A Comparative Study of the Antiemetic Effects of α2-Adrenergic Receptor Agonists Clonidine and Dexmedetomidine against Diverse Emetogens in the Least Shrew (Cryptotis parva) Model of Emesis

Abstract

In contrast to cats and dogs, here we report that the α2-adrenergic receptor antagonist yohimbine is emetic and corresponding agonists clonidine and dexmedetomidine behave as antiemetics in the least shrew model of vomiting. Yohimbine (0, 0.5, 0.75, 1, 1.5, 2, and 3 mg/kg, i.p.) caused vomiting in shrews in a bell-shaped and dose-dependent manner, with a maximum frequency (0.85 ± 0.22) at 1 mg/kg, which was accompanied by a key central contribution as indicated by increased expression of c-fos, serotonin and substance P release in the shrew brainstem emetic nuclei. Our comparative study in shrews demonstrates that clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) and dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, i.p.) not only suppress yohimbine (1 mg/kg, i.p.)-evoked vomiting in a dose-dependent manner, but also display broad-spectrum antiemetic effects against diverse well-known emetogens, including 2-Methyl-5-HT, GR73632, McN-A-343, quinpirole, FPL64176, SR141716A, thapsigargin, rolipram, and ZD7288. The antiemetic inhibitory ID50 values of dexmedetomidine against the evoked emetogens are much lower than those of clonidine. At its antiemetic doses, clonidine decreased shrews’ locomotor activity parameters (distance moved and rearing), whereas dexmedetomidine did not do so. The results suggest that dexmedetomidine represents a better candidate for antiemetic potential with advantages over clonidine.

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Citation: Sun, Y.; Darmani, N.A. A
Comparative Study of the Antiemetic
Effects of α2-Adrenergic Receptor
Agonists Clonidine and
Dexmedetomidine against Diverse
Emetogens in the Least Shrew
(Cryptotis parva) Model of Emesis. Int.
J. Mol. Sci. 2024,25, 4603. https://
doi.org/10.3390/ijms25094603
Academic Editor: Alain Couvineau
Received: 11 March 2024
Revised: 9 April 2024
Accepted: 19 April 2024
Published: 23 April 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Article
A Comparative Study of the Antiemetic Effects of
α2
-Adrenergic
Receptor Agonists Clonidine and Dexmedetomidine against
Diverse Emetogens in the Least Shrew (Cryptotis parva) Model
of Emesis
Yina Sun and Nissar A. Darmani *
Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University
of Health Sciences, 309 East Second Street, Pomona, CA 91766, USA; yinasun@westernu.edu
*Correspondence: ndarmani@westernu.edu; Tel.: +1-909-469-5654; Fax: +1-909-469-5535
Abstract: In contrast to cats and dogs, here we report that the
α2
-adrenergic receptor antagonist
yohimbine is emetic and corresponding agonists clonidine and dexmedetomidine behave as anti-
emetics in the least shrew model of vomiting. Yohimbine (0, 0.5, 0.75, 1, 1.5, 2, and 3 mg/kg, i.p.)
caused vomiting in shrews in a bell-shaped and dose-dependent manner, with a maximum frequency
(0.85
±
0.22) at 1 mg/kg, which was accompanied by a key central contribution as indicated by
increased expression of c-fos, serotonin and substance P release in the shrew brainstem emetic nuclei.
Our comparative study in shrews demonstrates that clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) and
dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, i.p.) not only suppress yohimbine (1 mg/kg, i.p.)-
evoked vomiting in a dose-dependent manner, but also display broad-spectrum antiemetic effects
against diverse well-known emetogens, including 2-Methyl-5-HT, GR73632, McN-A-343, quinpirole,
FPL64176, SR141716A, thapsigargin, rolipram, and ZD7288. The antiemetic inhibitory ID
50
values
of dexmedetomidine against the evoked emetogens are much lower than those of clonidine. At
its antiemetic doses, clonidine decreased shrews’ locomotor activity parameters (distance moved
and rearing), whereas dexmedetomidine did not do so. The results suggest that dexmedetomidine
represents a better candidate for antiemetic potential with advantages over clonidine.
Keywords:
α2
-adrenergic receptors; yohimbine; clonidine; dexmedetomidine; emesis; least shrew;
dorsal vagal complex
1. Introduction
Vomiting (emesis) is a protective reflex [
1
] which helps to remove ingested toxins from
the gastrointestinal tract (GIT) [
2
]. The emetic loci include the nucleus tractus solitarius
(NTS), the dorsal motor nucleus of the vagus (DMNX), and the area postrema (AP) in the
brainstem, as well as the enteric nervous system (ENS) and enterochromaffin cells (EC cells)
in the GIT [
3
]. The functional pathophysiology of vomiting indicates that emetic processes
are controlled via interactions between the gastrointestinal enteric nervous system, the
vagus, and the central nervous system (CNS) [
3
]. The emetic response involves multiple
neurotransmitters/mediators such as dopamine (DA), serotonin (5-HT), substance P (SP),
acetylcholine, histamine, prostaglandins, leukotrienes, and opiates, several of which have
been recognized as mediators of chemotherapy-evoked vomiting [
3
–
5
]. The act of emesis
may result from either direct activation of the brainstem dorsal vagal complex (DVC) emetic
nuclei including the AP, NTS, and DMNX via a blood-borne pathway, and/or indirect
stimulation of the DVC via activation of peripheral emetic loci such as the neurons of
the ENS and release of emetic neurotransmitters from the gastrointestinal EC cells which
subsequently activate the gastrointestinal vagal afferents to the brainstem [6,7].
α2
-Adrenergic receptors are expressed both presynaptically (auto-receptors) and post-
synaptically [
8
–
10
] in terminal fields in the nervous system, as well as on platelets which
Int. J. Mol. Sci. 2024,25, 4603. https://doi.org/10.3390/ijms25094603 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024,25, 4603 2 of 28
regulate several key physiological processes, including blood pressure, platelet aggre-
gation, insulin secretion, lipolysis, and neurotransmitter release [
11
]. Moreover, acti-
vation or blockade of presynaptic
α2
-adrenergic receptors respectively suppress or en-
hance norepinephrine (NE) release. In contrast, stimulation or antagonism of postsynaptic
α2
-adrenergic receptors exert direct inhibitory or excitatory postsynaptic effects, respec-
tively [
12
,
13
]. Systemic
α2
-adrenergic receptor perturbation can affect NE neurons in the
nucleus of the solitary tract [
14
,
15
], as well as many
α2
-adrenergic receptor-expressing
regions in the nervous system [16–18].
α2
-Adrenergic receptor agonists (e.g., clonidine, xylazine) have been shown to evoke
emesis in both cats and dogs [
19
–
26
]. Moreover, clonidine appears to be the most po-
tent emetic
α2
-adrenergic receptor agonist with an ED
50
of 25
µ
g/kg (i.m.) in dogs and
0.075
µ
g/kg (i.c.v.) in cats [
20
,
21
]. The evoked emesis is thought to be mediated by
α2
-adrenergic receptors, since it was blocked by the prototypical monoterpenoid indole
alkaloid
α2
-adrenergic receptor antagonist, yohimbine [
19
–
21
]. In addition, several stud-
ies also demonstrate that dexmedetomidine can evoke dose-dependent emesis in cats
and dogs [
26
–
29
]. However, in contrast to its emetic effects in cats and dogs, clonidine
failed to trigger vomiting even at large doses in ferrets [
30
], pigeons [
31
], or least shrews
(present study). On the contrary, clonidine was found to prevent vomiting induced by
type 4 phosphodiesterase (PDE4) inhibitors in ferrets, and reserpine-evoked emesis in
pigeons, whereas administration of yohimbine alone in both species produced unexpected
vomiting [
30
,
31
]. Moreover, similar emetic responses were observed in ferrets following
administration of either peripherally acting
α2
-adrenergic receptor antagonist MK-467, or
its brain-penetrating analog MK-912, indicating both a peripheral and a central locus of
action [
30
]. In humans, nausea and vomiting are also common side-effects of parenterally
administered yohimbine [
32
]. In the clinical setting, a number of meta-analysis studies
show that the incidence of post-operative nausea and vomiting (PONV) can be significantly
reduced by both clonidine [33–36] and dexmedetomidine [36,37] in patients.
Shrews are assigned to the order of Insectivora and are among the earliest animals.
Shrews are considered closer to primates than rodents, lagomorphs, and carnivores in the
phylogenetic system [
38
]. Unlike the large emetic animal models discussed above, the least
shrew (Cryptotis parva) is relatively much smaller (3–6 g in weight), and it is a well-validated
experimental emetic model that has been used to test the emetic and antiemetic potential of
diverse drugs [6,39].
Due to the discussed species differences, the present study sought to investigate the
emetic/antiemetic potential of
α2
-adrenergic receptor ligands in the least shrew animal
model of vomiting. Thus, we initially investigated whether intraperitoneal (i.p.) administra-
tion of varying doses of clonidine, dexmedetomidine, or yohimbine can evoke vomiting in
least shrews. We found that the
α2
-adrenergic receptor agonists clonidine and dexmedeto-
midine do not induce vomiting in least shrews, whereas its corresponding antagonist
yohimbine evoked emesis in a dose-dependent and bell-shaped manner. Secondly, we
examined via immunohistochemistry whether the dorsal vagal emetic loci in the least
shrew brainstem mediate the emetic effect of a maximally effective dose of yohimbine
(1.0 mg/kg, i.p.) through induction of c-fos, 5-HT-, and/or SP-release. Thereafter, we further
investigated whether clonidine and dexmedetomidine display broad-spectrum antiemetic
efficacy against diverse emetogens, such as selective agonists of serotonin 5-HT
3
(2-Methyl-
5-HT)-, substance P (SP) neurokinin NK
1
(GR73632)-, muscarinic M
1(McN-A-343)
, and
dopamine D
2
(quinpirole)-receptors, as well as the cannabinoid CB
1
receptor inverse ago-
nist/antagonist SR141716A, and Ca
2+
channel modulators, including the selective L-type
Ca
2+
channel (LTCC) agonist FPL6417 and the sarco/endoplasmic reticulum Ca
2+
-ATPase
(SERCA) inhibitor thapsigargin, which evoke pronounced vomiting in least shrews [
40
,
41
].
We also investigated the antiemetic potential of clonidine and dexmedetomidine against
the PDE4 inhibitor rolipram- [
30
,
42
] and the hyperpolarization-activated cyclic nucleotide-
gated (HCN) channel blocker ZD7288-induced vomiting [43].

Int. J. Mol. Sci. 2024,25, 4603 3 of 28
2. Results
2.1. Dose-Response Emetic Effect of Yohimbine in Least Shrews
Intraperitoneal injection of yohimbine (3 mg/kg) can evoke vomiting in all tested
ferrets [
30
]. In the present study, we initially assessed the emetic/antiemetic potential of
clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p., n= 6–10 per group), dexmedetomidine (0, 0.01,
0.05, 0.1, 0.5, and 1 mg/kg, i.p., n= 6–10 per group), and yohimbine (0, 0.5, 0.75, 1, 1.5, 2,
and 3 mg/kg, i.p., n= 7–14 per group) in the least shrew. Only yohimbine (0, 0.5, 0.75, 1, 1.5,
2, and 3 mg/kg, i.p.) increased both the frequency of emesis and the percentage of shrews
vomiting in a bell-shaped and dose-dependent manner (Figure 1A,B). A Kruskal–Wallis
(KW) non-parametric ANOVA test showed that, relative to the vehicle-pretreated control
group, yohimbine significantly increased the mean vomiting frequency (KW (6, 65) = 14.16;
p= 0.0279) with a maximum (0.85
±
0.22) occurring at 1 mg/kg (p= 0.0259; Dunn’s test).
At a dose of 3 mg/kg, few vomits were observed (Figure 1A). The chi-square test indicated
that the percentage of animals exhibiting emesis in response to yohimbine also showed a
bell-shaped and dose-dependent increase (
χ2
(6, 65) = 9.276, p= 0.1586), but a significant
difference was only observed at the 1 mg/kg dose, which evoked vomits in 61.54% of tested
shrews (p= 0.0445; chi-square test; Figure 1B).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 28
investigated the antiemetic potential of clonidine and dexmedetomidine against the PDE4
inhibitor rolipram- [30,42] and the hyperpolarization-activated cyclic nucleotide-gated
(HCN) channel blocker ZD7288-induced vomiting [43].
2. Results
2.1. Dose-Response Emetic Effect of Yohimbine in Least Shrews
Intraperitoneal injection of yohimbine (3 mg/kg) can evoke vomiting in all tested fer-
rets [30]. In the present study, we initially assessed the emetic/antiemetic potential of
clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p., n = 6–10 per group), dexmedetomidine (0, 0.01,
0.05, 0.1, 0.5, and 1 mg/kg, i.p., n = 6–10 per group), and yohimbine (0, 0.5, 0.75, 1, 1.5, 2,
and 3 mg/kg, i.p., n = 7–14 per group) in the least shrew. Only yohimbine (0, 0.5, 0.75, 1,
1.5, 2, and 3 mg/kg, i.p.) increased both the frequency of emesis and the percentage of
shrews vomiting in a bell-shaped and dose-dependent manner (Figure 1A,B). A Kruskal–
Wallis (KW) non-parametric ANOVA test showed that, relative to the vehicle-pretreated
control group, yohimbine significantly increased the mean vomiting frequency (KW (6,
65) = 14.16; p = 0.0279) with a maximum (0.85 ± 0.22) occurring at 1 mg/kg (p = 0.0259;
Dunn’s test). At a dose of 3 mg/kg, few vomits were observed (Figure 1A). The chi-square
test indicated that the percentage of animals exhibiting emesis in response to yohimbine
also showed a bell-shaped and dose-dependent increase (χ2 (6, 65) = 9.276, p = 0.1586), but
a significant difference was only observed at the 1 mg/kg dose, which evoked vomits in
61.54% of tested shrews (p = 0.0445; chi-square test; Figure 1B).
Figure 1. The pro-emetic effect of the α2-adrenergic receptor antagonist yohimbi ne and the correspond-
ing antiemetic efficacy of the α2-adrenergic receptor agonists clonidine and dexmedetomidine in least
shrews. Different groups of least shrews were given varying doses of yohimbine (0, 0.5, 0.75, 1, 1.5, 2,
3 mg/kg, i.p., n = 7–14 shrews per group (A,B). Emetic parameters were recorded for the next 30 min.
Figure 1. The pro-emetic effect of the
α2
-adrenergic receptor antagonist yohimbine and the corre-
sponding antiemetic efficacy of the
α2
-adrenergic receptor agonists clonidine and dexmedetomidine
in least shrews. Different groups of least shrews were given varying doses of yohimbine (0, 0.5, 0.75,
1, 1.5, 2, 3 mg/kg, i.p., n= 7–14 shrews per group (A,B). Emetic parameters were recorded for the
next 30 min. In drug interaction studies, different groups of least shrews were given an injection
(i.p.) of either the corresponding vehicle, or varying doses of clonidine (0.1, 1, 5, and10 mg/kg, i.p.,
n= 7–13 shrews per group (C,D) or dexmedetomidine (0.01, 0.05, and 0.1 mg/kg, i.p., n= 8–13 shrews
per group (E,F), 30 min prior to an injection of yohimbine (1 mg/kg, i.p.), and were observed for the
next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric one-way
ANOVA followed by Dunn’s post hoc test and presented as mean
±
SEM (A,C,E). The percentage
of shrews vomiting was analyzed with chi-square test and presented as mean (B,D,F). * p< 0.05,
** p< 0.01 vs. 0 mg/kg. The number of animals in each group is presented on the top of the
corresponding column.

