ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 1938-1949 © Pleiades Publishing, Ltd., 2024.
1938
Purmorphamine Alters Anxiety-Like Behavior
and Expression of Hedgehog Cascade Components
in Rat Brain after Alcohol Withdrawal
Danil I. Peregud
1,2,a
*, Nataliya I. Shirobokova
2
, Aleksei A. Kvichansky
2
,
Mikhail Yu. Stepanichev
2
, and Natalia V. Gulyaeva
2,3
1
Serbsky National Medical Research Center for Psychiatry and Drug Addiction,
Ministry of Health of the Russian Federation, 119034 Moscow, Russia
2
Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 117485 Moscow, Russia
3
Research and Clinical Center for Neuropsychiatry, Moscow City Health Department, 115419 Moscow, Russia
a
e-mail: peregud_d@yahoo.com
Received May 28, 2024
Revised July 13, 2024
Accepted August 6, 2024
Abstract— Disturbances in the Hedgehog (Hh) signaling play an important role in dysmorphogenesis of bone
tissue and central nervous system during prenatal alcohol exposure, which underlies development of fetal alco-
hol syndrome. The involvement of Hh proteins in the mechanisms of alcohol intake in adults remains obscure.
We investigated the role of the Hh cascade in voluntary ethanol drinking and development of anxiety-like
behavior (ALB) during early abstinence and assessed changes in the expression of Hh pathway components
in different brain regions of male Wistar rats in a model of voluntary alcohol drinking using the intermittent
access to 20% ethanol in a two-bottle choice procedure. Purmorphamine (Hh cascade activator and Smoothened
receptor agonist) was administered intraperitoneally at a dose of 5mg/kg body weight prior to 16-20 sessions
of alcohol access. Purmorphamine had no effect on the ethanol preference; however, rats exposed to ethanol
and receiving purmorphamine demonstrated changes in the ALB during the early abstinence period. Alcohol
drinking affected the content of the Sonic hedgehog (Shh) and Patched mRNAs only in the amygdala. In rats
exposed to ethanol and receiving purmorphamine, the level of Shh mRNA in the amygdala correlated negative-
ly with the time spent in the open arms of the elevated plus maze. Therefore, we demonstrated for the first
time that alterations in the Hh cascade induced by administration of purmorphamine did not affect alcohol
preference in voluntary alcohol drinking. It was suggested that Hh cascade is involved in the development
of anxiety after alcohol withdrawal through specific changes in the Hh cascade components in the amygdala.
DOI: 10.1134/S0006297924110087
Keywords: purmorphamine, alcohol, voluntary drinking, anxiety-like behavior, rats
Abbreviations: ALB, anxiety-like behavior; EPM,elevated plus maze; Gli,transcription factor; Hh,Hedgehog; Ptch,Patched
transmembrane protein; Shh, Sonic hedgehog protein; Smo, Smoothened G protein-coupled receptor.
* To whom correspondence should be addressed.
INTRODUCTION
Secreted hedgehog (Hh) ligands play a key role
in embryogenesis and functioning of stem cells, as
well as in the maintenance of tissue homeostasis due
to their involvement in the injury-induced regenera-
tion [1]. Mammals have three proteins of this family:
Sonic hedgehog(Shh), Desert hedgehog (Dhh), and In-
dian hedgehog (Ihh). The most studied of them is Shh.
The Hh signaling cascade is a multicomponent net-
work of molecular events that depend on many fac-
tors [2]. According to the simplified model of the Hh
pathway activation, (i) Patched transmembrane pro-
tein (Ptch) in its inactive state blocks the Smoothened
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BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
(Smo) G protein-associated receptor (GPCR); (ii) Hh li-
gand binds to Ptch thus abolishing its inhibitory effect
on Smo and leading to cascade activation; (iii) Smo
triggers an intracellular cascade resulting in the ac-
tivation of transcription factor Gli and expression of
target genes [2].
Because the Hh cascade plays a pivotal role in
cell proliferation and differentiation, its dysregula-
tion might lead to oncogenesis, which makes this sys-
tem a target in the development of pharmacological
agents for the treatment of malignant tumors [3].
Thus, drugs inhibiting the Hh pathway have been de-
veloped and approved for the treatment of certain
cancers. However, the Hh cascade is also essential for
the functioning of the central nervous system (CNS).
