A colony of adult least shrews from the Western University of Health Sciences Animal Facilities were housed in groups of 5–10 on a 14:10 light:dark cycle, and were fed and watered ad libitum. The experimental shrews were 45–60 days old and each weighed between 4 and 6 g. 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 animals suffering and to reduce the number of animals used in the experiments.

The following drugs were used: m-3M3FBS, U73122, U0126, GF109203X, FPL64176, 2-methyl-serotonin maleate salt (2-Methyl-5-HT), and GR73632 were purchased from Tocris (Minneapolis, MN); McN-A-343, quinpirole HCl and nifedipine from Sigma Sigma/RBI (St. Louis, MO); thapsigargin, dantrolene and 2-APB from Santa Cruz Biotechnology (Dallas, TX). Palonosetron and netupitant were kindly provided by Helsinn Health Care (Lugano, Switzerland). m-3M3FBS, U73122, nifedipine, U0126, netupitant were dissolved in a mixture of ethanol/Tween 80/saline at a volume ratio of 1:1:18. Dantrolene, 2-APB, GF109203X and FPL64176 were 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.

On the day of experimentation shrews were brought from the animal facility, separated into individual cages, and allowed to adapt for at least 2 hours (h). Daily food was withheld 2 h prior to the start of the experiment, but shrews were given 4 mealworms each prior to emetogen injection to aid in identifying wet vomits as described previously (Darmani, 1998). For systemic dose-response emesis studies, different groups of shrews were injected with varying doses of m-3M3FBS (0, 10, 20, and 50 mg/kg, i. p., n = 8 shrews per group). Each shrew was immediately placed in the observation cage and the frequency of emesis was recorded for the next 2 h m-3M3FBS at 50 mg/kg dose caused vomiting with maximal frequency in 87.5% of tested shrews. Thus, this dose was used for subsequent studies.

To evaluate drug interaction studies, different groups of shrews were pretreated for 30 min with an injection of either corresponding vehicle [(i.p.) or subcutaneously (s.c.)], or varying doses of the following antiemetic antagonists/inhibitors: 1) PLC inhibitor U73122 (2.5 and 10 mg/kg, i. p., n = 8 per group); 2) ERK1/2 inhibitor U1026 (1 and 5 mg/kg, i. p., n = 8 per group); 3) IP₃R antagonist 2-APB (0.5, 2.5 and 10 mg/kg, i. p., n = 8 per group); 4) RyR antagonist dantrolene (0.5, 2.5 and 10 mg/kg, i. p., n = 8 per group); 5) LTCC blocker nifedipine (0.5 and 1 mg/kg, s. c., n = 10 per group); 6) PKC inhibitor GF109203X (0.1, 1 and 10 mg/kg, i. p., n = 8); 7) 5-HT₃R antagonist palonosetron (0.1 and 0.5 mg/kg, s. c., n = 8 per group); 8) NK₁R antagonist netupitant (1, and 5 mg/kg, i. p., n = 8 per group). After 30 min pretreatment, each shrew was challenged with a single dose of m-3M3FBS at 50 mg/kg (i.p.) and emesis behaviors were recorded for the following 2 h. The selection of dosage levels of the above tested drugs were based up on our previous antiemetic studies (Zhong et al., 2019).

To determine the broad-spectrum dose-response antiemetic potential of the PLC inhibitor U73122, varying doses were injected (i.p.) into different groups of shrews at 0 min. Thirty minutes later, shrews were challenged for vomiting with a fully efficacious dose of one of the following emetogens (Darmani et al., 2014; Zhong et al., 2016; 2014): 1) the selective serotonin 5-HT₃R agonist 2-Methyl-5-HT (5 mg/kg, i. p., n = 8 per group), 2) the selective substance P neurokinin NK₁R agonist GR73632 (5 mg/kg, i. p., n = 6 per group), 3) the dopamine D_2/3 preferring receptor agonist quinpirole (2 mg/kg, i. p., n = 5 per group), 4) the selective muscarinic M₁R agonist McN-A-343 (2 mg/kg, i. p., n = 6 per group), 5) the LTCC agonist FPL64176 (10 mg/kg, i. p., n = 6 per group), 6) the SERCA inhibitor thapsigargin (0.5 mg/kg, i. p., n = 6 per group). The vomiting behavior (number of animals vomiting within groups and frequency of vomits) were then observed for 30 min.

In the forementioned emesis behavioral experiments, the observer was blinded to administration conditions. In all experiments each tested shrew was used once and then euthanized with isoflurane following the termination of each experiment.