Int. J. Mol. Sci. 2024,25, 4603 4 of 28
As shown in Figure 1C, the
α2
-adrenergic receptor agonist clonidine dose-depen-
dently attenuated the frequency of yohimbine (1 mg/kg, i.p.)-induced vomiting (KW
(4, 42) = 10.64; p= 0.0309) with an ID
50
value of 0.205 (0.01437–1.882) mg/kg. Clonidine
completely prevented the evoked vomiting at its 10 mg/kg dose (p= 0.0133). Clonidine
also reduced the percentage of shrews vomiting (
χ2
(4, 42) = 10.98, p= 0.0268) with an
ID
50
value of 0.2093 (0.02186–1.626) mg/kg, with significant reduction occurring at its
5 mg/kg (p= 0.0428) and complete protection at 10 mg/kg (p= 0.0048) (Figure 1D).
Likewise, dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg) reduced both the frequency
(KW (3, 34) = 8.073; p= 0.0445) and percentage (
χ2
(3, 34) = 8.78; p= 0.0324) of shrews
vomiting in response to yohimbine (1 mg/kg, i.p.) in a dose-dependent manner with
ID
50
values of 0.02113 (0.002887–0.0981) mg/kg and 0.02253 (0.004506–0.08576) mg/kg,
respectively (Figure 1E,F). Dexmedetomidine at 0.1 mg/kg completely suppressed both the
mean frequency (p= 0.0177) and the percentage (p= 0.0048) of shrews vomiting in response
to yohimbine (1 mg/kg, i.p.; Figure 1E,F).
2.2. Yohimbine Activates the Brainstem Emetic Nuclei
Since the 1 mg/kg (i.p.) dose of yohimbine caused maximal frequency of emesis in
the tested shrews, we conducted immunohistochemistry to determine c-fos responsiveness
following intraperitoneal administration of this dose of yohimbine. Figure 2A,B show that
very few c-fos positive cells were observed in the DVC emetic nuclei in shrew brainstem
sections from vehicle-treated controls, with mean values of 10.33
±
1.65, 56.5
±
4.99, and
9.67
±
1.15 in the AP, NTS, and DMNX, respectively (Figure 2E). Relative to the vehicle-
treated control group, a 1 mg/kg (i.p.) dose of yohimbine caused significant increases
in c-fos expression in the brainstem throughout the three DVC emetic nuclei. Following
vomiting induced by yohimbine, the average numbers of c-fos positive cells were increased
to 50.17
±
3.98 in the AP (p< 0.0001 vs. Vehicle), 166.3
±
15.41 in the NTS (p< 0.0001 vs.
Vehicle), and 36.17 ±4.73 in DMNX (p= 0.0003 vs. Vehicle), respectively (Figure 2C–E).
2.3. Effect of Yohimbine on 5-HT- and SP-Release in Brainstem Emetic Nuclei
Our previous studies have shown that emetogens (e.g., cisplatin, FPL64176, and
thapsigargin)-evoked emesis were accompanied by increases in 5-HT or SP immunoreactiv-
ity in brainstem emetic nuclei [
41
,
44
,
45
]. In the current study, we tested whether yohimbine
administration (1 mg/kg., i.p.) could also increase 5-HT and SP immunoreactivity in brain-
stem DVC containing emetic nuclei AP, NTS, and DMNX. Least shrews were euthanized at
15 min and 30 min post yohimbine treatment and were subjected to immunohistochemistry
to label 5-HT and SP. A representative image (Figure 3A) of the brainstem DVC emetic
nuclei after 5-HT immunolabelling shows that in vehicle-treated control group, the highest
density of 5-HT-positive fibers are in the dorsomedial subdivision of the NTS. A lower
density of the 5-HT immunoreactive profile is noted in the adjacent subnuclei of NTS
and DMNX. The AP had a few 5-HT-containing neurons. Figure 3D,J show that shrews
exhibited significant increases in 5-HT immunoreactivity in the AP (p= 0.0337 vs. vehicle),
NTS (p= 0.0298 vs. vehicle), and DMNX (p= 0.0446 vs. vehicle) at 15 min post yohimbine
treatment. Figure 3G,J indicate that at 30 min post-injection, 5-HT immunoreactivity in the
NTS, DMNX, and AP returned to basal levels.

Int. J. Mol. Sci. 2024,25, 4603 5 of 28
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 of 28
Figure 2. Immunohistochemical analysis of c-fos following emesis induced by systemic administra-
tion of the α2-adrenergic receptor antagonist yohimbine. Least shrews were sacrificed 90 min post
vehicle treatment, or after the first vomiting occurred post systemic administration of yohimbine (1
mg/kg, i.p., n = 6 shrews per group). Shrew brainstem sections (20 µm) were stained with rabbit c-
fos primary antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were
stained with DAPI in blue. Representative tile-scanned images (20×) show robust c-fos induction in
the area postrema (AP), the nucleus tractus solitarius (NTS), and the dorsal motor nucleus of the
vagus (DMNX) in response to yohimbine (1 mg/kg, i.p.; (C,D)) when compared to the vehicle-
treated group (A,B). Scale bar = 100 µm. Quantified data show the yohimbine-induced c-fos expres-
sion in the AP, NTS, and DMNX in least shrew brainstem (E). Values represent the mean number of
c-fos-positive nuclei of each region (AP/NTS/DMNV) per section and are presented as mean ± SEM
(n = 6 shrews per group). *** p < 0.001, **** p < 0.0001 vs. Vehicle, Unpaired t-test.
Representative images acquired from SP immunolabeling are presented as Figure
3B,E,H. In the brainstem DVC of the vehicle-treated control group (Figure 3B), SP-immu-
noreactive fibers were found in highest concentration within the DMNX and to a lesser
extent in the NTS, but rarely in the AP. Figure 3E,K show that yohimbine caused a signif-
icant increase in SP immunoreactivity only in the DMNX region of the shrew DVC (p =
0.0371 vs. vehicle) at 15 min post yohimbine treatment. Figure 3H,K show the intensity of
SP staining in the DMNX returned to basal level at 30 min post yohimbine injection.
Figure 2. Immunohistochemical analysis of c-fos following emesis induced by systemic administration
of the
α2
-adrenergic receptor antagonist yohimbine. Least shrews were sacrificed 90 min post vehicle
treatment, or after the first vomiting occurred post systemic administration of yohimbine (1 mg/kg,
i.p., n= 6 shrews per group). Shrew brainstem sections (20
µ
m) were stained with rabbit c-fos primary
antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were stained with DAPI
in blue. Representative tile-scanned images (20
×
) show robust c-fos induction in the area postrema
(AP), the nucleus tractus solitarius (NTS), and the dorsal motor nucleus of the vagus (DMNX) in
response to yohimbine (1 mg/kg, i.p.; (C,D)) when compared to the vehicle-treated group (A,B).
Scale bar = 100
µ
m. Quantified data show the yohimbine-induced c-fos expression in the AP, NTS,
and DMNX in least shrew brainstem (E). Values represent the mean number of c-fos-positive nuclei
of each region (AP/NTS/DMNV) per section and are presented as mean
±
SEM (n= 6 shrews per
group). *** p< 0.001, **** p< 0.0001 vs. Vehicle, Unpaired t-test.

Int. J. Mol. Sci. 2024,25, 4603 6 of 28
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 28
Following 5-HT and SP labeling, sections were counterstained with DAPI to visualize cel-
lular nuclei (Figure 3C,F,I).
Figure 3. Immunohistochemical analysis of 5-HT and SP involvement following emesis induced by
systemic administration of the α2-adrenergic receptor antagonist yohimbine. Shrews (n = 5–6 per
gro up) we re euthanized at 15 and 30 min post vehic le or yohim bine (1 mg/kg, i.p.) injection. C orona l
brain sections (20 µm) containing the brainstem DVC emetic nuclei [area postrema (AP), nucleus of
the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMNX)] were immunolabeled with
goat anti-5-HT antibody and rat anti-SP antibody overnight followed by Alexa Fluor 488 donkey
anti-goat and cy3-conjugated donkey anti-rat secondary antibody incubation. After counterstaining
with DAPI, images were acquired using a confocal microscope. Representative tile-scanned images
(20×) images (A–I) show sections from vehicle, 15, and 30 min post yohimbine treated groups la-
beled with anti-5-HT (green), anti-SP antibodies (red), and merged with DAPI (blue), respectively.
Scale bar = 100 µm. Quantified data show the yohimbine-induced release of 5-HT and SP in the AP,
NTS, and DMNX regions of least shrew brainstem, respectively (J,K). Values represent the mean
gray value of released 5-HT and SP of each region (AP/NTS/DMNV) per section and are presented
as mean ± SEM (n = 5–6 shrews per group). * p < 0.05 vs. Vehicle, one-way ANOVA followed by
Dunne’s post hoc test.
Figure 3. Immunohistochemical analysis of 5-HT and SP involvement following emesis induced by
systemic administration of the
α2
-adrenergic receptor antagonist yohimbine. Shrews (n= 5–6 per
group) were euthanized at 15 and 30 min post vehicle or yohimbine (1 mg/kg, i.p.) injection. Coronal
brain sections (20
µ
m) containing the brainstem DVC emetic nuclei [area postrema (AP), nucleus of
the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMNX)] were immunolabeled with
goat anti-5-HT antibody and rat anti-SP antibody overnight followed by Alexa Fluor 488 donkey
anti-goat and cy3-conjugated donkey anti-rat secondary antibody incubation. After counterstaining
with DAPI, images were acquired using a confocal microscope. Representative tile-scanned images
(20
×
) images (A–I) show sections from vehicle, 15, and 30 min post yohimbine treated groups labeled
with anti-5-HT (green), anti-SP antibodies (red), and merged with DAPI (blue), respectively. Scale
bar = 100
µ
m. Quantified data show the yohimbine-induced release of 5-HT and SP in the AP, NTS,
and DMNX regions of least shrew brainstem, respectively (J,K). Values represent the mean gray value
of released 5-HT and SP of each region (AP/NTS/DMNV) per section and are presented as mean
±
SEM (n= 5–6 shrews per group). * p< 0.05 vs. Vehicle, one-way ANOVA followed by Dunnett’s
post hoc test.
Representative images acquired from SP immunolabeling are presented as Figure 3B,E,H.
In the brainstem DVC of the vehicle-treated control group (Figure 3B), SP-immunoreactive
fibers were found in highest concentration within the DMNX and to a lesser extent in the
NTS, but rarely in the AP. Figure 3E,K show that yohimbine caused a significant increase in
SP immunoreactivity only in the DMNX region of the shrew DVC (p= 0.0371 vs. vehicle)
at 15 min post yohimbine treatment. Figure 3H,K show the intensity of SP staining in the
DMNX returned to basal level at 30 min post yohimbine injection. Following 5-HT and SP
labeling, sections were counterstained with DAPI to visualize cellular nuclei (Figure 3C,F,I).