In addition to being classical morphogens mediating
CNS development in embryogenesis, Hh proteins di-
rectly or indirectly regulate multiple processes, such
as neurogenesis, axonal growth, and neuroplasticity,
in mature brain [4]. Hh proteins are also involved in
the formation of response to the oxidative stress and
inflammation in the nervous tissue [5]. Impaired func-
tioning of the Hh signaling cascade is associated with
diseases of the nervous system, such as autism, depres-
sion, dementia, stroke, epilepsy, as well as neurodegen-
erative and demyelinating diseases [6]. This makes the
search and design of agents capable of influencing this
system a very promising direction in the development
of medications aimed at pharmacological correction of
CNS disorders [6].
Purmorphamine, a derivative of purine, deserves
special attention among compounds affecting the Hh
pathway. By acting as a Smo receptor agonist, it trig-
gers canonical Hh cascade accompanied by activation
of the Gli transcription factor [7]. Systemic adminis-
tration of purmorphamine is effective in a number of
experimental models of CNS diseases. Purmorphamine
alleviated brain tissue damage and reduced neurolog-
ical deficit [8] and memory impairment in the mouse
models of brain ischemia [9]. Purmorphamine de-
creased the neurotoxic effects of propionic acid and
ethidium bromide in rats, including neurodegenera-
tion, impairments in the motor activity and learning
in the Morris water maze test, and depressive-like
behavior [10, 11].
Alcohol consumption is accompanied by a com-
plex of molecular and cellular adaptations that might
underlie its transformation from a controlled process
to abuse and development of alcohol dependence
characterized by the withdrawal syndrome [12]. Me-
socorticolimbic structures, primarily, frontal cortex,
amygdala, hippocampus, and striatum, are the main
anatomical structures regulating various aspects of
alcohol drinking and formation of alcohol depen-
dence [13, 14]. Studying molecular mechanisms of
excessive alcohol consumption and alcohol depen-
dence, which are accompanied by neuroplastic pro-
cesses [15, 16], remains one of the priorities in neuro-
science.
It is well known that the Hh cascade is involved
in the developmental disorders of the bone tissue and
CNS in prenatal alcohol exposure and is one of the
molecular links in the development of fetal alcohol
syndrome [17, 18]. However, the role of Hh in the
regulation of alcohol consumption and accompanying
behavioral disorders in adults remains poorly studied
despite the relevance and prospects of this research.
There are indirect indications that the Hh cascade
participates in the mechanism regulating alcohol
drinking and concomitant changes in the CNS activity.
In particular, it was demonstrated that expression of
Ptch in the rat hippocampus during early abstinence
period in the model of voluntary drinking with the
intermittent access to 20% ethanol depended on the
pattern of alcohol consumption [19]. In this model,
rats demonstrated the ability to increase alcohol con-
sumption up to 10 g/kg body weight per day, which
corresponds to the excessive alcohol drinking in hu-
mans [20]. It should be noted that the intermittent ac-
cess to ethanol allowed rats to consume more alcohol
than protocols using continuous access to ethanol [20].
The abstinence period (up to 72 h, but not later) was
characterized by memory impairment, anxiety, and
depression-like symptoms accompanied by changes
in the CNS at the molecular level [20]. In the same
model, during the early abstinence period, there was a
significant increase in the Shh level in the hippocam-
pus and striatum of rats that had an access to alcohol
and received 7,8-dihydroxyflavone (brain-derived neu-
rotrophic factor mimetic) vs. animals that only had
access to alcohol [21].
As mentioned above, Hh participates in the for-
mation of various types of behavior. In regards of
alcohol consumption and accompanying impairments
it is important that activation of the Hh cascade at-
tenuated obsessive-compulsive disorders [22] and had
an anxiolytic effect [23, 24]. Abstinence after chronic
alcohol consumption can lead to the development of
anxiety-like disorders [25, 26], but the relationship be-
tween the Hh cascade and anxiety-like behavior (ALB)
during alcohol withdrawal has not been investigated
before.
Based on the published reports and our own data,
we suggested that activation of the Hh signaling may
alter alcohol consumption and/or behavior during the
early abstinence period. The main goal of this work
was to study the role of purmorphamine in alcohol
consumption in a model of voluntary alcohol drinking
with the intermittent access to 20% ethanol and in the
development of ALB in rats after alcohol withdrawal.
We also evaluated changes in the levels of Shh, Ptch,
Smo, and Gli mRNAs in the key mesocorticolimbic
PEREGUD et al.1940
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
Fig. 1. Scheme of the experiment. Voluntary alcohol drinking was modeled using the intermittent access to 20% ethanol in a
two-bottle choice procedure; alcohol was presented together with water for a period of 24 h, followed by alcohol replacement
with water for another 24 h. Purmorphamine was administered intraperitoneally at a dose of 5 mg/kg body weight before
sessions 16-20 of alcohol access. Alcohol intake was recorded during 20 sessions, after which the rats were accessed water
only. ALB was assessed in the open field and elevated plus maze tests one day and two days after alcohol withdrawal, re-
spectively. On the third day, the levels of mRNA for Shh, Ptch, Smo, and Gli (signaling cascade components) were determined
in the frontal cortex, amygdala, hippocampus, and striatum.
brain structures, since an increase in the expression
of Hh signaling pathway components is considered to
be a molecular marker of cascade activation in many
cell types [3].
