Anxiolytic-like effect of succinic acid in mice

Abstract

The putative anxiolytic activity of succinic acid was examined in male mice by using a number of experimental paradigms of anxiety and compared with that of the known anxiolytic compound diazepam. Use of the elevated plus-maze test revealed that diazepam (1.0, 2.0 and 4.0 mg/kg, PO) or succinic acid (3.0 or 6.0 mg/kg, PO) increased the percentage of entries into open arms and of time spent on open arms. In novel food consumption test, succinic acid (3.0, 6.0, and 12.0 mg/kg, IP) caused significant increases in food intake during 5 min when compared with the vehicle. In the stress-induced hyperthermia test, 40 min after drug administration rectal temperature was measured, succinic acid at dose of 1.5 mg/kg, inhibited stress-induced hyperthermia. Thus, these findings indicated that, in contrast with diazepam, succinic acid exhibits anxiolytic-like effect.

Full text

Anxiolytic-like effect of succinic acid in mice
Si Wei Chen*, Qin Xin
1
, Wei Xi Kong, Li Min, Jing Fang Li
Department of Pharmacology, Shenyang Pharmaceutical University, Box 41, 103 Wenhua Road, 110016 Shenyang, PR China
Received 28 February 2003; accepted 4 June 2003
Abstract
The putative anxiolytic activity of succinic acid was examined in male mice by using a number of
experimental paradigms of anxiety and compared with that of the known anxiolytic compound diazepam. Use
of the elevated plus-maze test revealed that diazepam (1.0, 2.0 and 4.0 mg/kg, PO) or succinic acid (3.0 or 6.0
mg/kg, PO) increased the percentage of entries into open arms and of time spent on open arms. In novel food
consumption test, succinic acid (3.0, 6.0, and 12.0 mg/kg, IP) caused significant increases in food intake
during 5 min when compared with the vehicle. In the stress-induced hyperthermia test, 40 min after drug
administration rectal temperature was measured, succinic acid at dose of 1.5 mg/kg, inhibited stress-induced
hyperthermia. Thus, these findings indicated that, in contrast with diazepam, succinic acid exhibits anxiolytic-
like effect.
D2003 Elsevier Inc. All rights reserved.
Keywords: Succinic acid; Anxiolytic; Elevated plus-maze; Novel food consumption; Hyperthermia; Mice
Introduction
Succinic acid is an endogenous metabolite of Krebs cycle and GABA-succinate shunt in the brain
(Sanders et al., 1969). It has been reported that succinic acid had sedative effect (4000 mg/kg) and
protective action against high pressure oxygen convulsion (1180 mg/kg) (Sanders et al., 1969; Gessa
et al., 1968). Jin et al. also reported that succinic acid at dose of 1180 mg/kg produced significant
0024-3205/$ - see front matter D2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2003.06.017
* Corresponding author. Tel.: +86-24-23909448; fax: +86-24-24513031.
E-mail address: c100sw@yahoo.com.cn (S.W. Chen).
1
Present address: Department of Pharmacology, Jining Medical College, 45 Jianshe Road, 272013 Jining, Shandong
Province, China.
www.elsevier.com/locate/lifescie
Life Sciences 73 (2003) 3257 – 3264

