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. 2024 May 17;17:58–64. doi: 10.1016/j.ibneur.2024.05.003

The additive effect between citalopram and muscimol upon induction of antinociceptive effect in male mice

Taha Shokrnejad-namin a,b, Elnaz Amini a,b, Fatemeh Khakpai a, Mohammad-Reza Zarrindast b,c,d,⁎
PMCID: PMC11725972  PMID: 39807389

Abstract

Previous investigations have revealed the role of GABAergic and serotonergic systems in the modulation of pain behavior. This research aimed to examine the effects of intracerebroventricular (i.c.v.) infusion of GABAA receptor agonist and antagonist as well as citalopram on pain behavior in male mice. For i.c.v. microinjection, a guide cannula was surgically implanted in the left lateral ventricle of male mice. Pain behavior was evaluated using a tail-flick test. Tail flick latency was measured in each experimental group of mice every 15 min (for 60 min). I.c.v. microinjection of muscimol (0.5 and 1 µg/mouse; GABAA receptor agonist) into the left lateral ventricle dose-dependently induced an antinociceptive effect. On the other hand, i.c.v. infusion of bicuculline (1 µg/mouse; GABAA receptor antagonist) induced a hyperalgesia response. Moreover, intraperitoneally (i.p.) administration of citalopram (8 mg/kg) produced an antinociceptive effect. Co-treatment of citalopram (8 mg/kg) along with muscimol (0.25 µg/mouse) or bicuculline (0.25 µg/mouse) potentiated the antinociceptive effect produced by citalopram. We found an additive antinociceptive effect of citalopram and muscimol in male mice. In conclusion, our results suggested an interaction between citalopram and GABAergic agents on the modulation of pain behavior in male mice.

Keywords: Citalopram, Muscimol, Bicuculline, Pain, Mice

Highlights

  • •

    I.c.v. infusion of muscimol and bicuculline-induced analgesia and hyperalgesia, respectively.

  • •

    I.p. administration of citalopram caused analgesia in male mice.

  • •

    Co-infusion of citalopram with muscimol or bicuculline potentiated analgesia caused by citalopram.

  • •

    There is an additive effect between citalopram and muscimol on the management of pain.

1. Introduction

Pain is a usual word utilized to refer to a wide range of physical and mental conditions (Papini et al., 2015). The well-known participant of nociceptive transmission and, so, a likely target for the analgesic, is the A-type gamma-aminobutyric acid (GABAA) receptor. GABAA receptors are ligand dependent ion (chloride) channels in the chemical synapses of the nervous system, which inhibit nervous excitation (D. A. Belinskaia, M. A. Belinskaia, O. I. Barygin, N. P. Vanchakova, & N. N. Shestakova, 2019). These receptors express in the brain areas related to the pain perception (Knabl et al., 2008) and induce antinociceptive effect (Jia et al., 2004). There are many reports that GABAergic transmission controls the nociceptive effect via reducing the activity of descending antinociceptive neurons in the periaqueductal grey (Harris and Westbrook, 1995) and rostral agranular insular cortex (Jasmin et al., 2003). Behaviorally, a GABA reuptake inhibitor decreases acute and tonic nociception, probably by facilitation of polysynaptic excitatory transmission (Laughlin et al., 2002).

Serotonin (5-HT) is a monoamine neurotransmitter that plays a main role in both nociception and mood modulation (Lowry et al., 2009, Patetsos and Horjales-Araujo, 2016). Serotonin has long been related to both central and peripheral modulation of the nociceptive signal (Millan, 1994). A main modulator of serotonin transmission is the serotonin transporter, which is important for defining the intensity and period of the serotoninergic signal (Hariri and Holmes, 2006). Antidepressants influencing the monoaminergic system are now part of the therapeutic approach for the treatment of numerous pain symptoms (Horjales-Araujo et al., 2013). Selective serotonin reuptake inhibitor (SSRI) is a family of antidepressants, which induces its effect by blocking the reuptake of serotonin in the presynaptic neuron (Hariri and Holmes, 2006). Citalopram is an SSRI that is effective for the treatment of depression diseases and pain symptoms (Lee et al., 2012, Varela et al., 2017). The analgesic property of citalopram was revealed in animal and human studies (Anderberg et al., 2000; D. A. Belinskaia et al., 2019; Bomholt et al., 2005; Campo et al., 2004; Patetsos and Horjales-Araujo, 2016; Roohafza et al., 2014). Some evidence suggested that serotonergic mechanisms may account for the analgesic effect of the antidepressants such as citalopram (Bomholt et al., 2005).