MATERIALS AND METHODS
Animals. Male Wistar rats were obtained from
the Stolbovaya Branch of the Scientific Center of Bio-
medical Technologies of the Federal Medical Biological
Agency (Moscow Region, Russia). The animals were 12
weeks old with the average body weight of 288 ± 4 g.
The adaptation period (from the moment the rats were
housed at the animal facility to their inclusion into the
experiment) was at least 7 days. The rats were kept
in individual cages under standard conditions with an
artificial 12/12-h light/dark illumination cycle at con-
stant temperature (21-23°C) with ad libitum access to
water and granulated chow.
Design of the experiment. The scheme of the
experiment and the number of animals in the exper-
imental and control groups are shown in Fig. 1.
Alcohol consumption in the model of intermit-
tent access to 20% ethanol. The model of voluntary
drinking with the intermittent access to 20% ethanol
in a two-bottle choice procedure was implemented ac-
cording to the protocol proposed by Simms etal. [27].
In the experimental groups, 20% ethanol solution and
water were presented to rats simultaneously in two
bottles for 24 h. Rats chose the bottle and the amount
of liquid to drink. A total of 20 sessions of alcohol ac-
cess were performed; the positions of the bottle in the
cage were alternated. The period between the sessions
was 24 h, during which the rats were presented with
two bottles of water. The control group had a continu-
ous access to two bottles of water. The ethanol access
sessions began during the dark phase of the light/dark
illumination cycle. Animals and bottles were weighed
each time before and after the drinking session. The
level of alcohol preference was estimated as the ratio
of the volume of alcohol intake per session (daily) to
the total volume of liquid consumed and expressed
in percent.
Purmorphamine administration. The animals
were injected intraperitoneally with a suspension of
purmorphamine (ab120933; Abcam, USA) in 10% di-
methyl sulfoxide, 40% PEG-400, and 50% isotonic sodi-
um chloride at a dose of 5 mg/kg body weight (3 ml/kg
body weight) 1 h before ethanol presentation during
sessions 16 to 20. It was previously demonstrated that
purmorphamine administered systemically at a dose
of 5 mg/kg affected various types of rat behavior [10,
11, 22]. Animals that did not receive purmorphamine
were injected with the vehicle (3 ml/kg body weight).
Prior to the first purmorphamine administration, an-
imals exposed to ethanol were randomly distribut-
ed according to the ethanol preference over the last
5 sessions. The following four groups were formed:
1) Control group with a constant access to water
only; 2) Control + purmorphamine group (control ani-
mals receiving purmorphamine injections); 3) Ethanol
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BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
group that was presented with alcohol and water
(according to the model described above); and 4) Eth-
anol + purmorphamine group (rats injected with
purmorphamine prior to the ethanol access).
Open field test was used for the assessment of
motor activity and ALB. The animals were tested in a
Circular Open Field for Rats (TS0501-R; Open Science
LLC, Russia) with a white round open arena (diame-
ter, 97 cm; wall height, 42 cm). The arena was divid-
ed into three equal concentric zones (center, middle,
and periphery) and illuminated at 500 lux. Rats were
placed in the central zone and allowed to explore the
arena for 5 min. After each test, the arena was puri-
fied with 20% ethanol. Animal behavior was recorded
with a DMK 23GV024 GigE digital monochrome video
camera (Imaging Source Europe GmbH, Germany) and
analyzed with the Ethovision XT11 software (Noldus,
Netherlands). The assessed parameters included the
distance traveled, movement speed, time spent in the
arena center, and number of entries into the center.
Elevated plus maze (EPM) test was used to
evaluate ALB. EPM was a cross-shaped platform
(TS0502-R3; Open Science LLC, Russia) consisting of
the central area (14×14 cm) and four open-top cross-
ing arms (50×14 cm): two opposing open arms with
a wall height of 1 cm and two opposing closed arms
with a wall height of 30 cm. The maze was placed
50 cm up from the floor and installed so that the open
arms were illuminated in the same way as the entire
experimental room (500 lux), while the closed arms
were darkened. The animal was placed into the cen-
tral area with its nose toward the open arm, and its
behavior was recorded with a DMK 23GV024 GigE dig-
ital monochrome video camera for 5 min. After each
test, the maze was cleaned with 20% ethanol. Animal
behavior was analyzed with Ethovision XT11 software.