decrease of the locomotor activity and hyperthermia and prolonged the pentobarbital sleeping time
significantly in mice. Moreover, succinic acid was found to protect rats against audiogenic seizure
and inhibit the electroshock seizure in mice, but not the convulsion induced by picrotoxin, strychnine
and semicarbazide (Jin and Zhang, 1980). Recently, Yue et al. reported that succinic acid (100–400
mg/kg) dose-dependently inhibited pentylenetetrazol chemical and amygdale kindled seizer (Yue et
al., 2002).
The purpose of the present work was to investigate the effects of succinic acid in the elevated plus-
maze, novel food consumption, and stress-induced hyperthermia tests. These animal models have been
validated as tests for anxiety, using behavioral and physiological measures, and have shown good
sensitivity to anxiolytic drugs.
Materials and methods
Animal
Male Swiss mice (Experimental Animal center of Shenyang Pharmaceutical University) weighing
20–22 g were used. For Elevated plus-maze test, mice were maintained under a 12 h reversed light
cycle (light off 07:00) at room temperature (22 F2jC) for at least 10 day (Ferrari et al., 1998)
For novel food consumption test and stress-induced hyperthermia test, mice were maintained on a
non-reversed 12L: 12D cycle conditions (lights on: 07:00). Five animals were housed in a cage
(25 14 12 cm). Food and water were freely available at all times. Each mouse was used only
once.
Drugs
Succinic acid was purchased from Shenyang Reagent Co. (Shenyang, China). Diazepam was obtained
from Hubei Pharmaceutical Factory (Hubei, China). Tween 80 was purchased from Shenyang Dongxing
Reagent Factory (Shenyang, China). Succinic acid was dissolved in 0.9% saline, then the solutions were
adjusted to pH 7.2 f7.5 with diluted NaOH. Diazepam was ultrasonically dispersed in distilled water
to which Tween 80 (2 drops/10 ml) had been added. Test drugs were administered PO or IP in a volume
of 10 ml/kg, control animals were administered with the corresponding vehicles. All drugs were freshly
prepared before each treatment trial.
Procedure
Elevated plus-maze test
The elevated plus-maze comprised two open arms (30 5 cm), facing each other, and two
closed arms (30 515 cm) which extended from a central platform (5 5 cm) to form a
plus sign. The open arms included a clear, slightly raised edge (0.25 cm height) to reduce the
likelihood of animals falling-over. The closed arms were enclosed with three roof-open walls (15
cm height) made from clear plexiglas (Lister, 1987; Cole and Rodgers, 1994). The maze floor was
constructed of black plexiglas. The apparatus was raised to a height of 45 cm by a single central
support (Kuribara et al., 1998). Four 25-W red fluorescent lights arranged as a cross at 100 cm
S.W. Chen et al. / Life Sciences 73 (2003) 3257–32643258