Immunohistochemistry of the least shrew brainstem and jejunal sections was conducted as previously reported (Zhong et al., 2019; Zhong et al., 2021). The jejunal segment of the least shrew small intestine was dissected as described by Ray et al. (2009). Following m-3M3FBS (50 mg/kg, i. p.) injection, vomiting shrews were subjected to c-Fos staining (n = 3–4 shrews/group). Thus, 90 min after the first emesis occurred, shrews were anesthetized with isoflurane and perfused with ice cold 4% paraformaldehyde in pH 7.4, 0.1 M phosphate-buffered saline (PBS) for 10 min. Brainstems and jejunum were removed and cryoprotected with 30% sucrose in 0.01 M PBS overnight. The OCT-embedded brainstem block and jejunum were cut on a freezing microtome (Leica, Bannockburn, IL, United States) into 20-μm and 25-μm sections respectively and stored in PBS with 0.03% sodium azide. Immunolabeling using rabbit anti-c-Fos polyclonal antibody (1:5,000, ab190289, Abcam) were performed. Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) was used as secondary antibody. Nuclei of cells were stained with DAPI. Images of the brainstem sections containing the dorsal vagal complex (AP/NTS/DMNV) and jejunal sections were taken by a confocal microscope (Zeiss LMS 880) with Zen software using ×20 objective. Cytoarchitectonic differences in the AP, NTS, and DMNV of the least shrew brainstem have been described in our previous report (Ray et al., 2009).

A Fos-expressing cell nucleus was only counted as positive if it retained its ovoid shape after high-pass filtering to eliminate variations in background as well as potential false positive and was fully within the defined region of interest. The numbers of Fos-positive nuclei of each region (AP/NTS/DMNV/jejunum) were counted by an observer blind to the animal’s treatment condition. For each region, the same number of sections were counted per animal: 3 brainstem sections at 90-μm intervals each through AP/NTS/DMNV and 3 jejunal sections. The mean value of each region per section, from an individual animal was used in statistical analysis.

Adult least shrews were administered m-3M3FBS (50 mg/kg, i. p.) or vehicle (n = 3 animals per group) and rapidly anesthetized with isoflurane and subjected to perfusion at 15 min post-treatment to evaluate phospho-ERK1/2 alteration. Brain sections (20 μm) observed under a light microscope and those containing the brainstem dorsal vagal complex (DVC) were subjected to immunostaining as described in our previous publication (Zhong et al., 2019). Immunostaining using rabbit anti-phospho-ERK1/2 (Thr202/Thr204) (1:500, 4,370, Cell Signaling) antibody was followed by Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) secondary antibody incubation. After washing with PBS 4 times, sections were mounted with anti-fade mounting medium containing DAPI staining nuclei (Vector Laboratories). Images were acquired under a confocal microscope (Zeiss) with Zen software using ×20 and ×60 objectives. Integrated density of phospho-ERK1/2 immunoreactivity in the brainstem dorsal vagal complex of 3 sections from each animal of both groups was determined with ImageJ and the mean value per section from individual animals was used in statistical analysis.

The vomit frequency data were analyzed using the Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test and expressed as the mean ± SEM. The percentage of animals vomiting across groups at different doses was compared using the chi-square test. Statistical significance for differences of c-Fos expression between two groups (vehicle controls vs. shrews vomiting induced by m-3M3FBS) was tested by unpaired t-test. p < 0.5 was considered statistically significant.

The emesis data for m-3M3FBS are depicted in Figure 1. Intraperitoneal administration of m-3M3FBS increased the frequency of emesis in the least shrew in a dose-dependent manner (KW (3, 28) = 19.51, p = 0.0002). Dunn’s multiple comparisons post hoc test showed that m-3M3FBS significantly increased the vomit frequency at its 50 mg/kg dose (p = 0.0004) (Figure 1A). In addition, the chi-square test indicated that the percentage of animals exhibiting emesis in response to m-3M3FBS also increased in a dose-dependent fashion (χ² (3, 28) = 20.25, p = 0.0002). However, only 87.5% of shrews vomited at its 50 mg/kg (p = 0.0004) (Figure 1B). We could not test larger doses of m-3M3FBS due to its insolubility.

As shown in Figure 1C, the PLC inhibitor U73122 attenuated the frequency of m-3M3FBS -induced vomiting [KW (2, 21) = 6.564, p = 0.0375], with a significant reduction (88%) seen at its 10 mg/kg dose (p = 0.0209). U73122 also reduced the percentage of shrews vomiting [χ² (2, 21) = 6.378; p = 0.0412], and significant protection (62.5%) occurred at 10 mg/kg (p = 0.0117) (Figure 1D).

We conducted immunohistochemistry to determine c-Fos responsiveness following systemic administration of m-3M3FBS. Figures 2A,B show very few of c-Fos-positive cells were observed in the dorsal vagal complex (DVC) emetic nuclei in shrew brainstem sections from vehicle-treated controls. Relative to the vehicle-treated control group, a 50 mg/kg (i.p.) dose of m-3M3FBS caused a significant increase in c-Fos expression in the brainstem throughout the three DVC emetic nuclei, the AP, NTS and DMNV (Figures 2C,D). The numbers of Fos-IR positive cell nuclei in each region are delineated in Figure 2I. In vehicle-treated shrews, the average values for Fos-positive cells were 12.9 ± 1.5, 27.8 ± 4.7, and 16.6 ± 2.6 in the AP, NTS, and DMNX, respectively. Following vomiting induced by m-3M3FBS, the average numbers of c-Fos-positive cells were increased to 26.3 ± 4.7 in the AP (p = 0.0361 vs. Control), 84.7 ± 3.6 in NTS (p < 0.0001), and 44.7 ± 1.8 in DMNX (p = 0.0001). The c-Fos expression following m-3M3FBS-induced vomiting was also examined in the shrew jejunum (Figures 2E–H). The mean number of c-Fos expressing cells in the enteric nervous system of the jejunum was 0.5278 ± 0.3938 in the vehicle-treated shrews, and 5.222 ± 2.164 after m-3M3FBS-evoked vomiting (p = 0.0768, vehicle vs. m-3M3FBS) (Figure 2I).