Some studies indicated the existence of a functional interaction between the GABAergic and serotonergic systems. For instance, the serotonergic and GABAergic neurons coexist in the raphe nuclei (Judge et al., 2006, Tao and Auerbach, 2003), the dorsal half of the midbrain (Griffiths and Lovick, 2002), and the amygdala (Donatti and Leite-Panissi, 2009). Furthermore, our previous study revealed an interaction between GABAergic and serotonergic systems on the induction of anxiolytic- and antidepressant-like effects in mice (Amini et al., 2024). Regarding this interaction as well as the analgesic properties of citalopram (Daria A Belinskaia et al., 2019) and muscimol’s potential effect for alleviating pain (Ramawad et al., 2023), this research was designed to examine the role of citalopram and the GABAergic system and their possible interaction on the control of pain behavior using tail-flick in male mice. Also in this study, only male mice were used to evade the effects of variable amounts of sexual hormones on pain behavior during the female sexual cycle.

2. Materials and methods

2.1. Animals

Male mice (Naval Medical Research Institute, NMRI) weighing 20–25 g were obtained from the Tehran University of Medical Sciences (Tehran, Iran). Mice were grouped housed in Plexiglas cages, 4 per cage, (length 60 cm, width 40 cm, and height 25 cm). They were kept in a room maintained at 24±1 ◦C, on a 12 h light/dark cycle (lights on at 07:00 h), with free access to water and food. The experiments were performed in compliance with the recommendations of the Research and Ethics Committee of Tehran University of Medical Sciences (NIH publications No. 80–23). All efforts were made to diminish animal suffering.

2.2. Stereotaxic surgery

For central microinjection of drugs, mice were implanted with a stainless-steel guide cannula (22 gauge) aimed at the lateral ventricle. Implantation was performed by intraperitoneally (i.p.) administration of ketamine hydrochloride (100000 µg/mouse) and xylazine (5000 µg/mouse) anesthesia and was done at least 1 week previous to the tests. Mice were located in a stereotaxic frame (Borj Sanat Company, Iran). The stereotaxic coordinates for the left lateral ventricle were: –0.9 mm posterior to the bregma, −1.5 mm lateral to the sagittal suture, and −2 mm ventral of the dorsal surface of the skull (Paxinos and Franklin, 2001). The cannula was fixed to the skull with one screw and acrylic dental cement. A stylet was implanted in the cannula to inhibit its patent before injection.

To minimize the effects of stereotaxic surgery, the vital signs of mice during surgery were monitored (for example, tail-pinch withdrawal reflexes, breathing rhythm and amplitude, and body temperature throughout the surgery). At the end of the stereotaxic surgery, a mouse was temporarily located in the recovery cage with an infrared spot above the cage. Notably, the light was located such that a mouse could move away from the hot spot when having recovered its righting reflex. Each mouse was returned to the home cage only after completely awakening from anesthesia (Ferry and Gervasoni, 2021).

2.3. Drugs and drug injection

The drugs used in this research were as follows: muscimol hydrobromide (GABAA receptor agonist), bicuculline (GABAA receptor antagonist), and citalopram HBR (Daroupakhsh, Tehran, Iran). Muscimol and citalopram were dissolved in sterile 0.9% saline. Bicuculline was dissolved in one drop of glacial acetic acid. Then, it was made up to a volume of 5 ml by sterile 0.9% saline. The pH of the final solution was neutral. Muscimol and bicuculline were infused intracerebroventricularly (i.c.v.; a volume of 1 μl/mouse) 5 min before the test. Citalopram was injected intraperitoneally (i.p.; a volume of 10 ml/kg) 15 min previous to the test. Drug microinjection in the left lateral ventricle was performed by an internal cannula (27 gauge), terminating 1 mm under the tip of the guide cannula, connected with polyethylene tubing to a Hamilton micro-syringe (volume 1 µl). To allow diffusion of the drug and to decrease the possibility of reflux, 1 µl solution was microinjected over 60 seconds in the left lateral ventricle. The experimental drug doses were chosen in a pilot study.