above the maze were used as the source of illumination (Ferrari et al., 1998; Hasenohrl et al.,
1996).
Mice were randomly assigned to eight experimental groups and then adjusted slightly to match the
average body weights of the groups (n = 10). All testing was conducted during the midportion of the
dark phase of light cycle and in an order counter balanced for treatment condition and commenced by
placing a mouse on the central platform of the maze facing an open arm. The number of entries into, and
the time spent in, each of the two types of arm, were counted during 5 min. Arm entry was defined as all
four paws having crossed the dividing line between an arm and the central area (Lin et al., 1998). The
maze was thoroughly cleaned with water after each trial to remove any trace of odour. The sessions were
recorded by an overhead video camera linked to a monitor and video recorder in an adjacent laboratory.
All behavioral recordings were carried out with the observer unaware of the treatment of the mice. Test
drugs and the vehicle were administered PO 40 min prior to the plus-maze test.
Novel food consumption in mice
This test was performed according to a procedure described by Keane (Keane et al., 1988). Mice
were subjected to food deprivation for 16 h before the test. Groups of 9–10 mice were administered
either succinic acid (IP) or diazepam (PO), and 30 min later the animals were placed individually in
plastic cages (25 14 14 cm) for 5 min. Each cage contained a weighed amount of a novel food
source (a mixture of one-third bean oil and two-thirds standard rodent food powder) in a Petri dish. At
the end of the 5 min test period the remaining food and crumb in the cage were collected and weighed
(Britton and Britton, 1981). Food consumption, defined as milligrams eaten in 5 min per gram of body
weight, was calculated for each mouse and the mean ( FSEM) was determined for each treatment
group.
Stress-induced hyperthermia test
The test procedure for stress-induced hyperthermia was similar to that described by Lecci et al. (Lecci
et al., 1990). Temperature measurements were carried out in the same room where mice were housed.
Rectal temperature was measured to the nearest 0.1 jC by a digital thermometer, model JM 624u. The
probe (2-mm diameter), dipped into liquid paraffin before insertion, was held in the rectum until steady
readings were obtained for 20 s and the mouse was reallocated to its cage after each temperature
measurement.
Eighteen animals were housed per cage (50 25 20 cm). The animals were administered orally
or intraperitoneally with either a drug or vehicle 40 min (succinic acid and its corresponding vehicle) or
50 min (diazepam and its vehicle) before temperature measurement. After diazepam, succinic acid were
given, the rectal temperature was recorded in the first and last three out of 18 mice allocated to each
cage, maintaining the same order as that of treatment, while mice numbers 4 through 15 inclusive were
simply removed and again returned to the cage. The whole procedure required about 10 min. All
experiments were performed between 8 and 12 a.m.
Statistical analyses
Data given represent mean FSEM values. In the elevated plus-maze and novel food consumption
test, data were analyzed by one-way ANOVA. In the stress-induced hyperthermia test, results were
analyzed by factorial analysis of variances (ANOVA). Whenever ANOVA was significant, further
comparisons between vehicle- and drug-treatment groups were performed using the Dunnett’s t-test. All
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analysis was performed using the software SPSS V11.0 for windows. The level of statistical significance
adopted was P< 0.05.
Results
Elevated plus-maze test
In the plus-maze test, conducted 40 min after administration of 3.0, 6.0, and 12.0 mg/kg, succinic acid
did not significantly alter the total number of arm entries made by mice [F(3, 36) = 0.62, P> 0.05, Table
1]. However, succinic acid at doses of 3.0 and 6.0 mg/kg resulted in a significant increase in the
percentage of entries into open arms respect to total arms entries [F(3,36) = 3.92, P< 0.05], as well as
the percentage of time spent in open arms [F(3,36) = 3.20, P< 0.05]. At 12.0 mg/kg, succinic acid-
Table 1
Effects of succinic acid on total arm entries by male mice on the elevated plus-maze
Drug Dose (mg/kg) Total arm entries
Vehicle 15.7 F1.0
Succinic acid 3 18.4 F1.2
6 18.0 F1.3
12 17.9 F2.4
Vehicle 16.3 F1.4
Diazepam 1 31.9 F2.2**
2 24.9 F1.3
4 25.4 F5.4
Values represent means FSEM from 10.
** P< 0.01, Dunnett’s t-test after ANOVA.
Fig. 1. Effects of succinic acid (A) and diazepam (B) in the elevated plus-maze in male mice (n = 10). Results are expressed as
means FSEM. The following parameters are shown: %EOA (percentage of entries into open arms respect to total entries into
the arms); %TOA (percentage of time spent in open arms respect to total time spent in the arms). Mice were given a 5 min test
40 min after PO with diazepam (1, 2, and 4 mg/kg) or succinic acid (3, 6, and 12 mg/kg). Significant differences calculated from
two-tailed Dunnett’ t-test are expressed by *P< 0.05, **P< 0.01 vs. respective vehicle after one – way ANOVA.
S.W. Chen et al. / Life Sciences 73 (2003) 3257–32643260