The assessed parameters were the distance traveled,
movement speed, number of entries into the open and
closed arms, time spent in the open and closed arms,
and the number of head dips and stretch-attend pos-
tures.
Polymerase chain reaction (PCR). The animals
were sacrificed by decapitation, the brains were re-
moved and washed in ice-cold isotonic sodium chlo-
ride solution. Frontal cortex, amygdala, hippocampus,
and striatum were isolated on ice and stored at –80°C.
Total RNA was extracted with ExtractRNA reagent
(#BC032; Evrogen, Russia) and treated with 2.5 U of
DNase I (#M0303; NEB, USA) to remove genomic DNA
impurities. Reverse transcription was performed with
100 U Protoscript II reverse transcriptase (#M0368;
NEB) in the reaction mixture containing 0.5 μg of
total RNA, 1 μM Oligo(dT)
15
(#SB001, Evrogen), and
1 μM random decanucleotide primers (#SB002, Evro-
gen). PCR was performed in a CFX96 thermal cycler
(Bio-Rad, USA) in two parallel repeats using 2 pmol
of synthetic oligonucleotide primers and qPCRmix-HS
SYBR PCR premix (#PK147S; Evrogen). The sequences
of primers used to amplify cDNAs for Shh [28], Ptch,
and Smo [29], Gli [30], and rpS18 [31] were described
previously. Relative mRNA levels were assessed as sug-
gested in [32].
Statistical analysis. The results were analyzed
and statistically processed with the STATISTICA 8.0
software package (StatSoft Inc., USA) and Prism 8.0
(GraphPad SoftwareInc., USA); the data were analyzed
for the normality of distribution using the Shapiro–
Wilk test and presented as mean ± standard error of
mean (SEM) or as median and interquartile range.
The differences between the levels of alcohol pref-
erence during the first and each following drinking
session were calculated using the mixed-effects model
(restricted maximum likelihood model) and Dunnett’s
method for multiple comparisons. The significance
of differences between the dependent samples was
evaluated with the repeated measures analysis of
variance (ANOVA) and Tukey’s test for multiple com-
parison of means. Comparison of several independent
samples was carried out with the two-factor ANOVA
and Tukey’s test or the Kruskal–Wallis test followed
by the post hoc analysis of multiple comparisons and
the Dunn’s test. Correlation analysis was performed by
estimating the Spearman correlation coefficient. The
differences were considered significant at p < 0.05.
RESULTS
Ethanol preference. According to the mixed-ef-
fects model, the level of alcohol preference varied sig-
nificantly between the sessions (F = 2.453; p = 0.034).
Based on the Dunnett’s test, the preference for alcohol
increased and was higher after sessions 7-9 and 12-13
compared to the alcohol preference after the first ses-
sion (Fig. 2).
The data on the effect of purmorphamine on
the alcohol preference are shown in Fig. 3. Accord-
ing to repeated measures ANOVA, ethanol drinking
session had a significant effect on the alcohol pref-
erence (F
(5,60)
= 5.269; p < 0.001). Purmorphamine ad-
ministration did not influence alcohol preference
(F
(1,12)
= 0.110; p = 0.745). The interaction of “ethanol
drinking session” and “purmorphamine administra-
tion” factors had not significant effect on the prefer-
ence for ethanol (F
(5,60)
= 0.237; p = 0.945).
Open field test was used to evaluate the ALB
parameters one day after alcohol withdrawal (Fig. 4).
The time spent in the center of the arena (Fig. 4a;
H
(3,29)
= 0.728; p = 0.867), number of entries to the
center (Fig. 4b; H
(3,29)
= 0.605; p = 0.895), traveled dis-
tance (data not shown), and movement speed (data
not shown) did not differ between the groups.
PEREGUD et al.1942
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
Fig. 2. Changes in ethanol preference in the model of vol-
untary drinking with the intermittent access to 20% ethanol
in a two-bottle choice procedure. The data are presented as
mean ± SEM; * p < 0.05; ** p < 0.005, significant differences
with the first session (mixed-effects model and Dunnett’s test
for multiple comparison).
Fig. 3. Effect of purmorphamine on ethanol preference in
the model of voluntary drinking with the intermittent access
to 20% ethanol in a two-bottle choice procedure. R,random-
ization by the preference for alcohol over 5 sessions before
the start of purmorphamine administration; P,intraperitone-
al administration of purmorphamine at a dose of 5 mg/kg
body weight 1 h before alcohol access (sessions 16-20). The
data are shown as mean ± SEM.