2.4. Tail-flick

Heat-induced tail-flick latency test was used to assess the thermal pain threshold of the mice. This test was performed upon the standard protocol (Shamsi Meymandi et al., 2019, Yang et al., 2022). A tail-flick apparatus was used for evaluating the nociceptive reaction to thermal stimulation (Borj Sanat Company, Iran). The tail-flick test was carried out in the light phase between 8 a.m. and 12 p.m. Response to the heat source was recorded as the tail-flick latency. Each mouse was slightly wrapped in a soft towel. The distal end of the tail was located in the apparatus every 15 min (for 60 min) after the drug/saline administration. Using a pedal, the heat source and timer were started concurrently. Both were ended automatically by a tail movement, which exposed a photocell below the tail, or by the blind experimenter at the end of a 10-cut-off time. This cut-off time was considered to evade tail damage (Miranda et al., 2011). The test was conducted using a double-blind approach. Individual tail withdrawal latencies were changed to the percentage of the maximum possible effect (%MPE) according to criteria described by (D’Amour and Smith, 1941) as follows: %MPE = [(test latency-baseline latency)/(cut-off latency-baseline latency)] × 100. Baseline measurements were recorded. Because there were no meaningful differences in baseline tail-flick latencies between the experimental groups before the administration of the drugs or saline, the baseline measurements were not reported. For all data, the area under the curve (AUC) of %MPE vs. time was calculated from 0 to 60 min by the trapezoidal rule to determine the overall magnitude and period of effect for the tail-flick latency.

2.5. Experiments

Eight mice were used in each experimental group. After recovery from surgery, male mice were divided into four experiments to test the effects of the drugs. In experiment 1, mice received saline (1 µl/mouse) and muscimol at doses of 0.25, 0.5, and 1 µg/mouse, as well as bicuculline at doses of 0.25, 0.5, and 1 µg/mouse. In experiment 2, mice received saline (1 µl/mouse) and citalopram at doses of 2, 4, and 8 mg/kg as well as co-administration of these doses along with muscimol (0.25 µg/mouse) or bicuculline (0.25 µg/mouse). In experiment 3, mice were injected with citalopram 2 mg/kg + muscimol 0.5 µg/mouse, citalopram 1 mg/kg + muscimol 0.25 µg/mouse, and citalopram 0.5 mg/kg + muscimol 0.125 µg/mouse. Table 1 clarifies the protocol and experimental groups.

Table 1.

explained the drug treatments and effects for each group.

Figure Panel Drug treatments Effect on pain
1 A Saline (1 µl/mouse; i.c.v.), muscimol (0.25, 0.5 and 1 µg/mouse; i.c.v.) Analgesia
B Vehicle (1 µl/mouse; i.c.v.), bicuculline (0.25, 0.5 and 1 µg/mouse; i.c.v.) Hyperalgesia
C (Left panel) Saline (10 ml/kg; i.p.), muscimol (0.25, 0.5 and 1 µg/mouse; i.c.v.) -
C (Right panel) Vehicle (1 µl/mouse; i.c.v.), bicuculline (0.25, 0.5 and 1 µg/mouse; i.c.v.) Hyperalgesia
2 A Saline (10 ml/kg; i.p.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
B Muscimol (0.25 µg/mouse; i.c.v.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
C Bicuculline (0.25 µg/mouse; i.c.v.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
D (Left panel) Saline (10 ml/kg; i.p.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
D (Middle panel) Muscimol (0.25 µg/mouse; i.c.v.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
D (Right panel) Bicuculline (0.25 µg/mouse; i.c.v.), citalopram (2, 4, and 8 mg/kg; i.p.) Analgesia
3 - Citalopram 2 mg/kg + muscimol 0.5 µg/mouse
Citalopram 1 mg/kg + muscimol 0.25 µg/mouse
Citalopram 0.5 mg/kg + muscimol 0.125 µg/mouse		 Additive analgesia
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2.6. Verification of cannula placements