Int. J. Mol. Sci. 2024,25, 4603 7 of 28
2.4. The Broad-Spectrum Antiemetic Potential of the α2-Adrenergic Receptor Agonists Clonidine
and Dexmedetomidine against Vomiting Evoked by Diverse Receptor-Selective Emetogens
Figures 4and 5demonstrate the broad-spectrum antiemetic potential of both clonidine
and dexmedetomidine against vomiting evoked by diverse receptor-selective emetogens, re-
spectively. In fact, pretreatment with clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 7–10 per group),
reduced both the frequency (KW (4, 40) = 26.40; p< 0.0001) and percentage (
χ2
(4, 40) = 26.52;
p< 0.0001) of shrews vomiting in a dose-dependent manner in response to the administration
of the 5-HT
3
receptor-selective agonist, 2-Methyl-5-HT (5 mg/kg, i.p.), with respective ID
50
values of 0.6431 (0.224–1.494) mg/kg and 1.353 (0.7354–2.368) mg/kg (Figure 4A,B). The mean
frequency of 2-Methyl-5-HT-induced emesis was significantly reduced at its 5 mg/kg dose
(p= 0.0003) and with complete suppression at its 10 mg/kg dose (p= 0.0003). Signifi-
cant decreases in the percentage of shrews vomiting were also noted at its 5 mg/kg dose
(90%; p< 0.0001) and complete protection at 10 mg/kg dose (100%; p< 0.0001). Like-
wise, shrews pretreated with dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, n= 8–10
per group) also reduced both the frequency (KW (3, 31) = 8.331; p= 0.0397) and percentage (
χ2
(3, 31) = 10.79; p= 0.0129) of shrews vomiting in a dose-dependent manner in response to 2-
Methyl-5-HT (5 mg/kg, i.p.) with ID
50
values of 0.02641 (0.007458–0.07622) mg/kg and 0.05137
(0.02349–0.1112) mg/kg, respectively (Figure 5A,B). Dexmedetomidine at 0.1 mg/kg signifi-
cantly suppressed both the mean frequency (p= 0.0124) and the percentage (75%; p= 0.005) of
shrews vomiting in response to 2-Methyl-5-HT (5 mg/kg, i.p.).
Next, the antiemetic effect of varying doses of clonidine and dexmedetomidine were
tested against vomiting caused by the NK
1
receptor selective agonist GR73632 (5 mg/kg,
i.p.). As shown in Figure 4C, clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 7–10 per group) also
caused dose-dependent decreases in the mean frequency (KW (4, 39) = 22.46;
p= 0.0002) of vomits induced by GR73632 with significant reductions occurring at its 1 mg/kg
(p= 0.0108)
, 5 mg/kg (p= 0.005), and 10 mg/kg (p= 0.0002) doses, and with an ID
50
value of
0.333 (0.1035–0.9538) mg/kg. The percentage of shrews vomiting was also reduced in a
dose-dependent fashion (
χ2
(4, 39) = 12.51; p= 0.0139) with significant reductions at its 5
(33.33%; p= 0.0466) and 10 mg/kg (71.43%; p= 0.0015) doses, and with an ID
50
value of 6.226
(2.959–13.5) mg/kg (Figure 4D). Figure 5C,D show that dexmedetomidine (0, 0.01, 0.05, and
0.1 mg/kg, n= 7–9 per group) dose-dependently suppressed vomiting caused by GR73632
(5 mg/kg, i.p.). The frequency of GR73632-induced emesis (KW (3, 28) = 8.195; p= 0.0421) was
significantly reduced at 0.1 mg/kg (p= 0.0156) with an ID
50
value of 0.04907 (0.0164–0.1427)
mg/kg (Figure 5C). Likewise, a significant decrease (
χ2
(3, 28) = 9.429; p= 0.0241) in the
percentage of animals vomiting were also noted at its 0.1 mg/kg dose (62.5%; p= 0.0048) with
an ID50 value of 0.08766 (0.04151–0.2017) mg/kg (Figure 5D).
Next, the antiemetic effect of clonidine and dexmedetomidine were assessed against
vomiting caused by the muscarinic M
1
receptor agonist, McN-A-343 (2 mg/kg, i.p.).
Figure 4E,F show that clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 6–9 per group) dose-
dependently suppressed vomiting (KW (4, 34) = 18.96; p= 0.0008) caused by McN-A-343
(2 mg/kg, i.p.). The frequency of McN-A-343-induced emesis was significantly reduced
at its 5 mg/kg dose (p= 0.0084) and completely reduced at its 10 mg/kg dose (p= 0.001)
with an ID
50
value of 0.6007 (0.1618–1.76) mg/kg (Figure 4E). Likewise, significant de-
creases (
χ2
(4, 34) = 20.92; p= 0.0003) in the percentage of animals vomiting were also
noted at its 5 mg/kg dose (75%; p= 0.0019) with complete protection at the 10 mg/kg
dose (100%; p= 0.0002), having an ID
50
value of 1.588 (0.744–3.202) mg/kg (Figure 4F).
Figure 5E,F show that dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, n= 7–8 per group)
also dose-dependently suppressed vomiting caused by McN-A-343 (2 mg/kg, i.p.). The
frequency of McN-A-343-induced emesis was significantly reduced (KW (3, 27) = 11.11;
p= 0.0111) at 0.05 mg/kg (p= 0.0166) and 0.1 mg/kg (p= 0.0089) with an ID
50
value
of 0.02732 (0.009468–0.06793) mg/kg (Figure 5E). However, dexmedetomidine failed to
significantly protect shrews from vomiting (
χ2
(3, 27) = 6.285; p= 0.0985) having an ID
50
value of 0.08931 (0.03922–0.2283) mg/kg (Figure 5F).

Int. J. Mol. Sci. 2024,25, 4603 8 of 28
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 28
Figure 4. The antiemetic effects of the α2-adrenergic receptor agonist clonidine against vomiting
evoked by diverse emetogens. Varying doses of clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) were in-
jected to different groups of shrews 30 min prior to an injection of a fully effective emetic dose of
selective serotonin 5-HT3 receptor agonist 2-Methyl-5-HT (5 mg/kg, i.p., n = 7–10 shrews per group)
(A,B), the selective neurokinin NK1 receptor agonist GR73632 (5 mg/kg, i.p., n = 7–10 shrews per
group) (C,D), the muscarinic M1 receptor agonist McN-A-343 (2 mg/kg, i.p., n = 6–9 shrews per
group) (E,F), the dopamine D2/3 receptor preferring agonist quinpirole (2 mg/kg, i.p., n = 10 shrews
per group) (G,H), or the cannabinoid CB1 receptor-selective inverse agonist/antagonist SR141716A
(20 mg/kg, i.p., n = 8–10 shrews per group) (I,J). Emetic parameters were recorded for the next 30
min. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric one-way ANOVA
followed by Dunn’s post hoc test and presented as mean ± SEM (A,C,E,G,I). The percentage of
shrews vomiting was analyzed with chi-square test and presented as the mean (B,D,F,H,J). * p <
0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 0 mg/kg (controls pretreated with vehicle of clonidine).
The number of animals in each group is presented on the top of the corresponding column.
Thereafter, the antiemetic effect of varying doses of clonidine (0, 0.1, 1, 5, and 10
mg/kg, n = 10 per group) were examined against vomiting caused by the dopamine D2/3
receptor agonist quinpirole (2 mg/kg, i.p.; Figure 4G,H). The mean frequency of
quinpirole-induced emesis (KW (4, 45) = 17.19; p = 0.0018) was significantly reduced by
clonidine at its 1 mg/kg (p = 0.025), 5 mg/kg (p = 0.0051) and 10 mg/kg (p = 0.0013) doses,
with an ID50 value of 0.7356 (0.1996–2.239) mg/kg (Figure 4G). However, there was not a
significant decrease (χ2 (4, 45) = 9.201; p = 0.0563) in the percentage of animals vomiting
with an ID50 value of 3.934 (1.304–10.17) mg/kg (Figure 4H). Pretreatment with dexme-
detomidine (0, 0.01, 0.05, and 0.1 mg/kg, n = 9–11 per group) also dose-dependently sup-
pressed the mean frequency of vomiting (KW (3, 36) = 11.31; p = 0.0102) caused by
quinpirole (2 mg/kg, i.p.; Figure 5G). Significant decreases in the frequency of vomiting
were noted at its 0.05 (p = 0.0188) and 0.1 mg/kg (p = 0.0079) doses with an ID50 value of
0.05819 (0.02685–0.1258) mg/kg (Figure 5G). The percentage of shrews vomiting (χ2 (3, 36)
Figure 4. The antiemetic effects of the
α2
-adrenergic receptor agonist clonidine against vomiting
evoked by diverse emetogens. Varying doses of clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) were
injected to different groups of shrews 30 min prior to an injection of a fully effective emetic dose
of selective serotonin 5-HT
3
receptor agonist 2-Methyl-5-HT (5 mg/kg, i.p., n= 7–10 shrews per
group) (A,B), the selective neurokinin NK
1
receptor agonist GR73632 (5 mg/kg, i.p., n= 7–10 shrews
per group) (C,D), the muscarinic M
1
receptor agonist McN-A-343 (2 mg/kg, i.p., n= 6–9 shrews
per group) (E,F), the dopamine D
2/3
receptor preferring agonist quinpirole (2 mg/kg, i.p., n= 10
shrews per group) (G,H), or the cannabinoid CB
1
receptor-selective inverse agonist/antagonist
SR141716A (20 mg/kg, i.p., n= 8–10 shrews per group) (I,J). Emetic parameters were recorded
for the next
30 min
. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric
one-way ANOVA followed by Dunn’s post hoc test and presented as mean
±
SEM (A,C,E,G,I).
The percentage of shrews vomiting was analyzed with chi-square test and presented as the mean
(B,D,F,H,J).
*p< 0.05,
** p< 0.01, *** p< 0.001, **** p< 0.0001 vs. 0 mg/kg (controls pretreated
with vehicle of clonidine). The number of animals in each group is presented on the top of the
corresponding column.
Thereafter, the antiemetic effect of varying doses of clonidine (0, 0.1, 1, 5, and
10 mg/kg,
n= 10 per group) were examined against vomiting caused by the dopamine D
2/3
receptor
agonist quinpirole (2 mg/kg, i.p.; Figure 4G,H). The mean frequency of quinpirole-induced
emesis (KW (4, 45) = 17.19; p= 0.0018) was significantly reduced by clonidine at its 1 mg/kg
(p= 0.025), 5 mg/kg (p= 0.0051) and 10 mg/kg (p= 0.0013) doses, with an ID
50
value of
0.7356 (0.1996–2.239) mg/kg (Figure 4G). However, there was not a significant decrease
(
χ2
(4, 45) = 9.201; p= 0.0563) in the percentage of animals vomiting with an ID
50
value
of 3.934 (1.304–10.17) mg/kg (Figure 4H). Pretreatment with dexmedetomidine (0, 0.01,
0.05, and 0.1 mg/kg, n= 9–11 per group) also dose-dependently suppressed the mean
frequency of vomiting (KW (3, 36) = 11.31; p= 0.0102) caused by quinpirole (2 mg/kg,
i.p.; Figure 5G). Significant decreases in the frequency of vomiting were noted at its 0.05
(p= 0.0188) and 0.1 mg/kg (p= 0.0079) doses with an ID
50
value of 0.05819 (0.02685–0.1258)

Int. J. Mol. Sci. 2024,25, 4603 9 of 28
mg/kg (Figure 5G). The percentage of shrews vomiting (
χ2
(3, 36) = 11.72; p= 0.0084) was
significantly reduced at its 0.1 mg/kg dose (36.36%, p= 0.0341) with an ID
50
value of 0.276
(0.1526–0.6753) mg/kg (Figure 5H).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 9 of 28
= 11.72; p = 0.0084) was significantly reduced at its 0.1 mg/kg dose (36.36%, p = 0.0341) with
an ID50 value of 0.276 (0.1526–0.6753) mg/kg (Figure 5H).
Figure 5. The antiemetic effects of the α2-adrenergic receptor agonist dexmedetomidine against
vomiting evoked by diverse receptor-selective emetogens. Varying doses of dexmedetomidine (0,
0.01, 0.05, and 0.1 mg/kg, i.p.) were injected to different groups of shrews 30 min prior t o an injection
of a fully effective emetic dose of the selective serotonin 5-HT3 receptor agonist 2-Methyl-5-HT (5
mg/kg, i.p., n = 8–10 shrews per group) (A,B), the selective neurokinin NK1 receptor agonist GR73632
(5 mg/kg, i.p., n = 7–9 shrews per group) (C,D), the muscarinic M1 receptor agonist McN-A-343 (2
mg/kg, i.p., n = 7–8 shrews per group) (E,F), the dopamine D2/3 receptor preferring agonist quinpirole
(2 mg/kg, i.p., n = 9–11 shrews per group) (G,H), or the cannabinoid CB1 receptor-selective inverse
agonist/antagonist SR141716A (20 mg/kg, i.p., n = 8–9 shrews per group) (I,J). Emetic parameters
were recorded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-
parametric one-way ANOVA followed by Dunn’s post hoc test and presented as mean ± SEM
(A,C,E,G,I). The percentage of shrews vomiting was analyzed with chi-square test and presented as
the mean (B,D,F,H,J). * p < 0.05, ** p < 0.01 vs. 0 mg/kg (controls pretreated with vehicle of dexme-
detomidine). The number of animals in each group is presented on the top of the corresponding
column.
We also investigated the antiemetic potential of clonidine and dexmedetomidine
against vomiting evoked by the CB1 receptor inverse agonist/antagonist SR141716A (20
mg/kg, i.p.). As shown in Figure 4I, clonidine (0, 0.1, 1, 5, and 10 mg/kg, n = 8–10 per group)
caused a dose-dependent decrease in the frequency of SR141716A-evoked vomits (KW (4,
41) = 23.22; p = 0.0001) with significant reductions occurring at its 1 mg/kg (p = 0.0244), 5
Figure 5. The antiemetic effects of the
α2
-adrenergic receptor agonist dexmedetomidine against
vomiting evoked by diverse receptor-selective emetogens. Varying doses of dexmedetomidine (0,
0.01, 0.05, and 0.1 mg/kg, i.p.) were injected to different groups of shrews 30 min prior to an injection
of a fully effective emetic dose of the selective serotonin 5-HT
3
receptor agonist 2-Methyl-5-HT
(5 mg/kg, i.p., n= 8–10 shrews per group) (A,B), the selective neurokinin NK
1
receptor agonist
GR73632 (5 mg/kg, i.p., n= 7–9 shrews per group) (C,D), the muscarinic M
1
receptor agonist
McN-A-343 (2 mg/kg, i.p., n= 7–8 shrews per group) (E,F), the dopamine D
2/3
receptor preferring
agonist quinpirole (2 mg/kg, i.p., n= 9–11 shrews per group) (G,H), or the cannabinoid CB
1
receptor-
selective inverse agonist/antagonist SR141716A (20 mg/kg, i.p., n= 8–9 shrews per group) (I,J).
Emetic parameters were recorded for the next 30 min. The frequency of emesis was analyzed with
Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented
as mean
±
SEM (A,C,E,G,I). The percentage of shrews vomiting was analyzed with chi-square test
and presented as the mean (B,D,F,H,J). * p< 0.05, ** p< 0.01 vs. 0 mg/kg (controls pretreated with
vehicle of dexmedetomidine). The number of animals in each group is presented on the top of the
corresponding column.
We also investigated the antiemetic potential of clonidine and dexmedetomidine
against vomiting evoked by the CB
1
receptor inverse agonist/antagonist SR141716A
(20 mg/kg, i.p.). As shown in Figure 4I, clonidine (0, 0.1, 1, 5, and 10 mg/kg,
n= 8–10
per group) caused a dose-dependent decrease in the frequency of SR141716A-evoked