treated mice were not different from vehicle control [Fig. 1(A)]. Diazepam at dose of 1.0 mg/kg
significantly increased the total number of arm entries made by mice [F(3,36) = 4.38, P< 0.01, Table 1].
The ANOVA showed that at doses of 1.0, 2.0, and 4.0 mg/kg, diazepam significantly elevated both the
percentage of entries into open arms and of time spent in open arms [F(3,36) = 3.90, P< 0.05; F(3,36) =
5.08, P< 0.01. Fig. 1(B)].
Novel food consumption test
Fig. 2. shows the mean values of food intake (milligrams per gram) in a 5 min period. Forty minutes
after PO administration, both succinic acid (3, 6, and 12 mg/kg) and diazepam (0.5, and 1 mg/kg)
Fig. 2. Effects of succinic acid and diazepam in the novel food consumption test. Experiment were carried out in groups of
9–10 mice, each mouse was subjected to food deprivation for 16 h before testing. The values represent the mean FSEM of
food intake (milligrams per gram) in a 5 min period. Significant differences from control: *P< 0.05, **P< 0.01.
Table 2
Effects of administration of succinic acid and diazepam on hyperthermia of mice removed at a later time
Drug Dose Route Time Rectal temperature DT ANOVA interaction
(mg/kg) (min) First three Last three
Vehicle – IP 40 37.5 F0.1 38.2 F0.2* 0.7
Succinic acid 1.5 IP 40 37.1 F0.2 37.2 F0.2
a
0.1 F(3, 64) = 1.68, P> 0.05
3.0 IP 40 37.6 F0.3 37.5 F0.3 0.1
6.0 IP 40 37.0 F0.2 37.6 F0.2* 0.6
Vehicle – PO 50 37.4 F0.1 38.3 F0.3** 0.9
Diazepam 1.25 PO 50 37.0 F0.2 37.3 F0.3 0.3 F(3, 64) = 3.31, P= 0.025
2.5 PO 50 36.9 F0.3 37.1 F0.3
a
0.2
5.0 PO 50 37.5 F0.2 36.7 F0.4
b
0.8
Values represent means FSEM from 9 mice. Time indicates the lag time between treatments and test. Dunnett’s t-test.
a
P< 0.05.
b
P< 0.01 vs corresponding vehicle; t-test.
*P< 0.05.
**P< 0.01 vs first-three.
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increased the amount of novel food consumption. On the average, the food consumption in the succinic
acid-treated group was significantly different from the vehicle-treated group [F(3,35) = 4.42, P< 0.01].
Diazepam as a reference drug for anxiolytic action, also increased the amount of food consumption [F(3,
35) = 3.27, P< 0.05].
Stress-induced hyperthermia test
The effects of succinic acid at doses of 1.5, 3.0 or 6.0 mg/kg and diazepam at doses of 1.25, 2.5, and
5.0 mg/kg are shown in Table 2. It was observed that the hyperthermia of the last three showed an
significant increase in the vehicle-group. The hyperthermia of the last three in the succinic acid-group
and diazepam-group were not significantly different from the first three except for the succinic acid 6
mg/kg group. Succinic acid (1.5 mg/kg), diazepam (5 mg/kg) inhibited the hyperthermia of the last three
[F(3, 32) = 3.28, p < 0.05; F(3, 32) = 4.65, P < 0.01]. Note that none of the treatments of succinic acid
significantly affected basal core body temperature.
Discussion
The present study is the first to demonstrate the anxiolytic effect of succinic acid on results from the
elevated plus-maze (a model of anxiety in an approach-avoidance conflict), novel food consumption, and
stress-induced hyperthermia (a model of anticipatory anxiety), which are animal models of anxiety that
have been subjected to through critical appraisal to demonstrate central nervous system activity of drugs.
The elevated plus-maze is an animal analogue that identifies clinically established anxiolytic and
anxiogenic drugs, and is currently used in the screening of drugs that act on the central nervous system
(Hasenohrl et al., 1996; Bhattacharya and Mitra, 1991; Pellow and File, 1986; Pellow et al., 1985).
Normally, mice spent most of their time in the closed arms and avoided the open arms (fear of the open
and high places possibly) (Ang and Hung, 1999). In the test the percentage of entries into open arms and
of time spent in open arms have generally been used as indices of the anxiety. These values were
increased by anxiolytic agents and reduced by anxiogenic agents (Pellow et al., 1985). In this study,
succinic acid at 3 and 6 mg/kg have anxiolytic effects, as indicated by increased the amount of open time
and the number of open entries. At dose of 12 mg/kg, succinic acid did not influence the mice behavior
on the elevated plus-maze. The anxiolytic profile of succinic acid revealed in the present experiment
showed some differences with regard to the known anxiolytic profile of benzodiazepins in this model.
Succinic acid did not affect motor activity. However, diazepam at 1 mg/kg increased motor activity. This
effect of diazepam might be correlated with disinhibition and with ataxia and motor impairment,
respectively.
Animals generally show neophobia when they are presented with novel food or are placed in a novel
environment (Miyamoto et al., 1992). Anxiolytics facilitate the ‘‘resolution’’ of that apparent neophabia
by increasing the amount of food eaten (Britton and Britton, 1981). In the novel food consumption test,
succinic acid had significantly increased the amount of food intake in mice compared with control. This
finding suggests that succinic acid has reduced fear of eating novel food in mice, and produced
anxiolytic effect.
Various stressors such as handling, restraint, exposure to a novel environment, and foot shock are
known to cause increases in body temperature (Pechnick and Morgan, 1987; York and Regan, 1982).
S.W. Chen et al. / Life Sciences 73 (2003) 3257–32643262