At the end of the experiments, mice were deeply anesthetized by an i.p. administration of urethane (1 g/Kg). Methylene blue solution (1%, 1 µl/mouse) was injected within the guide cannula for the confirmation of the cannula location. The brain of each mouse was removed and placed in a formaldehyde solution (10%). After 7–10 days, the brain sections (40 µm) were observed under a microscope to detect the locations of the stimulated sites according to the atlas of Paxinos and Franklin (Paxinos and Franklin, 2001). Only mice whose microinjections reached the target site were used for statistical analysis. A cannula was fixed into the left ventricle of a total of 170 mice, but, only the data from 160 mice with correct cannula implants were used in the statistical analyses.

2.7. Data analysis

Results were assessed for normal distribution using the Kolmogorov–Smirnov test and homogeneity of variances by Levene’s test. All results are expressed as means ± standard error of the mean (SEM). Data were examined by repeated measures ANOVA considering the time intervals as the repeated measures factor and treatments as between subject’s factor. Also, one-way ANOVA was carried out for comparison of the effects of different dosages of drugs with control groups. Two-way ANOVA was carried out for the assessment of interactions between drugs. After a significant F value, post hoc analysis (Tukey test) was carried out to evaluate exact group comparisons. The level of statistical significance is set at p < 0.05.

Furthermore, isobolographic analysis was carried out to determine the interactions following the co-administration of the two drugs (Khakpai et al., 2021, Nejati et al., 2020). The ED50 of each drug (2 mg/kg for citalopram and 0.5 µg/mouse for muscimol) was analyzed by linear regression analysis and a combination of the two drugs was injected in a constant dosage ratio upon the ED50 values. For drug combinations, the theoretic ED50 is citalopram ED50/2 + muscimol ED50/2. Additionally, experimental values of drug combinations from a constant ratio considered were measured by the regression analysis, after which the experimental ED50 value of the drug combinations was detected (%50 tail-flick latency). The statistical significance of the difference between theoretical ED50 and experimental ED50 of drug combinations was determined using. the one-sample t-test. Once the experimental ED50 was significantly lower than the theoretical ED50, a synergistic interaction between citalopram and muscimol could be determined. Nonetheless, there was no difference between them presenting additive interaction rather than a synergistic effect (Khakpai et al., 2021, Nejati et al., 2020). Differences with P < 0.05 between the experimental mice at each point were indicated statistically significantly.

3. Results

3.1. The effects of GABAA agents on pain behavior

The effects of muscimol and bicuculline infused into the left lateral ventricle on tail-flick latency are shown in Fig. 1. Repeated measures and two-way ANOVA indicated no significant interaction between muscimol doses and time intervals on %MPE [time intervals × muscimol interaction F (3, 56) = 1.737, P = 0.170; Fig. 1A]. Concerning the time interval effect and muscimol effect, Tukey’s multiple comparisons showed that microinjection of muscimol at doses of 0.5 and 1 µg/mouse into the left lateral ventricle increased %MPE at the time interval 60 min after microinjection (Fig. 1A).

Fig. 1.

Fig. 1

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The effects of different doses of muscimol (0.25, 0.5, and 1 µg/mouse; i.c.v.) and bicuculline (0.25, 0.5 and 1 µg/mouse; i.c.v.) on nociceptive responses in the tail-flick test. Data presented as mean ± S.E.M. (n = 8). Two-way ANOVA followed by repeated measures was performed for analysis of MPE% of the tail-flick test (A and B). One-way ANOVA followed by Tukey's post hoc test was performed for AUC of MPE% analysis (C). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

Furthermore, repeated measures and two-way ANOVA revealed no significant interaction between bicuculline doses and time intervals on %MPE [time intervals × muscimol interaction F (3, 56) = 1.017, P = 0.392; Fig. 1B].

As presented in Fig. 1C, one-way ANOVA followed by Tukey's Post hoc test for normalized AUC values revealed that muscimol (0.5 and 1 µg/mouse) increased the AUC of %MPE [F (3, 28) = 10.270, P < 0.0001; Fig. 1C, left panel] while bicuculline (1 µg/mouse) decreased the AUC of %MPE [F (3, 28) = 3.219, P = 0.038; Fig. 1C, right panel].