Fig. 4. Effect of purmorphamine on the ALB in the open field test after implementation of the voluntary drinking model with
the intermittent access to 20% ethanol in a two-bottle choice procedure. a) Time spent in the center; b) number of entries
tothe center. The data are presented as median and interquartile range. Purmorphamine was administered intraperitoneally
at a dose of 5 mg/kg body weight one hour before ethanol drinking sessions 16-20. The test was performed one day after
alcohol withdrawal.
Elevated plus maze (EPM) test. Two days after
alcohol withdrawal, rat behavior was assessed in the
EPM test (Fig.5). According to the Kruskal–Wallis test,
experimental groups differed in the number of head
dips (Fig. 5a; H
(3,30)
= 11.800; p = 0.008), time spent
in the open arms (Fig. 5c; H
(3,30)
= 9.440; p = 0.024),
and number of entries to the closed arms (Fig. 5d;
H
(3,30)
=8.121; p = 0.044), but not in the number of en-
tries to the open arms (data not shown; H
(3,30)
=1.985;
p = 0.576) and number of stretch-attend postures
(Fig. 5b; H
(3,30)
= 1.604; p = 0.659). Post hoc analysis of
multiple comparisons using the Dunn’s test showed
that the number of head dips in the Ethanol + pur-
morphamine group was significantly lower compared
to the Control group (Fig. 5a). The Ethanol + purmor-
phamine group also showed a trend (p < 0.1) toward
an increase in the time spent in the open arms com-
pared to the Control + purmorphamine (p = 0.084)
and Ethanol (p = 0.053; Fig. 5c) groups, as well as
adecrease in the number of entries to the closed arms
compared to the Control (p = 0.088) and Control + pur-
morphamine (p = 0.095; Fig. 5d) groups. The distance
traveled and the speed of movement did not differ
between the groups (data not shown).
Expression of Shh, Ptch, Smo, and Gli mRNAs in
brain structures. Three days after alcohol withdraw-
al, the relative content of mRNAs for Shh, Ptch, Smo,
and Gli in the frontal cortex, amygdala, hippocampus,
and striatum was evaluated (Table 1). According to
ANOVA, the factor “ethanol” had a considerable effect
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BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
Fig. 5. Effect of purmorphamine on rat behavior in the EPM test after implementation of the voluntary drinking model with
the intermittent access to 20% ethanol in a two-bottle choice procedure. a)Number of head dips; b)number of stretch-attend
postures; c) time spent in the open arms; d) number of entries to the closed arms (Kruskal–Wallis test followed by posthoc
analysis of multiple comparisons with the Dunn’s test). The data are presented as median and interquartile range. Purmor-
phamine was administered intraperitoneally at a dose of 5 mg/kg body weight one hour before ethanol drinking sessions
16-20. The test was carried out two days after alcohol withdrawal.
on the relative level of Shh mRNA in the amygdala,
while the effects of the factor “purmorphamine” and
interaction of “ethanol”and“purmorphamine” factors
did not reach the level of significance (Table 1). The
factor “ethanol” (but not “purmorphamine” or interac-
tion of “ethanol” and “purmorphamine” factors) had a
considerable effect on the relative Ptch mRNA level in
the amygdala (Table 1). Subsequent multiple compar-
ison of means revealed no significant differences be-
tween the groups. Nevertheless, it should be noted that
the Ethanol + purmorphamine group showed a trend
(p < 0.1) toward a decrease in the Shh mRNA level in
the amygdala compared to the Control (p = 0.068) and
Control + purmorphamine (p = 0.062) groups (Fig. 6).
At the same time, in the Ethanol + purmorphamine
group, the level of Shh mRNA in the amygdala exhibit-
ed a significant inverse correlation with the time spent
in the open arms (r = −0.786; p = 0.036), whereas in the
Control (r = 0.167; p = 0.693), Control + purmorphamine
(r = −0.071; p = 0.879) and Ethanol (r = 0.357; p = 0.432)
groups, this correlation did not reach the level of sig-
nificance. There were no considerable changes in the
content of Ptch, Smo, and Gli mRNAs in the amygdala,
as well as in the content of all studied mRNAs in the
frontal cortex, hippocampus, and striatum (Table1).
Fig. 6. Effect of purmorphamine on the relative Shh mRNA
content in the amygdala after the voluntary drinking model
with the intermittent access to 20% ethanol in a two-bottle
choice procedure. The data are presented as mean ± SEM.
Purmorphamine was administered intraperitoneally at a
dose of 5 mg/kg one hour before the ethanol drinking ses-
sions 16-20. mRNA levels were assessed by PCR three days
after alcohol withdrawal.