Int. J. Mol. Sci. 2024,25, 4603 10 of 28
vomits (KW (4, 41) = 23.22; p= 0.0001) with significant reductions occurring at its 1 mg/kg
(p= 0.0244), 5 mg/kg (p= 0.0022), and 10 mg/kg (p< 0.0001) doses, and with an ID
50
value of 0.0875 (0.02819–0.2546) mg/kg. The chi-square test indicates that the number
of shrews vomiting in response to SR141716A was also significantly attenuated by cloni-
dine (
χ2
(4, 41) = 19.1; p= 0.0008) with 40% protection at its 0.1 mg/kg (p= 0.0253) and
1 mg/kg (p= 0.0253) doses, 62.5% protection at 5 mg/kg (p= 0.0033), and complete protec-
tion at 10 mg/kg (p< 0.0001) with an ID
50
value of 1.291 (0.3871–3.31) mg/kg (Figure 4J).
Figure 5I,J show that dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, n= 8–9 per group) also
dose-dependently suppressed vomiting caused by SR141716A (20 mg/kg, i.p.). The mean
frequency of SR141716A-induced emesis was significantly reduced (KW
(3, 29) = 13.21
;
p= 0.0042) at 0.1 mg/kg (p= 0.004) with an ID
50
value of 0.02453 (0.007966–0.06693) mg/kg
(Figure 5I). The percentage of shrews vomiting (
χ2
(3, 29) = 17.81; p= 0.0005) was signifi-
cantly reduced at its 0.05 mg/kg (62.5%, p= 0.007)- and 0.1 mg/kg (75%, p= 0.0019) doses
with an ID50 value of 0.04303 (0.02993–0.08087) mg/kg (Figure 5J).
2.5. The Antiemetic Potential of the α2-Adrenergic Receptor Agonists Clonidine and
Dexmedetomidine against Vomiting Evoked by Ca2+ Channel Regulators
We then investigated the antiemetic effect of clonidine and dexmedetomidine against
vomiting evoked by Ca
2+
channel regulators, such as the LTCC agonist FPL64176 (
10 mg/kg,
i.p.) and the SERCA inhibitor thapsigargin (0.5 mg/kg, i.p.) (Figures 6and 7).
Pretreatment with clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 6–9 per group) significantly
attenuated the mean frequency of FPL64176-induced vomiting in a dose-dependent manner
(KW (4, 34) = 19.15; p= 0.0007) with significant reductions occurring at its 5 mg/kg
(p= 0.01) and 10 mg/kg (p= 0.0008; ID
50
value: 0.404 (0.1078–1.332) mg/kg; Figure 6A)
doses. In addition, the percentage of shrews vomiting in response to FPL64176 was
also suppressed by clonidine (
χ2
(4, 34) = 18.34; p= 0.0011) at its 5 mg/kg dose (66.67%,
p= 0.0042) and completely protected at its 10 mg/kg dose (100%, p= 0.0001) with ID
50
value
of 1.661 (0.6772–3.796) mg/kg (Figure 6B). Dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg,
n= 7–11 per group) also dose-dependently suppressed vomiting caused by FPL64176 (KW
(3, 35) = 16.43; p= 0.0009) with significant reductions occurring at its 0.05 (p= 0.0028) and
0.1 mg/kg (p= 0.0009) doses, with an ID
50
value of 0.006993 (0.001346–0.02088) mg/kg
(Figure 7A). Likewise, significant decreases (
χ2
(3, 35) = 11.46; p= 0.0095) in the percentage
of animals vomiting were also noted at its 0.05 (54.55%; p= 0.0057) and 0.1 mg/kg (71.43%;
p= 0.0015) doses with an ID50 value of 0.03667 (0.01645–0.0796) mg/kg (Figure 7B).
Figure 6C,D show that clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 7–9 per group) dose-
dependently suppressed vomiting caused by thapsigargin (0.5 mg/kg, i.p.). The frequency
of thapsigargin-induced emesis (KW (4, 36) = 25.05; p< 0.0001) was significantly reduced
at 1 mg/kg (p= 0.0457), 5 mg/kg (p= 0.0013), and 10 mg/kg (p= 0.0003) doses, with
an ID
50
value of 0.1488 (0.04487–0.4809) mg/kg (Figure 6C). Significant decreases in the
percentage of animals vomiting (
χ2
(4, 36) = 16.38; p= 0.0026) occurred at its 5 mg/kg
(55.56%; p= 0.0121) and 10 mg/kg (66.67%; p= 0.0041) doses with an ID
50
value of 4.691
(2.37–9.161) mg/kg (Figure 6D). As shown in Figure 7C,D, dexmedetomidine (0, 0.01, 0.05,
and 0.1 mg/kg, n= 8–9 per group) also caused a dose-dependent decrease in the mean
frequency of vomits (KW (3, 30) = 8.296; p= 0.0403) induced by the thapsigargin (0.5 mg/kg,
i.p.) with a significant reduction at its 0.1 mg/kg dose (p= 0.0135; ID
50
value: 0.05317
(0.017–0.2617) mg/kg; Figure 7C), but no significant decrease (
χ2
(3, 30) = 5.542; p= 0.1362)
in the percentage of animals vomiting was observed (ID
50
value: 0.1372 (0.06515–0.363)
mg/kg; Figure 7D).

Int. J. Mol. Sci. 2024,25, 4603 11 of 28
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 28
mg/kg (p = 0.0022), and 10 mg/kg (p < 0.0001) doses, and with an ID50 value of 0.0875
(0.02819–0.2546) mg/kg. The chi-square test indicates that the number of shrews vomiting
in response to SR141716A was also significantly aenuated by clonidine (χ2 (4, 41) = 19.1;
p = 0.0008) with 40% protection at its 0.1 mg/kg (p = 0.0253) and 1 mg/kg (p = 0.0253) doses,
62.5% protection at 5 mg/kg (p = 0.0033), and complete protection at 10 mg/kg (p < 0.0001)
with an ID50 value of 1.291 (0.3871–3.31) mg/kg (Figure 4J). Figure 5I,J show that dexme-
detomidine (0, 0.01, 0.05, and 0.1 mg/kg, n = 8–9 per group) also dose-dependently sup-
pressed vomiting caused by SR141716A (20 mg/kg, i.p.). The mean frequency of
SR141716A-induced emesis was significantly reduced (KW (3, 29) = 13.21; p = 0.0042) at
0.1 mg/kg (p = 0.004) with an ID50 value of 0.02453 (0.007966–0.06693) mg/kg (Figure 5I).
The percentage of shrews vomiting (χ2 (3, 29) = 17.81; p = 0.0005) was significantly reduced
at its 0.05 mg/kg (62.5%, p = 0.007)- and 0.1 mg/kg (75%, p = 0.0019) doses with an ID50
value of 0.04303 (0.02993–0.08087) mg/kg (Figure 5J).
2.5. The Antiemetic Potential of the α2-Adrenergic Receptor Agonists Clonidine and
Dexmedetomidine against Vomiting Evoked by Ca2+ Channel Regulators
We then investigated the antiemetic effect of clonidine and dexmedetomidine against
vomiting evoked by Ca2+ channel regulators, such as the LTCC agonist FPL64176 (10
mg/kg, i.p.) and the SERCA inhibitor thapsigargin (0.5 mg/kg, i.p.) (Figures 6 and 7).
Figure 6. The antiemetic effects of the α2-adrenergic receptor agonist clonidine against vomiting
caused by Ca2+ channel regulators, the LTCC agonist FPL64176 and the SERCA inhibitor thapsigar-
gin. Varying doses of clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) were injected to different groups of
shrews 30 min prior to an injection of a fully effective emetic dose of FPL64176 (10 mg/kg, i.p., n =
Figure 6. The antiemetic effects of the
α2
-adrenergic receptor agonist clonidine against vomiting caused
by Ca
2+
channel regulators, the LTCC agonist FPL64176 and the SERCA inhibitor thapsigargin. Varying
doses of clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) were injected to different groups of shrews 30 min
prior to an injection of a fully effective emetic dose of FPL64176 (10 mg/kg, i.p., n= 6–9 per group)
(A,B) or thapsigargin (0.5 mg/kg, i.p., n= 7–9 per group) (C,D). Emetic parameters were recorded
for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric one-
way ANOVA followed by Dunn’s post hoc test and presented as mean
±
SEM (A,C). The percentage
of shrews vomiting was analyzed with chi-square test and presented as the mean (B,D). * p< 0.05,
** p< 0.01, *** p< 0.001 vs. 0 mg/kg (controls pretreated with vehicle of clonidine). The number of
animals in each group is presented on the top of the corresponding column.

Int. J. Mol. Sci. 2024,25, 4603 12 of 28
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 11 of 28
6–9 per group) (A,B) or thapsigargin (0.5 mg/kg, i.p., n = 7–9 per group) (C,D). Emetic parameters
were recorded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-
parametric one-way ANOVA followed by Dunn’s post hoc test and presented as mean ± SEM (A,
C). The percentage of shrews vomiting was analyzed with chi-square test and presented as the mean
(B, D). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 mg/kg (controls pretreated with vehicle of clonidine).
The number of animals in each group is presented on the top of the corresponding column.
Figure 7. The antiemetic effects of the α2-adrenergic receptor agonist dexmedetomidine against
vomiting caused by Ca2+ channel regulators, the LTCC agonist FPL64176 and the SERCA inhibitor
thapsigargin. Varying doses of dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, i.p.) were injected to
different groups of shrews 30 min prior to an injection of a fully effective emetic dose of FPL64176
(10 mg/kg, i.p., n = 7–11 per group) (A,B) or thapsigargin (0.5 mg/kg, i.p., n = 8–9 per group) (C,D).
Emetic parameters were recorded for the next 30 min. The frequency of emesis was analyzed with
Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented
as mean ± SEM (A,C). The percentage of shrews vomiting was analyzed with chi-square test and
presented as the mean (B,D). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. 0 mg/kg (controls pretreated with
vehicle of dexmedetomidine). The number of animals in each group is presented on the top of the
corresponding column.
Pretreatment with clonidine (0, 0.1, 1, 5, and 10 mg/kg, n = 6–9 per group) significantly
aenuated the mean frequency of FPL64176-induced vomiting in a dose-dependent man-
ner (KW (4, 34) = 19.15; p = 0.0007) with significant reductions occurring at its 5 mg/kg (p
= 0.01) and 10 mg/kg (p = 0.0008; ID50 value: 0.404 (0.1078–1.332) mg/kg; Figure 6A) doses.
In addition, the percentage of shrews vomiting in response to FPL64176 was also
Figure 7. The antiemetic effects of the
α2
-adrenergic receptor agonist dexmedetomidine against
vomiting caused by Ca
2+
channel regulators, the LTCC agonist FPL64176 and the SERCA inhibitor
thapsigargin. Varying doses of dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, i.p.) were injected to
different groups of shrews 30 min prior to an injection of a fully effective emetic dose of FPL64176
(10 mg/kg, i.p., n= 7–11 per group) (A,B) or thapsigargin (0.5 mg/kg, i.p., n= 8–9 per group) (C,D).
Emetic parameters were recorded for the next 30 min. The frequency of emesis was analyzed with
Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented
as mean
±
SEM (A,C). The percentage of shrews vomiting was analyzed with chi-square test and
presented as the mean (B,D). * p< 0.05, ** p< 0.01, *** p< 0.001 vs. 0 mg/kg (controls pretreated with
vehicle of dexmedetomidine). The number of animals in each group is presented on the top of the
corresponding column.
2.6. The Antiemetic Potential of Clonidine and Dexmedetomidine against Vomiting Evoked
by Rolipram, the Inhibitor of PDE4 in Shrews
In the ferret, clonidine was found to prevent emesis induced by PDE4 inhibitors,
such as PMNPQ (i.e., 6-(4-pyridylmethyl)-8-(3-nitrophenyl) quinoline, rolipram, and CT–
2450 (i.e., (R)-N-{4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4- pyridyl)ethyl]phenyl}N9-
ethylurea) [
30
]. In the present study, we explored the antiemetic potential of the
α2
-
adrenergic receptor agonists clonidine and dexmedetomidine against rolipram (1 mg/kg,
i.p.)-induced vomiting in shrews [42].
As shown in Figure 8A, clonidine (0, 0.1, 1, 5, and 10 mg/kg, n= 8–10 per group) caused
dose-dependent decreases in the frequency of rolipram-evoked vomits ((KW (4, 41) = 10.9;
p= 0.0277) with significant reductions occurring at its 5 mg/kg (p= 0.0332) and 10 mg/kg
(p= 0.0182) doses, with an ID
50
value of 1.024 (0.1497–4.052) mg/kg. The percentage of
animals vomiting in response to rolipram (
χ2
(4, 41) = 11.21; p= 0.0243) was also significantly
reduced at its 0.1 mg/kg (40%, p= 0.0253), 1 mg/kg (60%, p= 0.0034), 5 mg/kg (62.5%,
p= 0.0033) and 10 mg/kg (62.5%, p= 0.0033) doses with an ID
50
value of 0.7897
(0.1143–3.64)