3.2. The effects of citalopram alone or with GABAA agents on pain behavior

Moreover, repeated measures and two-way ANOVA exhibited no significant interaction between citalopram doses and time intervals on %MPE [time intervals × citalopram interaction F (3, 56) = 1.283, P = 0.289; Fig. 2A]. Concerning the time interval effect and citalopram effect, Tukey’s multiple comparisons displayed that i.p. injection of citalopram at a dose of 8 mg/kg increased %MPE at the time interval of 30 and 60 min after injection and citalopram at a dose of 4 mg/kg enhanced %MPE at the time interval of 60 min after administration (Fig. 2A). Thus, citalopram caused a time-dependent antinociceptive effect in mice. The maximal effect (% MPE) was achieved 30 and 60 min after administration but decreased to baseline within 45 min (Fig. 2A).

Fig. 2.

Fig. 2

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The effects of alone administration of citalopram (2, 4, and 8 mg/kg; i.p.) and co-administration of it along with muscimol (0.25 µg/mouse; i.c.v.) or bicuculline (0.25 µg/mouse; i.c.v.) on nociceptive responses in the tail-flick test. Data presented as mean ± S.E.M. (n = 8). Two-way ANOVA followed by repeated measures was performed for analysis of MPE% of the tail-flick test (A, B, and C). One-way ANOVA followed by Tukey's post hoc test was performed for AUC of MPE% analysis (D). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

As seen in Fig. 2B, repeated measures and two-way ANOVA showed no significant interaction between drug combination (citalopram plus muscimol) doses and time intervals on %MPE [time intervals × drugs-administration interaction F (3, 56) = 0.255, P = 0.857; Fig. 2B]. Regarding the time interval effect and drugs-administration effect, Tukey’s multiple comparisons exhibited that co-administration of citalopram (8 mg/kg) and muscimol (0.25 µg/mouse) enhanced %MPE at the time intervals of 30, 45 and 60 min after co-administration (Fig. 2B).

Additionally, repeated measures and two-way ANOVA revealed no significant interaction between drug combination (citalopram along with bicuculline) doses and time intervals on %MPE [time intervals × drugs-administration interaction F (3, 56) = 0.970, P = 0.413; Fig. 2C]. Concerning the time interval effect and drugs-administration effect, Tukey’s multiple comparisons displayed that co-injection of citalopram (8 mg/kg) and bicuculline (0.25 µg/mouse) increased %MPE at the time intervals of 15, 30, 45 and 60 min after co-injection (Fig. 2C).

Also, one-way ANOVA and post hoc analysis exhibited that alone injection of citalopram (8 mg/kg) [F (3, 28) = 3.148, P = 0.041; Fig. 2D, left panel], and co-injection of citalopram (8 mg/kg) and muscimol (0.25 µg/mouse) [F (3, 28) = 3.854, P = 0.038; Fig. 2D, middle panel], as well as co-injection of citalopram (8 mg/kg) and bicuculline (0.25 µg/mouse) [F (3, 28) = 4.248, P = 0.014; Fig. 2D, right panel], increased the AUC of %MPE.

3.3. The additive effect between citalopram and muscimol on the antinociceptive effect

To confirm whether citalopram and muscimol co-injection would induce additive or synergistic effects on their induced antinociceptive response, the isobolographic analysis was carried out to compare the theoretical and experimental ED50 of the drugs when co-injected. The theoretical additive line presented that at all points, the citalopram and muscimol combination produced an effect of theoretical %50 tail-flick latency according to an additive interaction (Fig. 3). One sample t-test revealed that there is no significant difference between experimental ED50 and theoretical ED50. Our data suggested an additive effect of citalopram and muscimol co-administration upon induction of antinociceptive responses [t (31) =1.689, P = 0.101; Fig. 3] in mice.

Fig. 3.

Fig. 3

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The isobologram analysis of the effects of drug treatment revealed the additive effect of citalopram and muscimol on the induction of antinociceptive effect in male mice. Statistical analysis showed that there is no significant difference between experimental ED50 and theoretical ED50 points, showing an additive effect of the administration of the drug for tail-flick latency. ED50, effective dose 50.