PEREGUD et al.1944
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
Table 1. Relative content of mRNAs for the Shh signaling cascade components in different brain regions after
implementation of the voluntary drinking model with the intermittent access to 20% ethanol in a two-bottle
choice procedure
Brain
area
mRNA
Control,
n=8
Control +
Purmor-
phamine,
n=7
Ethanol,
n=7
Ethanol +
Purmor-
phamine,
n=8
ANOVA: F; p
Ethanol
Purmor-
phamine
Ethanol ×
Purmor-
phamine
Frontal
cortex
Shh 1.03 ± 0.11 1.17 ± 0.09 1.21 ± 0.14 1.03 ± 0.16 0.02; 0.89 0.02; 0.89 1.46; 0.24
Ptch 1.03 ± 0.10 1.13 ± 0.08 1.08 ± 0.10 1.00 ± 0.16 0.11; 0.74 0.01; 0.91 0.69; 0.42
Smo 1.05 ± 0.13 1.10 ± 0.08 1.00 ± 0.12 1.03 ± 0.10 0.34; 0.57 0.17; 0.68 0.01; 0.92
Gli 1.09 ± 0.18 1.20 ± 0.22 1.54 ± 0.36 1.06 ± 0.26 0.32; 0.58 0.48; 0.49 1.24; 0.28
Amygdala
Shh 1.01 ± 0.04 1.02 ± 0.10 0.89 ± 0.07 0.76 ± 0.04 7.58; 0.01 0.76; 0.39 1.10; 0.30
Ptch 1.02 ± 0.07 1.05 ± 0.06 0.93 ± 0.07 0.81 ± 0.11 4.28; 0.05 0.30; 0.59 0.90; 0.35
Smo 1.02 ± 0.07 1.10 ± 0.07 0.96 ± 0.06 0.94 ± 0.12 1.55; 0.22 0.10; 0.75 0.33; 0.57
Gli 1.22 ± 0.29 1.21 ± 0.14 1.04 ± 0.11 1.05 ± 0.27 0.60; 0.45 0.00; 0.99 0.00; 0.98
Hippo-
campus
Shh 1.02 ± 0.08 0.86 ± 0.13 0.98 ± 0.07 0.85 ± 0.06 0.09; 0.77 2.82; 0.11 0.05; 0.83
Ptch 1.03 ± 0.09 0.91 ± 0.13 0.97 ± 0.10 0.73 ± 0.08 1.29; 0.27 3.21; 0.08 0.34; 0.56
Smo 1.03 ± 0.09 0.99 ± 0.17 1.10 ± 0.13 0.82 ± 0.09 0.18; 0.68 1.82; 0.19 1.07; 0.31
Gli 1.05 ± 0.11 0.84 ± 0.23 0.92 ± 0.07 0.75 ± 0.07 0.69; 0.41 2.02; 0.17 0.02; 0.88
Striatum
Shh 1.09 ± 0.16 1.43 ± 0.17 1.09 ± 0.17 1.28 ± 0.14 0.20; 0.66 2.76; 0.11 0.22; 0.64
Ptch 1.05 ± 0.10 1.07 ± 0.09 1.12 ± 0.18 1.09 ± 0.07 0.17; 0.68 0.00; 0.98 0.04; 0.85
Smo 1.04 ± 0.10 0.95 ± 0.10 1.17 ± 0.19 1.03 ± 0.11 0.70; 0.41 0.78; 0.39 0.05; 0.83
Gli 1.17 ± 0.25 1.08 ± 0.11 1.13 ± 0.27 1.03 ± 0.12 0.05; 0.83 0.21; 0.65 0.00; 0.99
Note. Statistically significant differences (two-factor ANOVA) are shown in bold.
DISCUSSION
Hh cascade and alcohol drinking. The role of
Hh proteins in the mechanisms of alcohol consump-
tion in adults and development of disorders associat-
ed with chronic intoxication is still poorly understood.
Our work is the first one to assess the effect of pur-
morphamine on the voluntary alcohol drinking in
adult rats. According to the obtained results, purmor-
phamine had no significant effect on the preference
for ethanol. However, we should mention the earlier
reported data on the effectiveness of purmorphamine
in the model of obsessive-compulsive disorder, which
might also be involved in the formation of patholog-
ical forms of alcohol drinking. In particular, it was
demonstrated that systemic administration of pur-
morphamine attenuated obsessive-compulsive behav-
ior caused by the injection of serotonin 1A receptor
agonist into the raphe nuclei [22]. Furthermore, the
authors of this work observed a decrease in the Shh
content in the cerebrospinal fluid, blood plasma, and
brain tissue, i.e., purmorphamine stabilized the level
of Shh without affecting its content [22].