Int. J. Mol. Sci. 2024,25, 4603 13 of 28
mg/kg (Figure 8B). Dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg, n= 10 per group)
also dose-dependently suppressed the mean vomiting frequency evoked by rolipram (KW
(3, 36) = 10.7; p= 0.0135) with significant reductions at its 0.05 mg/kg (p= 0.0305) and
0.1 mg/kg (p= 0.0084) doses, and with an ID
50
value of 0.01748 (0.004243–0.05612) mg/kg
(Figure 8C). Significant decreases (
χ2
(3, 36) = 9.167; p= 0.0272) in the percentage of animals
vomiting were also noted at its 0.01 mg/kg (50%; p= 0.0098), 0.05 mg/kg (50%; p= 0.0098)
and 0.1 mg/kg (60%; p= 0.0034) doses with an ID
50
value of 0.03517 (0.011–0.09391) mg/kg
(Figure 8D).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 13 of 28
Figure 8. The antiemetic effects o f the α2-adrenergic receptor agonists clonidine and dexmedetomidine
against vomiting caused by the PDE4 inhibitor rolipram (1 mg/kg, i.p.). Varying doses of clonidine (0,
0.1, 1, 5, and 10 mg/kg, i.p., n = 8–10 per group; (A,B)) or dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg,
i.p., n = 10 per group; (C,D)) were injected to different groups of shrews 30 min prior to an injection of
a fully effective emetic dose of PDE4 inhibitor rolipram (1 mg/kg, i.p.). Emetic parameters were rec-
orded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric
one-way ANOVA followed by Dunn’s post hoc test and presented as mean ± SEM (A,C). The percent-
age of shrews vomiting was analyzed with chi-square test and presented as the mean (B,D). * p < 0.05,
** p < 0.01 vs. 0 mg/kg (controls pretreated with vehi cle of clonidine or dexmedetomidine). The number
of animals in each group is presented on the top of the corresponding column.
2.7. The Antiemetic Effect of Clonidine and Dexmedetomidine against Vomiting Evoked by the
HCN Channel Blocker ZD7288
Our previous study has shown that the HCN channel blocker ZD7288 induces vom-
iting in a dose-dependent manner, with maximal efficacy of 100% at 1 mg/kg (i.p.) [43]. In
the current study, the antiemetic potential of clonidine and dexmedetomidine on ZD7288
(1 mg/kg, i.p.)-induced vomiting was also examined (Figure 9). Pretreatment with
clonidine (0, 0.1, 1, 5, and 10 mg/kg, n = 10 per group) significantly aenuated the mean
frequency of ZD7288-induced vomiting in a dose-dependent manner (KW (4, 40) = 29.08;
p < 0.0001) with significant reductions occurring at its 5 mg/kg (p = 0.0003) and 10 mg/kg
(p < 0.0001; ID50 value: 0.4023 (0.1636–0.8948) mg/kg; Figure 9A) doses. Significant de-
creases in the percentage of animals vomiting (χ2 (4, 40) = 17.22; p = 0.0017) also occurred
at its 5 mg/kg (44.44%; p = 0.0233) and 10 mg/kg (55.56%; p = 0.0085) doses with an ID50
Figure 8. The antiemetic effects of the
α2
-adrenergic receptor agonists clonidine and dexmedetomidine
against vomiting caused by the PDE4 inhibitor rolipram (1 mg/kg, i.p.). Varying doses of clonidine (0,
0.1, 1, 5, and 10 mg/kg, i.p., n= 8–10 per group; (A,B)) or dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg,
i.p., n= 10 per group; (C,D)) were injected to different groups of shrews 30 min prior to an injection
of a fully effective emetic dose of PDE4 inhibitor rolipram (1 mg/kg, i.p.). Emetic parameters were
recorded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-parametric
one-way ANOVA followed by Dunn’s post hoc test and presented as mean
±
SEM (A,C). The percentage
of shrews vomiting was analyzed with chi-square test and presented as the mean (B,D). * p< 0.05,
** p< 0.01 vs. 0 mg/kg (controls pretreated with vehicle of clonidine or dexmedetomidine). The number
of animals in each group is presented on the top of the corresponding column.
2.7. The Antiemetic Effect of Clonidine and Dexmedetomidine against Vomiting Evoked by the
HCN Channel Blocker ZD7288
Our previous study has shown that the HCN channel blocker ZD7288 induces vomit-
ing in a dose-dependent manner, with maximal efficacy of 100% at 1 mg/kg (i.p.) [
43
]. In
the current study, the antiemetic potential of clonidine and dexmedetomidine on ZD7288
(1 mg/kg, i.p.)-induced vomiting was also examined (Figure 9). Pretreatment with clonidine
(0, 0.1, 1, 5, and 10 mg/kg, n= 10 per group) significantly attenuated the mean frequency
of ZD7288-induced vomiting in a dose-dependent manner (KW (4, 40) = 29.08; p< 0.0001)

Int. J. Mol. Sci. 2024,25, 4603 14 of 28
with significant reductions occurring at its 5 mg/kg (p= 0.0003) and 10 mg/kg (p< 0.0001;
ID
50
value: 0.4023 (0.1636–0.8948) mg/kg; Figure 9A) doses. Significant decreases in the
percentage of animals vomiting (
χ2
(4, 40) = 17.22; p= 0.0017) also occurred at its 5 mg/kg
(44.44%; p= 0.0233) and 10 mg/kg (55.56%; p= 0.0085) doses with an ID
50
value of 7.819
(4.363–14.69) mg/kg (Figure 9B). Administration of dexmedetomidine (0, 0.01, 0.05, and
0.1 mg/kg, n= 7–10 per group) also suppressed the mean frequency of vomiting evoked by
ZD7288 (KW (3, 30) = 8.11; p= 0.0438) with a significant reduction at its 0.1 mg/kg dose
(p= 0.0155), and with an ID
50
value of 0.08931 (0.03732–0.2473) mg/kg (Figure 9C). How-
ever, dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg) failed to significantly protect shrews
from vomiting (χ2(3, 30) = 3.974; p= 0.2643; Figure 9D).
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 14 of 28
value of 7.819 (4.363–14.69) mg/kg (Figure 9B). Administration of dexmedetomidine (0,
0.01, 0.05, and 0.1 mg/kg, n = 7–10 per group) also suppressed the mean frequency of vom-
iting evoked by ZD7288 (KW (3, 30) = 8.11; p = 0.0438) with a significant reduction at its
0.1 mg/kg dose (p = 0.0155), and with an ID50 value of 0.08931 (0.03732–0.2473) mg/kg (Fig-
ure 9C). However, dexmedetomidine (0, 0.01, 0.05, and 0.1 mg/kg) failed to significantly
protect shrews from vomiting (χ2 (3, 30) = 3.974; p = 0.2643; Figure 9D).
Figure 9. The antiemetic effects of the α2-adrenergic receptor agonists clonidine and dexmedetomi-
dine against vomiting caused by the HCN channel blocker ZD7288 (1 mg/kg, i.p.). Varying doses of
clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p., n = 9 per group; (A,B)) or dexmedetomidine (0, 0.01, 0.05,
and 0.1 mg/kg, i.p., n = 7–10 per group; (C,D)) were injected to different groups of shrews 30 min
prior to an injection of a fully effective emetic dose of ZD7288 (1 mg/kg, i.p.). Emetic parameters
were recorded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis non-
parametric one-way AN OVA followed by Dunn’s post hoc test and presented as mean ± SEM (A,C).
The percentage of shrews vomiting was analyzed with chi-square test and presented as the mean
(B,D). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. 0 mg/kg (controls pretreated with vehicle
of clonidine or dexmedetomidine). The number of animals in each group is presented on the top of
the corresponding column.
2.8. Open-Field Locomotor Studies
Figure 10 shows the effects of corresponding vehicle, varying doses of clonidine (0.01,
0.1, 1, 5, and 10 mg/kg, i.p.), and dexmedetomidine (0.01, 0.05, 0.1, and 0.5 mg/kg, i.p.) on
the locomotor activities of shrews using the open-field locomotor test. A one-way ANOVA
analysis demonstrated that clonidine significantly decreased (F 5, 39 = 5.255; p = 0.0009) the
Figure 9. The antiemetic effects of the
α2
-adrenergic receptor agonists clonidine and dexmedetomi-
dine against vomiting caused by the HCN channel blocker ZD7288 (1 mg/kg, i.p.). Varying doses of
clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p., n= 9 per group; (A,B)) or dexmedetomidine (0, 0.01, 0.05,
and 0.1 mg/kg, i.p., n= 7–10 per group; (C,D)) were injected to different groups of shrews 30 min
prior to an injection of a fully effective emetic dose of ZD7288 (1 mg/kg, i.p.). Emetic parameters
were recorded for the next 30 min. The frequency of emesis was analyzed with Kruskal–Wallis
non-parametric one-way ANOVA followed by Dunn’s post hoc test and presented as mean
±
SEM
(A,C). The percentage of shrews vomiting was analyzed with chi-square test and presented as the
mean (B,D). * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001 vs. 0 mg/kg (controls pretreated with
vehicle of clonidine or dexmedetomidine). The number of animals in each group is presented on the
top of the corresponding column.
2.8. Open-Field Locomotor Studies
Figure 10 shows the effects of corresponding vehicle, varying doses of clonidine (0.01,
0.1, 1, 5, and 10 mg/kg, i.p.), and dexmedetomidine (0.01, 0.05, 0.1, and 0.5 mg/kg, i.p.) on
the locomotor activities of shrews using the open-field locomotor test. A one-way ANOVA
analysis demonstrated that clonidine significantly decreased (F
5, 39
= 5.255; p= 0.0009)

Int. J. Mol. Sci. 2024,25, 4603 15 of 28
the total distance moved by the shrews at its 1 mg/kg (p= 0.0015), 5 mg/kg (p= 0.0225),
and 10 mg/kg (p= 0.0114) doses in the 30 min observation period (Figure 10A). Like-
wise, the frequencies of rearing behavior were also significantly attenuated (F
5, 39
= 5.223;
p= 0.0009) by clonidine at its 1 mg/kg (p= 0.0299), 5 mg/kg (p= 0.0013), and
10 mg/kg
(p= 0.0014; Figure 10B) doses. Relative to the corresponding vehicle-pretreated control
group, dexmedetomidine only significantly decreased the total distance moved
(F
4, 32
= 6.877; p= 0.0004) and the frequency of rearing behavior (F
4, 32
= 4.758;
p= 0.004) at its 0.5 mg/kg dose (p= 0.0002 for distance moved, Figure 10C; p= 0.0009 for
rearing frequency, Figure 10D), which is larger than its antiemetic doses.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 15 of 28
total distance moved by the shrews at its 1 mg/kg (p = 0.0015), 5 mg/kg (p = 0.0225), and
10 mg/kg (p = 0.0114) doses in the 30 min observation period (Figure 10A). Likewise, the
frequencies of rearing behavior were also significantly aenuated (F 5, 39 = 5.223; p = 0.0009)
by clonidine at its 1 mg/kg (p = 0.0299), 5 mg/kg (p = 0.0013), and 10 mg/kg (p = 0.0014;
Figure 10B) doses. Relative to the corresponding vehicle-pretreated control group, dex-
medetomidine only significantly decreased the total distance moved (F 4, 32 = 6.877; p =
0.0004) and the frequency of rearing behavior (F 4, 32 = 4.758; p = 0.004) at its 0.5 mg/kg dose
(p = 0.0002 for distance moved, Figure 10C; p = 0.0009 for rearing frequency, Figure 10D),
which is larger than its antiemetic doses.
Figure 10. The effect of the α2-adrenergic receptor agonists clonidine and dexmedetomidine on
open-field locomotor behaviors. Different groups of shrews were injected with either vehicle or var-
ying doses of clonidine (0.1, 1, 5, and 10 mg/kg, i.p., n = 6–10; (A,B))/dexmedetomidine (0.01, 0.05,
and 0.1 mg/kg, i.p., n = 5–10; (C,D)) at 0 min. Thirty minutes later, each shrew was individually
placed in a white observation cage (27.5 × 27.5 × 28 cm), and the locomotor activity [distance moved
(A,C) and rearing frequency (B,D)] were recorded for 30 min by a computerized video tracking,
motion analysis, and behavior recognition system (Ethovision, version XT 9). The dose-response
effects of clonidine and dexmedetomidine on different groups of least shrews were analyzed using
the Noldus software. Significant difference relative to the corresponding vehicle group is indicated
as * p < 0.05, ** p < 0.01, *** p < 0.001. The number of animals in each group is presented on the top
of the corresponding column.
3. Discussion Software
3.1. Significance of the Present Study
Per the Introduction Section, currently there is significant confusion in the published
literature on the ability of α2-adrenergic receptor ligands evoking emetic/antiemetic ef-
fects, which are probably due to: (i) species variation in the array of animal models (e.g.,
cats, dogs, ferrets, pigeons, least shrews, humans) employed for emesis studies in different
laboratories, (ii) lack of published comprehensive studies investigating the emetic/antie-
metic potential of α2-adrenergic receptor agonists and antagonists in a given laboratory
using one emesis model, and (iii) full pharmacological assessment of α2-adrenergic
Figure 10. The effect of the
α2
-adrenergic receptor agonists clonidine and dexmedetomidine on
open-field locomotor behaviors. Different groups of shrews were injected with either vehicle or
varying doses of clonidine (0.1, 1, 5, and 10 mg/kg, i.p., n= 6–10; (A,B))/dexmedetomidine (0.01,
0.05, and 0.1 mg/kg, i.p., n= 5–10; (C,D)) at 0 min. Thirty minutes later, each shrew was individually
placed in a white observation cage (27.5
×
27.5
×
28 cm), and the locomotor activity [distance moved
(A,C) and rearing frequency (B,D)] were recorded for 30 min by a computerized video tracking,
motion analysis, and behavior recognition system (Ethovision, version XT 9). The dose-response
effects of clonidine and dexmedetomidine on different groups of least shrews were analyzed using
the Noldus software. Significant difference relative to the corresponding vehicle group is indicated as
*p< 0.05, ** p< 0.01, *** p< 0.001. The number of animals in each group is presented on the top of the
corresponding column.
3. Discussion
3.1. Significance of the Present Study
Per the Introduction Section, currently there is significant confusion in the published
literature on the ability of
α2
-adrenergic receptor ligands evoking emetic/antiemetic effects,
which are probably due to: (i) species variation in the array of animal models (e.g., cats,
dogs, ferrets, pigeons, least shrews, humans) employed for emesis studies in different labo-
ratories, (ii) lack of published comprehensive studies investigating the emetic/antiemetic
potential of
α2
-adrenergic receptor agonists and antagonists in a given laboratory using one
emesis model, and (iii) full pharmacological assessment of
α2
-adrenergic receptor ligands
against diverse emetogens. In the present study, we investigated the emetic/antiemetic