4. Discussion

This study aimed to examine the effects of citalopram and GABAA agents on pain behavior in male mice using a tail-flick apparatus. Our results revealed that i.c.v. infusion of muscimol displayed a significant dose-dependent enhancement in %MPE and AUC of %MPE, presenting an antinociceptive effect. Furthermore, i.c.v. microinjection of bicuculline did not exhibit a significant effect on %MPE while reducing the AUC of %MPE, showing a hyperalgesia response. Consistent with previous investigations, we found that the GABAergic system participated in the modulation of pain behavior. In this context, analgesia and hyperalgesia induced by muscimol and bicuculline have been reported previously (Zarrindast and Mahmoudi, 2001). For example, several studies demonstrated that GABAA receptors are involved in the regulation of many physiological functions in the brain mechanisms that are associated with pain transmission and pain relief (Benson et al., 2015, Froestl, 2011, Li et al., 2020, Rashmi et al., 2018, Sałat et al., 2015). Moreover, the role of GABAA receptors in pain modulation is supported by evidence indicating that intrathecal (Dirig and Yaksh, 1995) or local injection of muscimol in the amygdala (Pedersen et al., 2007), or the hippocampus (Favaroni Mendes and Menescal-de-Oliveira, 2008) modulate pain process. There are reports that GABAA receptor agonists cause the analgesic effects in animal models of acute, inflammatory and neuropathic pain (Di Lio et al., 2011, Zeilhofer et al., 2009). There is a report revealing that GABAergic neurotransmission mediates the nociceptive reaction by reducing the activity of descending antinociceptive fibers in the periaqueductal grey (Harris and Westbrook, 1995) and the rostral insular cortex (Jasmin et al., 2003). Additionally, Hwang et al. (2001) reported that GABAA receptor agonists maybe induce theirs antinociceptive effects via affecting different neurotransmitter systems (Hwang et al., 2001). Furthermore, some investigations revealed that the enhancement of GABAergic tone might participate in a therapeutic effect on disorders such as pain (Froestl, 2011, Rashmi et al., 2018, Sałat et al., 2015).

The obtained results indicated that i.p. injection of citalopram increased %MPE and AUC of %MPE, thus inducing an antinociceptive effect. Consistent with our results, it has been reported that citalopram elicited analgesic effects in rodents and humans (Anderberg et al., 2000; D. A. Belinskaia et al., 2019; Bomholt et al., 2005; Campo et al., 2004; Patetsos and Horjales-Araujo, 2016; Roohafza et al., 2014). Citalopram is a very selective and powerful SSRI with a somewhat partial effect on the neuronal reuptake of norepinephrine and dopamine. It has been revealed that treatment with citalopram produced an acute enhancement in the extracellular serotonin levels in animals which in turn induced stimulation of the somatodendritic 5-HT1A auto-receptors. The result of the acute activation is feedback inhibition in the raphe nuclei along with down-regulation of auto-receptors and the following enhancement in the serotonergic neurotransmission (Misrani and Long, 2021). Consequently, enhancement of serotonin neurotransmission may account for the antinociceptive effect of citalopram. In this research, the % MPE was differentially increased by citalopram at the time intervals of 30 and 60 min but not 45 min. The failure of citalopram to affect pain-related behavior in the time intervals of 45 min was unexpected. The failure of citalopram to modulate mechanical hypersensitivity in the chronic constriction injury model reported by Bomholt and co-workers (2005). Although, they indicated the antinociceptive effect of citalopram in animal models of acute, persistent, and neuropathic pain (Bomholt et al., 2005). It was reported that SSRIs, including citalopram, were more effective in pain models of the hot-plate test. Because, in the hot-plate test, both supra-spinal mechanisms (emotional perception) and spinal reactions are triggered. Additionally, it would be helpful to include information on the antinociceptive effects and/or absence of effects of citalopram in mice, as assessed in the tail flick test. On the other hand, it has been reported that serotonin transporter polymorphism changes citalopram effects on pain behavior. If 5-HTTLPR modulates pain treatment, s/s and l/l homozygotes show differential citalopram-induced variations on pain-correlated brain responses. Precisely, as l/l homozygotes display greater pain sensitivity (Lindstedt et al., 2011) and enhanced 5-HTT expression (Lesch et al., 1996), which exhibit higher pain-correlated brain activity compared to s/s homozygotes. Additionally, given that l/l relative to s/s homozygotes indicate stronger SSRI functions in clinical (Serretti et al., 2007) and laboratory studies (Whale et al., 2000), citalopram treatment should induce stronger reductions in pain-correlated brain activity in l/l homozygotes. Thus, an interaction between genotype and treatment on pain-correlated brain responses is important for pain relief (Ma et al., 2016).