Hh cascade and ALB. Anxiety is a typical affec-
tive symptom associated with the alcohol withdrawal
syndrome [25, 26]. Alcohol intake in voluntary drink-
ing models, including the model of intermittent ac-
cess to 20% ethanol in a two-bottle choice procedure,
has been accompanied by changes in the ALB. It was
shown that alcohol withdrawal after the intermittent
access to 20% ethanol led to the pronounced mani-
festations of ALB in the open field and EPM tests in
mice [33] and rats [34]. However, some researchers
failed to demonstrate the effect of alcohol intake on
INTERACTION OF PURMORPHAMINE AND ALCOHOL 1945
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
ALB in the voluntary drinking models. Thus, providing
free access to 10% alcohol for 4 h during the dark
phase of the day/night cycle for 12 days did not affect
the number of entries to the center in the open field
test 8 days after alcohol withdrawal [35]. No behavior
changes in the EPM test were reported one day [36]
or one week [37] after stopping the intermittent access
to 20% ethanol. Similarly, continuous access to alco-
hol in the voluntary drinking model had no effect on
animal behavior in the EPM test [38]. Hence, the indi-
cations that voluntary alcohol drinking in rodents can
modify their behavior in the anxiety tests are rather
contradictory. Here, we found no behavioral changes
in the open field and EPM tests after alcohol with-
drawal.
The Hh cascade is known to participate in the
molecular mechanisms of ALB. Direct involvement of
the Hh family proteins in the regulation of ALB was
demonstrated in Dhh-deficient mice [39]. The authors
investigated different forms of behavior, including mo-
tor activity, learning, and memory formation, as well
as anxious- and depressive-like behavior, and found
an increase in the depressive-like behavior (in the
forced swim test) and ALB (in the Vogel conflict test)
in Dhh-deficient male mice compared to the wild-type
controls [39]. In the model of fetal alcohol syndrome
in Danio rerio, upregulation of Shh expression in fish
exposed to ethanol in early embryogenesis prevented
CNS dysmorphogenesis and ALB at the late develop-
ment stages [40]. Conditional Smo knockout in neural
stem cells disrupted neurogenesis in the mature hip-
pocampus, which was accompanied by exacerbation
of anxiety- and depressive-like behavior, without af-
fecting motor activity and learning [23].
Here, we demonstrated for the first time that al-
terations in the animal behavior in the EPM test might
be caused by the interaction between voluntary alco-
hol drinking and systemic administration of purmor-
phamine leading to the activation of the Shh cascade.
These results are consistent with the concept that stim-
ulation of the Hh cascade produces the anxiolytic ef-
fect upon access to alcohol, as evidenced by the trend
toward an increase in the time spent in the open arms
and decrease in the number of entries to the closed
arms in the EPM test.
Biochemical markers of the Hh signaling activa-
tion in the brain and ALB after alcohol with drawal.
According to the ANOVA, alcohol drinking affected the
content of only Shh and Ptch mRNAs and only in the
amygdala, i.e., in the brain structure responsible for
the formation of emotions associated with anxiety.
Since activation of genes coding for the Hh signaling
components (primarily Ptch and Gli) is considered to
be a marker of the Hh cascade activation in many
cell types [3], including glia [41] and neurons [42, 43],
we expected to demonstrate upregulation of the corre-
sponding mRNAs in the brain of animals treated with
purmorphamine. Contrary to our expectations, we did
not detect it. Instead, there was an obvious trend to-
wards a decrease in the content of Shh mRNA in the
animals that had an access to alcohol and received
purmorphamine.
In experimental models, expression of the Hh sig-
naling cascade components in the brain may reflect
the neuroprotective properties of certain compounds
or their impact on behavior, including ALB. In this
case, the direct pharmacological activity of these sub-
stances might not necessarily be associated with the
Hh cascade.
Thus, in the rat model of chronic unpredictable
stress, the flavanone naringenin prevented the devel-
opment of depressive-like behavior in the forced swim
test and cognitive impairment in the Morris water
maze test, as well as normalized the content of Shh
and Gli mRNAs previously reduced by the stress in the
hippocampus [44]. In the same model, exacerbation of
anxiety was accompanied by a decrease in the content
of Shh and Gli mRNAs in the hippocampus, while nic-
otine was able to abolish behavioral impairments and
changes in gene expression [45].
