (2017) (in press).
[54] M. Alkondon, E.X. Albuquerque, Diversity of nicotinic acetylcholine receptors in rat
hippocampal neurons. I. Pharmacological and functional evidence for distinct
structural subtypes, J. Pharmacol. Exp. Ther. 265 (1993) 1455–1473.
[55] M. Griguoli, E. Cherubini, Regulation of hippocampal inhibitory circuits by nico-
tinic acetylcholine receptors, J. Physiol. 590 (2012) 655–666.
[56] M. Ardalan, A.H. Rafati, J.R. Nyengaard, G. Wegener, Rapid antidepressant effect of
ketamine correlates with astroglial plasticity in the hippocampus, Br. J. Pharmacol.
174 (2017) 483–492.
[57] R. Zwart, R.G. van Kleef, C. Gotti, C.J. Smulders, H.P. Vijverberg, Competitive
potentiation of acetylcholine effects on neuronal nicotinic receptors by acet-
ylcholinesterase-inhibiting drugs, J. Neurochem. 75 (2000) 2492–2500.
[58] F.J. Barrantes, Structural basis for lipid modulation of nicotinic acetylcholine re-
ceptor function, Brain Res. Rev. 47 (2004) 71–95.
[59] B.I. Kalappa, V.V. Uteshev, The dual effect of PNU-120596 on α7 nicotinic acet-
ylcholine receptor channels, Eur. J. Pharmacol. 718 (2013) 226–234.
[60] M. Kaniakova, K. Skrenkova, S. Adamek, F. Vyskocil, J. Krusek, Different effects of
lobeline on neuronal and muscle nicotinic receptors, Eur. J. Pharmacol. 738 (2014)
352–359.