Int. J. Mol. Sci. 2024,25, 4603 16 of 28
potential of two
α2
-adrenergic receptor agonists (clonidine and dexmedetomidine), as
well as an
α2
-adrenergic receptor antagonist yohimbine, in the least shrew animal model
of vomiting. Only yohimbine increased both the mean frequency of emesis and the per-
centage of shrews vomiting. The evoked vomiting was bell-shaped and occurred in a
dose-dependent manner with a maximal efficacy of 1 mg/kg. However, only 61.5% of
least shrews vomited at the 1 mg/kg dose, whereas larger doses of yohimbine were less
efficacious. Our immunohistochemical findings demonstrated that yohimbine (1 mg/kg,
i.p.) causes significant increases in both c-fos expression and release of 5-HT and SP in the
shrew brainstem DVC emetic nuclei. Furthermore, both
α2
-adrenergic receptor agonists
not only suppressed yohimbine-induced vomiting in a dose-dependent manner, but also
emesis evoked by: (1) selective agonists of diverse emetic receptors including serotonin
5-HT
3
(2-Methyl-5-HT), SP neurokinin NK
1
(GR73632), muscarinic M
1
(MCN-A-343), and
dopamine D
2/3
(quinpirole); (2) the selective CB
1
receptor inverse agonist/antagonist
(SR141716A); (3) Ca
2+
channel modulators including the LTCC agonist FPL64176 and the
SERCA inhibitor thapsigargin; (4) the PDE4 inhibitor rolipram; and 5) the HCN channel
blocker ZD7288. Clonidine at antiemetic doses (1, 5, and 10 mg/kg, i.p.) decreased the
locomotor activity of shrews in the open-field test, whereas dexmedetomidine did not
influence shrew locomotion at its tested antiemetic doses.
3.2. Emetic Effect of Yohimbine in the Least Shrew
Existing experimental evidence suggests a role for
α2
-adrenergic receptor in emesis.
The
α2
-adrenergic receptor agonist clonidine has been shown to evoke vomiting in a
dose-dependent fashion in both cats and dogs in an
α2
-adrenergic receptor antagonist
(yohimbine)-sensitive manner [
19
–
21
]. However, clonidine was found to prevent vomiting
caused by either rolipram (an inhibitor of PDE4) in ferrets [
30
], or reserpine (a monoamine
releaser) in pigeons [
31
]. Moreover, yohimbine not only induced dose-dependent emesis in
both ferrets and pigeons, but also potentiated reserpine-induced vomiting and reversed
the inhibitory effect of clonidine against reserpine-evoked vomiting [
30
,
31
]. However, the
discussed emetic studies in pigeons and ferrets were limited in scope since yohimbine’s
full dose-response emetic effects were not evaluated, but 100% of both species vomited
in response to an i.p. injection of its 0.5 or 3 mg/kg doses, respectively [
30
,
31
]. In the
present study, yohimbine evoked vomiting in a dose-dependent, but bell-shaped manner,
with a maximal efficacy of 0.85
±
0.22 vomits at 1 mg/kg dose in 61.54% of tested shrews,
whereas its larger doses were less efficacious. A similar bell-shaped dose-response effect
has also been observed in aggression studies where smaller doses of yohimbine increased,
and larger doses decreased aggressive behavior in rats [
46
]. The bell-shaped dose-response
effects may be due to the non-selective nature of yohimbine since it does not exclusively
bind to
α2
-adrenergic receptors as it also (i) has moderate to weak affinity for dopamine D
2
-,
adrenergic
α1
-, and serotonergic 5-HT
1A
receptors [
47
–
49
], and (ii) can release monoamines
(NE, DA, and 5-HT) both in the periphery and the CNS [50,51].
3.3. Yohimbine-Induced Expression of c-fos and Release of 5-HT and SP in the DVC Central
Emetic Loci
Induction of c-fos immunoreactivity is an indirect but classical tool to evaluate neu-
ronal activation following peripheral agonist administration [
52
]. Our lab has also utilized
c-fos induction in the least shrew brainstem DVC emetic nuclei to demonstrate central
responsiveness to peripheral administration of diverse emetogens [
41
,
53
]. In the current
study, relative to vehicle injection, vomiting induced by yohimbine (i.p.) was followed by
increased expression of c-fos in all the DVC emetic nuclei (the AP, NTS, and DMNV) in
the shrew brainstem. Likewise, we have previously shown that the nonspecific emetogen
cisplatin and the more selective 5-HT
3
receptor agonist 2-Methyl-5-HT can evoke vomiting
in least shrews and c-fos expression in their corresponding DVC emetic nuclei AP, NTS, and
DMNV [
53
]. Such a pattern of c-fos expression in the DVC emetic nuclei is not surprising
since the AP, NTS, and DMNX are populated by neurons containing a number of emetic

Int. J. Mol. Sci. 2024,25, 4603 17 of 28
neurotransmitters (e.g., 5-HT, DA) and corresponding receptors, and systemic administra-
tion of yohimbine promotes their release both in the periphery and brainstem [
50
,
51
]. In
fact, prior depletion of emetic monoamines with reserpine can completely prevent vomiting
caused by yohimbine or reserpine in pigeons, suggesting both yohimbine and reserpine
may induce emesis by releasing monoamines, albeit via different mechanisms [31].
5-HT is an important gastrointestinal signaling molecule. In the CNS it regulates
appetite and emesis, and in the gastrointestinal tract 5-HT is an important mediator of
sensation (e.g., nausea and emesis) between the intestine and the brainstem [
54
]. It can
evoke vomiting via peripheral stimulation of 5-HT
3
receptors in least shrews, whereas
its brain penetrant analog 2-Methyl-5-HT involves both central and peripheral compo-
nents
[3,55]
. We have previously demonstrated that the LTCC agonist FPL64176-induced
emesis was accompanied by an increase in 5-HT immunoreactivity in the dorsomedial
subdivision of the NTS [
45
]. In the present study, compared to vehicle pretreatment, a 15
min exposure to yohimbine resulted in a moderate enhancement of 5-HT immunoreac-
tivity in the AP, NTS, and DMNX. Indeed, yohimbine has been shown to release emetic
monoamines, including 5-HT, via blockade of presynaptic
α2
-adrenergic receptors [
55
–
59
].
In the current study, the yohimbine-evoked increases in 5-HT-positive fibers were most
abundant in the least shrew dorsomedial area of the NTS, followed by smaller increases in
the adjacent subnuclei of the NTS and DMNX, but the AP contained fewer 5-HT-containg
neurons. In the current study, yohimbine also evoked an increase in SP immunoreactivity in
the DMNX at 15 min post treatment, which is not surprising, since release of stimuli-evoked
SP in the rat or rabbit spinal dorsal horn can be modulated by yohimbine [
60
,
61
]. The
current finding agrees with our published study which demonstrated that a 15–30 min
thapsigargin exposure (0.5 mg/kg, i.p.), can increase the SP tissue content up to ~3 times
over the basal level in the least shrew DMNX [
41
]. The DMNX is known to send appropriate
output to the gastrointestinal tract to alter gastric motility [
53
,
62
]. Thus, yohimbine-evoked
enhancements in 5-HT and SP immunoreactivity in the DVC suggests that both 5-HT and
SP are likely to be involved in the evoked emesis.
3.4. Effects of α2-Adrenergic Receptor Agonists Clonidine and Dexmedetomidine
on Locomotor Activity
A major concern regarding
α2
-adrenergic receptor agonists is the possibility of se-
dation [
63
]. Although the affinity of dexmedetomidine is eight times greater than cloni-
dine [
64
], similar low doses of both
α2
-adrenergic agonists (0.03–0.1 mg/kg, i.p.) reduce
spontaneous locomotor activity in rodents [
65
–
68
]. Thus, we examined the motor suppres-
sive effects of both clonidine and dexmedetomidine. Clonidine at 0.01 and 0.1 mg/kg doses
did not significantly affect either spontaneous locomotor activity (i.e., distance moved) or
the frequency of rearing behavior in least shrews, but significantly decreased both behaviors
at its antiemetic doses (1, 5, and 10 mg/kg) in the 30 min observation period. This should
not be surprising since, unlike rodents, least shrews are constantly on the move and rarely
remain motionless. Some clinical findings suggest that although sedation is a well-known
side-effect of clonidine [69], it does not appear to be related to its antiemetic property. For
example, Kumar et al. [
70
] have shown that in elderly patients undergoing intraocular
surgery, clonidine had no effect on emesis despite the fact that it induced more seda-
tion. In contrast to clonidine, the tested antiemetic doses of dexmedetomidine (0.01, 0.05,
and 0.1 mg/kg) in the current study did not significantly decrease either shrew motor
parameters. Overall, our data provide evidence for the safe use of dexmedetomidine as an
antiemetic drug at human doses equivalent to less than 0.1 mg/kg in least shrews.
3.5. Antiemetic Effects of α2-Adrenergic Receptor Agonists Clonidine and Dexmedetomidine
against Yohimbine and Diverse Emetogens
In contrast to cats and dogs (see introduction), both the non-selective (clonidine)
and the more selective
α2
-adrenergic receptor agonist dexmedetomidine failed to trigger
vomiting in shrews even at doses 333–400 times greater than their corresponding ED
50
values reported in dogs (clonidine: 25
µ
g/kg, i.m.; dexmedetomidine: 0.003 mg/kg,