In the next section of this research, we evaluated the involvement of GABAA receptor agents in the antinociceptive response induced by citalopram in the tail-flick test. Certain evidence indicated that acute serotonin release could directly stimulate the GABAergic interneurons by stimulation of 5-HT2 receptors and raise the frequency of inhibitory synaptic actions in projection neurons, in the amygdala, and other regions of the central nervous system (Donatti and Leite-Panissi, 2009, Stutzmann and LeDoux, 1999). Furthermore, the expression of 5-HT2 receptors on interneurons of the amygdala proposes that inhibition of cell firing also might be facilitated via direct excitation of the GABAergic interneurons (Stutzmann and LeDoux, 1999). A similar interaction happens in the prefrontal cortex which receives the serotoninergic innervations from the dorsal raphe nucleus (Graeff, 1990). This raphe-cortical projection exerts activation of the GABA interneurons via the 5-HT2A receptors and inhibition of the glutamatergic pyramidal neurons through the 5-HT1A receptors in the prefrontal cortex (Aghajanian and Marek, 2000). Our results exhibited that co-treatment of different doses of citalopram along with a sub-threshold dose of muscimol or bicuculline potentiated the antinociceptive response induced by citalopram. Furthermore, our findings revealed an additive antinociceptive effect between citalopram and muscimol in male mice. We proposed these findings due to the functional interaction between the GABAergic and serotonergic systems (Bailey and Nutt, 2008). As mentioned above, the serotonergic and GABAergic fibers coexist in the brain (Griffiths and Lovick, 2002, Judge et al., 2006, Tao and Auerbach, 2003). In agreement with our results, Rodrigues Tavares et al. (2023) reported that 5-HT3 receptors and GABAergic mechanisms in the amygdala are involved in the pain hypersensitivity produced via the empathy for pain model (Rodrigues Tavares et al., 2023). Furthermore, Donatti and Leite-Panissi (2009) indicated the interaction between GABAergic and serotoninergic mechanisms on the induction of tonic immobility behavior which is mediated by GABAA and 5-HT2 receptors of the basolateral nucleus of the amygdala in guinea pigs (Donatti and Leite-Panissi, 2009). Moreover, Bailey and Nutt (2008) reported the interaction between serotonergic and GABAergic systems on the modulation of anxiogenic behaviors (Bailey and Nutt, 2008). Additionally, some evidence showed that serotonin could directly stimulate the GABAergic interneurons through stimulation of 5-HT2 receptors, hence raising the rate of inhibitory synaptic processes in different areas of the brain (Donatti and Leite-Panissi, 2009, Stutzmann and LeDoux, 1999).

In summary, our experiments revealed that there is an interaction between the serotonergic and GABAergic systems in the modulation of pain processes. Treatment with GABAergic agents potentiated the antinociceptive effect induced by citalopram. However, further examinations are needed to describe the exact mechanism of interaction between citalopram and muscimol on the modulation of pain behavior in male mice.

Compliance with ethical standards

The research was performed under ethical standards in all aspects.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Elnaz Amini: Data curation. Fatemeh Khakpai: Software, Resources, Methodology. Taha Shokrnejad-namin: Data curation. Mohammad-Reza Zarrindast: Supervision, Resources, Project administration, Methodology, Investigation, Conceptualization.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

We are thankful to all contributors for their participation. We did not receive any funding for our work.

Author agreement

T. Shokrnejad-namin and E. Amini acquired the animal data. F. Khakpai analyzed the data and wrote the manuscript. M.R. Zarrindast was responsible for the study concept, and design, and assisted with the interpretation of findings. The authors critically reviewed the content and approved the final version for publication.

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