Among animals of the same cage, mice removed last from their home cage always have higher rectal
temperatures than mice removed first from this cage (Lecci et al., 1990; Zethof et al., 1995; Borsini et al.,
1989). This phenomenon is called stress-induced hyperthermia and has been interpreted as being caused
by anticipatory fear of an aversive event (handling). Interestingly, interactions between body temperature
and emotional states seen to exist in man also. The stress-induced rise in rectal temperature can be
blocked by various mechanisms. Benzodiazepine and non-benzodiazepine anxiolytics were capable of
antagonizing stress-induced hyperthermia. The effect of succinic acid on stress-induced hyperthermia
can be considered as specific, because the compound did not affect the core temperature per se:
obviously, succinic acid counteracted the anxiety-dependent variable, and produced anxiolytic activity
similar to diazepam.
Our preliminary study showed that succinic acid did not produce the anxiolytic effect at doses lower
than 1.5 mg/kg or higher than 12 mg/kg such as 24 and 48 mg/kg on the elevated plus-maze
(unpublished data). Succinic acid exhibited an inverted U-shaped dose-response curve for the percentage
of open arm entries based on the present and preliminary set of data. Consistent with many reports
(Pellow and File, 1986; Dawson and Tricklebank 1995), diazepam resulted in a dose-dependent
anxiolytic effect. Succinic acid at 1.5 f12 mg/kg elicited significant anxiolytic effect and did not
cause the change of motor activity. Gessa et al. reported that succinic acid induced a central depressant
action at high doses, for example sedation at 4000 mg/kg (Gessa et al., 1968), this dose are 2666 times
higher than the minimum dose for significant anxiolytic effect (1.5 mg/kg, IP). Thus it is expected that
when the anxiolytic effect is considered as the main effect, the central depressant effect of succinic acid
is very weak.
Succinic acid is widely distributed in the nervous systems. The distribution of succinate in rabbit
revealed that there are regional differences in succinate concentrations within the CNS. Concentrations
of succinate in cerebral cortex, cerebral white matter, and cerebellum are 0.19, 0.24 and 0.35 Amol/g
respectively (Petroff et al., 1988). Succinic acid mainly produces from the tricarboxylic acid cycle and
GABA-succinate shunt in the brain. The possible physiological importance of the GABA-shunt is seen if
one recognizes that succinate markedly stimulates respiration and oxidative phosphorylation. GABA
could serve as a rapidly available source of succinate under physiological stress conditions. It was
reported that succinate oxidation could monopolize the respiratory-electron transport chain which is the
major souce of ATP production. But the effect of low milligram doses of succinic acid on endogenous
levels of this substance in the brain has not been reported.
Although the behavioural effects of succinic acid have been repeatedly reported, the neural
mechanisms underlying these effects have not been clearly elicited. Yue et al. suggested GABAergic
involvement in the anticonvulsion effect of succinic acid (Yue et al., 2002). Given that the role of
GABA
A
receptors in mediation of the anxiolytic effects of benzodiazepines has been well established, it
seems reasonable to propose a GABAergic mechanism for the effects of succinic acid on the results from
the plus-maze, novel food consumption, and stress-induced hyperthermia. Furthermore, we recently
found that the noncompetitive GABA
A
receptor antagonist, picrotoxin selectively antagonized the
anxiolytic-like effects of succinic acid on the percentage of open arm entries and of time spent in the
open arms of plus-maze anxiety (unpublished data). This suggests that the interaction between succinic
acid and GABA
A
receptors may be important for the anxiolytic effects of succinic acid.
In conclusion, this study supported the suggestion that succinic acid possesses anxiolytic effects in the
elevated plus-maze, food neophobia and stress-induced hyperthermia models. Furthermore, only low
doses succinic acid were needed to induce an anxiolytic action.
S.W. Chen et al. / Life Sciences 73 (2003) 3257–3264 3263

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