Int. J. Mol. Sci. 2024,25, 4603 18 of 28
s.c.; [
20
,
26
]). Subsequently, we investigated the antiemetic potential of clonidine and
dexmedetomidine, and found that both
α2
-adrenergic receptor agonists dose-dependently
reduce to varying degrees both the frequency and percentage of shrews vomiting caused
by diverse emetogens, including the following.
1.
The
α2
-adrenergic receptor antagonist yohimbine (1 mg/kg, i.p.): Both clonidine and
dexmedetomidine reduced the mean frequency and percentage of shrews vomiting
in response to yohimbine in a dose-dependent manner with respective ID
50
values
ranging between 0.21 mg/kg and 0.021 mg/kg, respectively. Thus, dexmedetomidine
appears to be ten times more potent antiemetic than clonidine against yohimbine-
evoked emesis, and both agents completely protected shrews from vomiting at their
maximal tested dose of 0.1 and 10 mg/kg, respectively. Likewise, dexmedetomi-
dine is reported to be eight times more potent than clonidine in the clinic [
71
]. It
is suggested that yohimbine-induced vomiting could be due to blockade of the in-
hibitory
α2
-adrenergic receptors, which is associated with the enhanced release of
monoamines NE, DA, and 5-HT [
50
]. Clonidine overrides the ability of yohimbine to
release monoamines, and thus prevents vomiting evoked by the monoamine releaser,
reserpine [
31
]. These maximal tested doses of clonidine and dexmedetomidine in
least shrews were subsequently used as their utmost tested doses against vomiting
produced by the following emetogens.
2.
The more selective and centrally/peripherally-acting 5-HT
3
receptor agonist, 2-methyl-
5-HT (5 mg/kg, i.p.): 5-HT
3
receptor selective antagonists, such as tropisetron [
72
] or
palonosetron [
40
], can suppress vomiting caused by 2-Methyl-5-HT. Here, clonidine
and dexmedetomidine suppressed the mean frequency of vomiting and protected
shrews from emesis in a dose-dependent manner. In the latter case, dexmedetomidine
was 25 times more potent than clonidine in suppressing 2-Methyl-5-HT-evoked vom-
iting. However, clonidine completely protected shrews from vomiting at its highest
tested dose (10 mg/kg), whereas the largest dose of dexmedetomidine (0.1 mg/kg)
only prevented 75% of shrews from vomiting. This difference probably reflects the
nonselective nature of clonidine since the most potent and selective antagonist of
5-HT
3
receptors, palonosetron (10 mg/kg, i.p.), could not completely prevent least
shrews from 2-Methyl-5-HT-induced vomiting [
40
]. Ca
2+
mobilization via extracellu-
lar Ca
2+
influx through 5-HT
3
receptors and L-type Ca
2+
channels, and intracellular
Ca
2+
release via ryanodine receptors (RyRs) present on the endoplasmic reticulum
(ER), initiate Ca
2+
-dependent sequential activation of CaMKIIa and signal-regulated
kinase 1/2 (ERK
1/2
), which contribute to 2-Methyl-5-HT-evoked emesis [
73
]. The
most studied signaling pathway of
α2
-adrenergic receptor is through G protein-
coupled receptors, which consists of direct inhibition (“membrane-delimited”) by
G protein
βγ
(G
βγ
) subunit complexes of Ca
2+
entry through voltage-gated Ca
2+
(Cav) channels [
74
]. For
α2
-adrenergic receptor, the L-type Cav channel is the main
effector [
75
]. Typically, L-type calcium currents are maximally inhibited by 50% upon
α2
-adrenergic agonist application [
74
]. We suggest that the antiemetic effects of cloni-
dine and dexmedetomidine on 2-Methyl-5-HT-evoked emesis is probably related to
the inhibition of the L-type calcium currents.
3.
The selective SP neurokinin NK
1
receptor agonist GR73632 (5 mg/kg, i.p.): At this
dose, GR73632 causes robust vomiting in least shrews through the activation of SP
neurokinin NK
1
receptors [
76
], which can be completely blocked by the selective and
potent NK
1
receptor antagonist netupitant (10 mg/kg, i.p.) in least shrews [
77
]. In
the present study, both clonidine and dexmedetomidine prevented GR73632-evoked
emesis in a dose-dependent manner. However, the largest tested dose of clonidine
(10 mg/kg, i.p.) reduced the frequency of GR73632-evoked vomiting by 94% and
the percentage of least shrews vomiting by 71.4%. Also, dexmedetomidine at its
maximum tested dose (0.1 mg/kg) significantly but partially attenuated both the
mean vomit frequency (76.3%) and the percentage of least shrews vomiting (62.5%)
in response to GR73632. Their ID
50
values indicate that dexmedetomidine is more

Int. J. Mol. Sci. 2024,25, 4603 19 of 28
potent than clonidine in preventing GR73632-evoked vomiting. The NK
1
receptor is
G-protein coupled and can increase cytoplasmic Ca
2+
concentration [
78
–
80
]. In fact,
GR73632 evokes an increase in intracellular Ca
2+
concentration via both Ca
2+
release
from intracellular Ca
2+
stores, and extracellular Ca
2+
influx through the transient
receptor potential channels [
79
]. In addition, we have shown that LTCC blockers
amlodipine and nifedipine also suppress vomiting caused by GR73632 in a dose-
dependent manner [
40
]. Per our discussion above, we suggest that the antiemetic
effects of clonidine and dexmedetomidine on GR73632-induced emesis may involve
suppression of intracellular Ca2+ concentration.
4.
The cholinergic M
1
receptor agonist McN-A-343 (2 mg/kg, i.p.): Clonidine reduced
both the mean frequency and the percentage of least shrews vomiting in response to
McN-A-343 in a dose-dependent fashion, with complete emesis protection occurring at
its 10 mg/kg dose. However, dexmedetomidine significantly but partially reduced the
mean frequency of vomiting (66.7%) at its tested maximal dose of 0.1 mg/kg but failed
to significantly protect shrews from vomiting. McN-A-343 can activate PKC and PKA
phosphorylates to enhance Ca
2+
influx through LTCC channels [
81
,
82
]. Moreover, the
L-type calcium antagonist nifedipine can significantly and completely prevent in a
potent and dose-dependent manner both the percentage of shrews vomiting and the
mean frequency of emesis evoked by McN-A-343 (2 mg/kg, i.p.) in the least shrew [
83
].
The antiemetic effects of clonidine and dexmedetomidine on McN-A-343-induced
emesis may also involve suppression of intracellular Ca2+ mobilization.
5.
The dopamine D
2/3
receptor preferring agonist quinpirole (2 mg/kg, i.p.): Clonidine
only significantly but partially reduced the mean frequency of quinpirole-evoked
vomiting by 80.4% at its 10 mg/kg maximal tested dose but failed to completely
protect shrews from vomiting. Likewise, dexmedetomidine significantly reduced
both the mean frequency (57.8% reduction at 0.1 mg/kg) and the percentage of least
shrews vomiting (36.4% protection at 0.1 mg/kg) in response to quinpirole, but
these reductions were only partial. Although the dopamine D
2
receptor-preferring
antagonist sulpiride can completely prevent apomorphine (2 mg/kg, i.p.)-induced
vomiting in least shrews at 2 mg/kg (s.c.), but it cannot fully protect shrews from
quinpirole (2 mg/kg, i.p.)-evoked emesis even up to 8 mg/kg [
84
]. Thus, it appears
that quinpirole-evoked emesis cannot be easily subdued by sulpiride. Mechanistically,
we have previously demonstrated the involvement of the PI3K/mTOR/Akt signaling
pathway in dopamine D
2
receptor-evoked vomiting [
85
]. Since the
α2
-adrenergic
receptor agonist tizanidine can reduce the expression levels of PI3K/Akt [
86
], it is
possible that clonidine and dexmedetomidine may also affect this signaling system to
suppress the evoked vomiting, but this remains to be investigated.
6.
The cannabinoid CB
1
receptor-selective inverse agonist/antagonist SR141716A
(20 mg/kg, i.p.): SR141716A can induce vomiting in the least shrew at large doses
(20–40 mg/kg, i.p.) which can be fully prevented by CB
1
receptor agonists, includ-
ing
∆9
-THC [
87
]. Clonidine significantly reduced both the mean frequency and the
percentage of shrews vomiting in response to SR141716A in a dose-dependent manner
with complete emesis protection at it 10 mg/kg dose. Dexmedetomidine reduced the
mean frequency of vomiting by 88% and protected shrews from vomiting by 75% at
its maximal tested dose of 0.1 mg/kg. SR141716A increases the release and turnover
of monoamines DA, NE, and 5-HT [
88
] in several brain regions of rodents and least
shrews, and can enhance capsaicin-evoked release of SP in the mouse spinal cord [
89
].
As discussed earlier, since clonidine can prevent vomiting caused by the monoamine
releaser reserpine [
31
], it probably prevents SR141716A-evoked vomiting via a similar
mechanism.
7.
The selective LTCC agonist FPL64176 (10 m/kg, i.p.): The LTCC regulates extracel-
lular Ca
2+
influx into the cytosol [
90
]. FPL64176 is an extracellular Ca
2+
-mobilizing
agent and evokes vomiting in all tested shrews at 10 mg/kg [
40
,
83
]. Clonidine sig-
nificantly and completely reduced the mean vomiting frequency and the percentage

Int. J. Mol. Sci. 2024,25, 4603 20 of 28
of shrews vomiting in response to FPL64176 challenge in a dose-dependent manner.
Dexmedetomidine significantly and dose-dependently reduced the mean vomit fre-
quency by 92.3% following its maximal tested dose (0.1 mg/kg), and protected 71.4%
of shrews from FPL64176 (10 mg/kg, i.p.)-induced vomiting. However, their ID
50
values indicate that dexmedetomidine is more potent than clonidine in preventing
FPL64176-evoked vomiting. As was discussed earlier, the antiemetic effects of cloni-
dine and dexmedetomidine on FPL64176-evoked emesis may also involve inhibition
of L-type calcium currents.
8.
Specific inhibitor of the SERCA pump thapsigargin (0.5 mg/kg, i.p.): The SERCA
pump is a main mechanism that transports free cytosolic Ca
2+
into endoplasmic retic-
ulum (ER) Ca
2+
stores. Release of Ca
2+
from the ER stores into the cytosol occurs
through the inositol trisphosphate (IP
3
)- and ryanodine (RyR)-receptor ion chan-
nels localized on the ER membrane [
91
,
92
]. Thapsigargin is a specific and potent
inhibitor of SERCA pumps and causes a rapid elevation in cytosolic Ca
2+
concentra-
tions. Thapsigargin can also release Ca
2+
from the ER stores into the cytosol via IP
3
and RyR calcium channels [
93
–
95
]. Thapsigargin causes vomiting by triggering an
initial elevation in the cytoplasmic Ca
2+
concentration by inhibiting the SERCA as
well as releasing Ca
2+
from the ER into the cytoplasm via both RyR- and IP
3
-receptors
(IP3Rs), which is followed by an extracellular Ca
2+
influx through LTCCs prior to
the intracellular activation of the Ca
2+
-CaMKII-ERK1/2 cascade [
41
]. In the present
study, clonidine significantly reduced the mean frequency of thapsigargin-evoked
vomiting by 93.3% at its 10 mg/kg dose, but only partially protected 66.7% of shrews
at this maximal dose. Moreover, dexmedetomidine only reduced the mean frequency
of the evoked vomiting by 70.8% at its 0.1 mg/kg maximal dose but failed to sig-
nificantly protect shrews from vomiting. Since the LTCC inhibitor nifedipine can
dose-dependently inhibit thapsigargin-induced vomiting [
41
], we speculate that cloni-
dine and dexmedetomidine may partially suppress extracellular Ca
2+
influx through
LTCCs, which could then attenuate the above-discussed signaling cascade, leading to
a limited reduction in thapsigargin-evoked vomiting.
9.
The PDE4 inhibitor rolipram (1 mg/kg, i.p.): PDE4 inhibitors prevent metabolism
of second messengers such as cAMP and increase their tissue levels. They have
procognitive and antidepressant properties [
96
]. The emetic effect of some PDE4
inhibitors is thought to be a consequence of inhibition of PDE4 and the subsequent
increase in cAMP levels in the brainstem DVC [
30
,
42
]. PDE4 inhibitors may mimic
the pharmacological effect of
α2
-adrenergic receptor antagonists, which elevate in-
tracellular levels of cAMP in noradrenergic neurons [
30
]. In contrast,
α2
-adrenergic
receptor activation decreases intracellular levels of cAMP in noradrenergic neurons.
PDE4 inhibitors are thought to modulate the release of emetic mediators including
5-HT, SP, and noradrenaline which are involved in the onset of the emetic reflex [30].
We have previously shown that the PDE4 inhibitor rolipram (1 mg/kg, i.p.) evokes
both vomiting as well as significant increases in shrew brainstem cAMP levels, while
pretreatment with SQ22536, an inhibitor of adenylyl cyclase, prevented the evoked
emesis [
42
]. In the present study, clonidine dose-dependently and significantly, albeit
partially, reduced both the mean vomit frequency (80.3%) and the percentage (62.5%)
of shrews vomiting in response to rolipram at its highest tested dose, 10 mg/kg.
Likewise, dexmedetomidine produced similar dose-dependent but partial reductions
in both the mean vomit frequency (73.7%) and the percentage of shrews vomiting
(60%) at its maximal tested dose, 0.1 mg/kg. The antiemetic effect of clonidine and
dexmedetomidine against rolipram-induced vomiting might be due to suppression
of increased intracellular tissue levels of cAMP evoked by rolipram in the shrew
brainstem. In fact, administration of cAMP analogs such as 8-chloro-cAMP cause
vomiting in cancer patients [97].

Int. J. Mol. Sci. 2024,25, 4603 21 of 28
10.
The HCN blocker ZD7288 (1 mg/kg, i.p.): The HCN channels are a class of voltage-
gated ion-channels permeable to Na
+
and K
+
and constitutively open at voltages near
the resting membrane potential [
98
,
99
]. The hyperpolarization-activated currents (I
h
)
mediated by HCN channels elicit membrane depolarization toward a threshold for
action potential generation, which plays a pivotal role in controlling neuronal excitabil-
ity [
98
,
100
]. The HCN channel blocker ZD7288 can reduce apomorphine-induced
conditioned taste aversion to saccharin preference and depress the excitability of the
AP since it blocks HCN channel activation [
101
]. We have recently demonstrated that
ZD7288 (1 mg/kg, i.p.) evokes both vomiting in a dose-dependent manner as well
as a robust expression of c-fos and ERK
1/2
phosphorylation in the shrew brainstem
DVC, indicating a central contribution to the evoked vomiting [
43
]. In the present
study, clonidine significantly and dose-dependently reduced ZD7288-evoked mean
vomit frequency (92.4% reduction at 10 mg/kg), but only partially protected shrews
from vomiting (55.6% protection at 10 mg/kg). Dexmedetomidine only significantly
reduced the mean frequency (63.1% protection at 0.1 mg/kg) and failed to signifi-
cantly protect shrews from ZD7288-evoked vomiting. Given the well-known negative
coupling of
α2
-adrenergic receptors to adenylate cyclase via a heterotrimeric G pro-
tein [
102
], any reduction in the cAMP level would decrease I
h
. Dexmedetomidine
via
α2
-adrenergic receptors activates G-protein-coupled K
+
channels and inhibits
I
h
, which leads to membrane hyperpolarization [
103
]. In addition, clonidine can
directly inhibit I
f
current [
11
]. Hence, we speculate that administration of clonidine
and dexmedetomidine reverse the inhibitory effect of ZD7288 on the HCN channels
and consequently suppresses ZD7288-induced vomiting in shrews.
As discussed in the introduction section, although both clonidine and dexmedetomi-
dine are used as antiemetics for suppression of PONV, to date no basic or clinical study
has compared their antiemetic potential against diverse emetogens. In the present study
their antiemetic ID
50
values are comparatively determined against various emetogens, and
dexmedetomidine appears to be 3–69 times more potent than clonidine, demonstrating
that dexmedetomidine has more efficacious antiemetic potential at non-sedating doses.
4. Materials and Methods
4.1. Animals
A colony of adult least shrews between 45–60 days old and weighing between 4 and
6 g from the Western University of Health Sciences Animal Facilities were employed in
this study. Shrews were housed in groups of 5–10 on a 14:10 light: dark cycle and were
fed and watered ad libitum. Animal experiments were conducted in accordance with the
principles and procedures of the National Institutes of Health Guide for the Care and Use
of Laboratory Animals. All protocols were approved by the Institutional Animal Care and
Use Committee of Western University of Health Sciences (Protocol number R20IACUC018).
All efforts were made to minimize animal suffering and to reduce the number of animals
used in the experiments.
4.2. Chemicals
The following drugs were used in the present study: clonidine, dexmedetomidine,
yohimbine, GR73632, SR141716A, FPL64176, thapsigargin, and ZD7288 were purchased
from Tocris (Minneapolis, MN, USA); McN-A-343, quinpirole HCl, and rolipram were
purchased from Sigma-Aldrich (St. Louis, MO, USA); 2-methyl-serotonin maleate salt (2-
Methyl-5-HT) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). SR141716A
was dissolved to twice the stated drug dose in a 1:1:18 solution of ethanol:Emulphor™:0.9%
saline and was then diluted further with an equal volume of saline. FPL64176 was dissolved
in 25% DMSO in water. Thapsigargin was dissolved in 10% DMSO in distilled water.
Other drugs were dissolved in distilled water. All drugs were administered at a volume
of 0.1 mL/10 g of body weight. The doses and routes used for the emetogens were based
upon previous publications from our laboratory [6,43,87].