Intoxication of rats with propionic acid or ethid-
ium bromide was accompanied by a reduction in the
Shh content in the brain, while systemic administra-
tion of purmorphamine had a neuroprotective effect
in both these models, which was accompanied by nor-
malization of the Shh levels [10, 11]. Chronic systemic
administration of the Smo agonist SAG (Smoothened
agonist), a derivative of benzothiophene, alleviated
anxiety in the open field and EPM tests in the high-
fat diet-fed mice [24]. Moreover, SAG normalized the
reduced content of Shh in these animals without af-
fecting the amount of Shh in the neocortex [24]. Sys-
temic administration of purmorphamine in mice sub-
jected to the middle cerebral artery occlusion reduced
the brain infarction area and decreased the develop-
ment of neurological deficits, which was accompanied
by a decrease in the apoptotic death of neurons [8].
Interestingly, occlusion of the middle cerebral artery
upregulated expression of mRNAs for the Hh cascade
components (Shh, Ptch, Smo, and Gli) in the neocortex.
However, despite its physiological activity, purmor-
phamine did not affect expression of these proteins
[8]. Therefore, purmorphamine activity does not nec-
essarily reflected by changes in the expression of the
Hh signaling components.
We found that in the Ethanol + purmorphamine
group. behavior in the EPM test and the Shh content
in the amygdala showed changes in opposite direc-
tions. This pattern was confirmed by the existence
of the inverse correlation between the time spent
in the open arms and relative content of the Shh
mRNA in the amygdala only in this group of animals.
PEREGUD et al.1946
BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024
The amygdaloid complex plays a key role in the for-
mation of stress response and anxiety [46-48]. The ac-
tivity of this brain region was found to change during
withdrawal after chronic intermittent intoxication
with alcohol vapors and might be associated with
manifestations of the withdrawal [49, 50]. The data
on the Hh functioning in the amygdala are scarce,
with the exception of few works showing Hh involve-
ment in memory formation. The manifestation of the
conditional fear response to a combination of sound
signal and electric shock in mice was accompanied
by the activation of cell proliferation and increase in
the levels of Shh, Ptch, and Gli in the amygdala [51].
At the same time, suppression of Shh in mitotic neu-
rons of the basolateral amygdala by injection of a ret-
rovirus with an interfering RNA attenuated formation
of conditional fear response and neurogenesis [51].
Moreover, by regulating neurogenesis in the amygda-
la, Shh normalized attenuation of the conditional fear
response over time. Shh overexpression in the amyg-
dala stimulated neurogenesis and attenuated fear
response, while interfering RNAs had the opposite
effect [52].
CONCLUSION
This work is the first one to investigate the activ-
ity of purmorphamine in the voluntary ethanol drink-
ing in rats and to study their behavior in the open
field and EPM tests after alcohol withdrawal. Although
purmorphamine did not modify alcohol preference,
it altered rat behavior in the EPM test after alcohol
withdrawal. Rat behavior in the EPM test, as well as
the Shh mRNA content in the amygdala demonstrated
changes in opposite directions and correlated inverse-
ly only if the access to alcohol was combined with
purmorphamine administration. The data obtained
suggest the involvement of the Shh signaling in the
formation of mechanisms of alcohol withdrawal-
induced emotional disturbances in the amygdala.
Further studies are needed to reveal specific details
of this involvement.
Contributions. D.I.P., M.Yu.S., and N.V.G. devel-
oped the study concept; D.I.P. and N.V.G., supervised
the study; D.I.P., N.I.S., and A.A.K. conducted the exper-
iments; D.I.P., analyzed the results and wrote the text
of the article; M.Yu.S. edited the manuscript; N.V.G.
prepared the final version of the manuscript.
Funding. This work was supported by the Min-
istry of Science and Higher Education of the Russian
Federation, within the framework of the State Task
“Neurophysiological and biochemical mechanisms of
nervous system pathology, neurodegeneration” (Regis-
tration number: 1021062411628-8-3.1.4.).
Ethics declarations. All applicable institutional
guidelines for the care and use of animals (Guide for
the Care and Use of Laboratory Animals, Eighth Edi-
tion, 2010), the requirements of the European Conven-
tion for the Protection of Vertebrate Animals Used for
Experiments or for Other Scientific Purposes (Stras-
bourg, 1986 with Appendix dated 15.06.2006), princi-
ples of Good Laboratory Practice (Order of the Minis-
try of Health of the Russian Federation no.199n dated
01.04.2016, GOSTR 53434-2009), as well as “Guidelines
for Working with Animals” approved by the Bioethical
Committee of the Institute of Higher Nervous Activi-
ty and Neurophysiology were followed. The authors
of this work declare that they have no conflicts of
interest.
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