Int. J. Mol. Sci. 2024,25, 4603 22 of 28
4.3. Behavioral Emesis Studies
On the day of the experiment, both male and female shrews [
43
] were brought from
the animal facility, randomly separated into individual cages, and allowed to adapt to the
experimental condition for at least 2 h. Daily food was withheld 2 h prior to the start of the
experiment, but shrews were given 4 mealworms each 30 min prior to emetogen injection
to help identify wet vomits as described previously [
39
]. Experiments were performed
between 8:00 a.m. and 5:00 p.m.
To identify the emetic/antiemetic potential of either the
α2
-adrenergic receptor ago-
nists clonidine and dexmedetomidine, or the corresponding antagonist yohimbine, different
groups of shrews were injected with varying doses of either the corresponding vehicle,
clonidine (0.1, 1, 5, and 10 mg/kg, i.p., n= 6–10), dexmedetomidine (0.01, 0.05, 0.1, 0.5,
and 1 mg/kg, i.p., n= 6–10), or yohimbine (0.5, 0.75, 1, 1.5, 2, and 3 mg/kg, i.p., n= 7–14).
Immediately following the injection, each shrew was placed in the observation cage and
the frequency of emesis was recorded for the next 30 min. Based on the results obtained,
the corresponding vehicles, clonidine, or dexmedetomidine at all tested doses failed to
evoke vomiting in least shrews. However, the i.p. administration of yohimbine produced
vomiting in shrews in a dose-dependent and bell-shaped response manner, and a 1 mg/kg
dose of yohimbine caused maximal mean frequency of emesis in shrews, and thus was
chosen for subsequent immunofluorescence staining and drug interaction studies.
Since the
α2
-adrenergic receptor antagonist yohimbine caused vomiting, we initially
tested the antiemetic potential of its corresponding agonists clonidine and dexmedetomi-
dine in the following manner: different groups of shrews were pretreated with an injection
of either corresponding vehicles or varying doses of clonidine (0.1, 1, 5, and 10 mg/kg, i.p.)
or dexmedetomidine (0.01, 0.05, and 0.1 mg/kg, i.p.) at 0 min. Following 30 min, the pre-
treated shrews were challenged for vomiting with a maximally effective dose of yohimbine
(1 mg/kg, i.p.). Each shrew was then placed in the observation cage and the frequency of
vomiting was recorded for the next 30 min. Using the same procedure, we subsequently
investigated the antiemetic potential of these
α2
-adrenergic receptor agonists against fully
efficacious emetic dose of the following emetogens in separate experiments [
40
,
41
,
43
]:
(1) the selective serotonin 5-HT
3
receptor agonist 2-Methyl-5-HT (5 mg/kg, i.p.) [
73
];
(2) the selective SP neurokinin NK
1
receptor agonist GR73632 (5 mg/kg, i.p.) [
76
,
77
];
(3) the muscarinic M
1
receptor agonist McN-A-343 (2 mg/kg, i.p.) [
83
]; (4) the dopamine
D
2/3
preferring receptor agonist quinpirole (2 mg/kg, i.p.) [
84
]; (5) the CB
1
receptor in-
verse agonist/antagonist SR141716A (20 mg/kg, i.p.) [
87
]; (6) the LTCC agonist FPL64176
(10 mg/kg, i.p.) [
45
]; (7) the SERCA inhibitor thapsigargin (0.5 mg/kg, i.p.) [
41
];
(8) the
PDE4 inhibitor rolipram (1 mg/kg, i.p.) [
42
]; (9) the HCN channel blocker ZD7288
(1 mg/kg, i.p.) [
43
]. In the emesis studies the observer was unaware of treatment conditions.
Each shrew was used once, then euthanized with isoflurane (3%) in the anesthesia chamber
following the termination of each behavioral experiment.
4.4. Immunohistochemistry and Image Analysis
4.4.1. c-fos Staining and Image Analysis
Immunohistochemistry of the least shrew brainstem (20
µ
m) was conducted as previ-
ously reported [
43
,
77
]. Following vehicle or yohimbine (1 mg/kg, i.p.) injection, vomiting
shrews were subjected to c-fos staining (n= 6 shrews per group). Following 90 min after
the first emesis occurred, shrews were deeply anesthetized with isoflurane (3%), then
transcardially perfused with 0.01 M phosphate buffered saline (PBS) followed by ice cold
4% paraformaldehyde (pH 7.4) in 0.01 M PBS for 10 min. Brainstems were post-fixed in
the same fixative for 2 h, then placed in 0.1 M PB containing 30% sucrose at 4°C until they
sank. The brainstem was cut in 20
µ
m sections using a cryostat (Leica, Bannockburn, IL,
USA), and pre-incubated in the blocking buffer (0.01 M PBS containing 10% normal donkey
serum and 0.3% Triton X-100) for 1 h at room temperature. The slices were then incubated
in a rabbit anti-c-fos primary antibody (1:1000, ab190289, Abcam, Cambridge, UK) in 0.01M
PBS containing 5% normal donkey serum, 0.05% sodium azide, and 0.3% Triton X-100 at

Int. J. Mol. Sci. 2024,25, 4603 23 of 28
4°C overnight. The sections were washed 3 times (10 min each) in PBS and incubated in
an Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:500, A-21207, Invitrogen,
Waltham, USA) in 0.01 M PBS containing 0.3% Triton X-100 for 2 h at room temperature,
then washed 3 times (10 min each) and were mounted and coverslipped with an anti-fade
mounting medium containing DAPI (Vector Laboratories, Newark, USA). Tile-scanning
images of the brainstem sections containing the brainstem emetic nuclei (AP/NTS/DMNV)
were taken by a confocal microscope (Zeiss LMS 880, Oberkochen, Germany) at 1024
×
1024
pixels with Zen software using Plan-Apochromat 20
×
/0.8 M27 objective. Cytoarchitectonic
differences in the AP, NTS, and DMNV of the least shrew brainstem have been described in
our published studies [
41
,
45
,
76
]. For each animal, c-fos positive cells in the AP and both
sides of NTS and DMNX from 3 sections at 90
µ
m intervals were counted manually by
an experimenter blind to the experimental conditions. The average value was used in
statistical analysis.
4.4.2. 5-HT and SP Immunohistochemistry
Shrews (n= 5–6 shrews per group) were treated with either vehicle or yohimbine
(1 mg/kg, i.p.) and rapidly anesthetized with isoflurane and subjected to perfusion at
15 min and 30 min post-treatment to examine 5-HT and SP immunoreactivity. The experi-
mental procedure prior to staining was performed as described above for Section 4.4.1. c-fos
Staining and Image Analysis. Coronal brainstem sections (20
µ
m) were blocked with 0.1 M
PBS containing 10% donkey serum and 0.3% Triton X-100, then incubated overnight at 4
◦
C
with a mix of goat anti-5-HT primary antibody (1:1000, ab66047, Abcam) and rat anti-SP
primary antibody (1:400, MAB356, EMD Millipore, Burlington, VT, USA) in 0.1 M PBS
containing 5% donkey serum and 0.3% Triton X-100. Sections were washed 3 times (10 min
each) in PBS and incubated in a mix of Alexa Fluor 488 donkey anti-goat (1:500, ab150133,
Abcam) and cy3-conjugated donkey anti-rat (1:500, AP189C, EMD Millipore) secondary
antibody in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature. After
washing with PBS 3 times (10 min each), sections were mounted with anti-fade mounting
medium containing DAPI (Vector Laboratories). Images for the DVC were acquired using
a confocal microscope (Zeiss LMS 880) as described above. Fluorescence intensity (mean
gray value) of 5-HT and SP values were acquired using ImageJ software, as described
previously [45].
4.5. Locomotor Activity Studies
Because
α2
-adrenergic receptor agonists have potent analgesic, sedative/hypnotic
properties in humans and in experimental animals [
104
–
107
], the locomotor activity param-
eters (i.e., total distance moved and rearing behavior) were measured in shrews following
injection of varying doses of clonidine (0, 0.1, 1, 5, and 10 mg/kg, i.p.) or dexmedetomidine
(0, 0.01, 0.05, 0.1, and 0.5 mg/kg, i.p.). The Ethovision (version XT 9) locomotion analysis and
behavior recognition system (Noldus Information Technology, Wageningen, The Netherlands)
was used, as reported previously [108]. The parameters of Ethovision were set to record two
locomotor activities: (1) total distance moved in centimeters (spontaneous locomotor activity),
and (2) rearing frequency, which was recorded when the shrew was standing upright with a
5% reduction in body surface area as seen by the overhead video camera.
On the day of the experiment, both male and female shrews were brought in their home
cages to the experimental room and were allowed to acclimate to a semi-dark environment
for 1 h. Each shrew was further acclimated in an empty white plastic observation cage
(27.5
×
27.5
×
28 cm) for 1 h before testing. Different groups of shrews were injected with
either corresponding vehicle or varying doses of clonidine (0.1, 1, 5, and 10 mg/kg, i.p.,
n= 6–10) or dexmedetomidine (0.01, 0.05, 0.1, and 0.5 mg/kg, i.p., n= 6–10) at 0 min.
Following 30 min, each shrew was individually placed in a white observation cage of the
same dimension, and the two motor parameters were recorded for 30 min by an overhead
camera and data were analyzed using the Noldus software. The chamber was thoroughly

Int. J. Mol. Sci. 2024,25, 4603 24 of 28
cleaned with 70% ethanol and dried to eliminate animal odors between test sessions. Each
shrew was used once and euthanized using isoflurane at the end of the experiment.
4.6. Statistical Analysis
Statistical analyses were done using Graphpad Prism 8 (Graphpad software Inc., San
Diego, CA, USA). The frequencies of vomits were analyzed using the Kruskal–Wallis non-
parametric one-way analysis of variance (ANOVA) followed by Dunn’s post hoc test and
expressed as the mean
±
SEM. The percentage of animals vomiting across treatment groups
at different doses was compared using the chi-square test. The locomotor activities and the
differences of 5-HT/SP mean gray values among groups were compared with an ordinary
ANOVA test followed by Dunnett’s post hoc test. The numbers of c-fos-positive cells
between two groups were tested by unpaired t-test. p< 0.05 was considered statistically
significant. Sample size calculations were determined as described by Chow [109].
5. Conclusions
Our results demonstrate that the
α2
-adrenergic receptor antagonist yohimbine is pro-
emetic in least shrews, and its corresponding agonists clonidine and dexmedetomidine
possess broad-spectrum antiemetic potential. In fact, yohimbine caused vomiting in a
bell-shaped and dose-dependent manner, accompanied by increased c-fos expression and
release of 5-HT and SP in the brainstem DVC emetic nuclei. Clonidine and dexmedeto-
midine not only suppressed yohimbine-evoked emesis in a dose-dependent manner, but
also vomiting caused by other well-investigated emetogens of varied mechanism of actions.
The broad-spectrum antiemetic effects of dexmedetomidine occur at much lower doses
than those of clonidine. Furthermore, the antiemetic doses (1, 5, and 10 mg/kg, i.p.) of
clonidine decreased the least shrew locomotor activity parameters, whereas dexmedetomi-
dine lacked motor suppressive behaviors. The current results suggest both clonidine and
dexmedetomidine possess broad-spectrum antiemetic potential, but dexmedetomidine’s
antiemetic ability occurs at non-sedative doses.
Author Contributions: N.A.D. and Y.S. conceived and designed the project, contributed to tissue
dissection and collection, completed immunohistochemical and behavioral experiments, contributed
toward statistical analyses, and wrote the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was funded in part by the NIH-NCI grant (CA207287) and the WesternU
intramural startup fund (1395) to N.A.D.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors, without undue reservation.
Conflicts of Interest: The authors declare no conflicts of interest.
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