To determine the involvement of ERK1/2 in m-3M3FBS-induced emesis, we performed immunohistochemistry to detect changes in ERK1/2 phosphorylation expression evoked by m-3M3FBS (50 mg/kg, i. p.). Representative images and statistical graph in Figure 3 demonstrate that following systemic administration of m-3M3FBS, a significant increase in ERK1/2 phosphorylation (p = 0.0246) occurred throughout the dorsal vagal complex including the emetic nuclei, AP, NTS and DMNV.

Next, we examined the antiemetic potential of the ERK1/2 inhibitor U0126 against m-3M3FBS-induced emesis. The vehicle or U0126 (1 and 5 mg/kg, i. p.) were administered to different groups of shrews 30 min prior to m-3M3FBS (50 mg/kg, i. p.) injection. U0126 pretreatment reduced the frequency of m-3M3FBS-induced vomiting (KW (2, 21) = 11.4, p = 0.0034), with a significant reduction in the vomit frequency at its 5 mg/kg dose (p = 0.0015) (Figure 4A). The chi-square test showed U0126 pretreatment also reduced the percentage of shrews vomiting (χ² (2, 21) = 9.399, p = 0.0091) in response to m-3M3FBS, and achieved significance at 5 mg/kg dose (p = 0.0027) (Figure 4B).

The antiemetic potential of the PKC inhibitor GF109203X (0.1, 1 and 10 mg/kg) was tested against m-3M3FBS (50 mg/kg, i. p.)-induced vomiting. GF109203X pretreatment reduced both the mean vomit frequency [KW (3, 28) = 12.22, p = 0.0067] (Figure 5A) and percentage of shrews vomiting [χ² (3, 28) = 8.635; p = 0.0346] (Figure 5B) in response to m-3M3FBS in a dose-dependent manner. Significant reduction in the mean vomit frequency occurred at the 10 mg/kg dose of GF109203X, whereas the percentage of shrews vomiting was significantly reduced from its 1 mg/kg dose.

Different groups of shrews were pretreated with either the 5-HT₃R antagonist palonosetron (0.025, 0.1, and 0.5 mg/kg, s. c.), the NK₁R antagonist netupitant (0.25, 1 and 5, i. p.), or their corresponding vehicle, for 30 min prior to m-3M3FBS (50 mg/kg, i. p.) injection. Palonosetron pretreatment reduced the mean vomit frequency [KW (3, 28) = 8.218, p = 0.0417] in response to m-3M3FBS (Figure 7A), with significant reduction occurring at its 0.1 (p = 0.0460) and 0.5 (p = 0.0410) doses. However, palonosetron failed to completely protect shrews from vomiting [χ² (3, 28) = 5.333; p = 0.1490] (Figure 7B).

The substance P receptor antagonist netupitant also caused a dose-dependent decrease in the frequency of evoked vomits [(KW (3, 28) = 14.78, p = 0.0020)] with significant reductions occurring at its 1 mg/kg (p = 0.0038) and 5 mg/kg doses (p = 0.0020) (Figure 7C). The chi-square test indicates that the number of shrews vomiting in response to m-3M3FBS was also significantly attenuated by netupitant [(χ² (3, 28) = 12.83, p = 0.0050)] (Figure 7D) with ∼80% protection at 1 mg/kg and 5 mg/kg doses (p = 0.0027).

Figure 8 demonstrates the antiemetic potential of U73122 against vomiting evoked by diverse emetic receptor agonists. In fact, pretreatment with U73122 (5 and 10 mg/kg), reduced both the frequency (KW (2, 21) = 8.768, p = 0.0125) and percentage (χ² (2, 21) = 12.69; p = 0.0018) of shrews vomiting in response to the administration of the 5-HT₃R-selective agonist, 2-Methyl-5-HT (5 mg/kg, i. p.) (Figures 8A,B). A significant reduction in vomit frequency occurred at its 10 mg/kg dose (p = 0.0062). In addition, the percentage of shrews vomiting was reduced by 87.5% at 10 mg/kg dose (p = 0.0004). As shown in Figures 8C,D, U73122 (5 and 10 mg/kg) also caused a dose-dependent decrease in the mean frequency of vomits induced by the NK₁R selective agonist GR73632 (KW (2, 15) = 8.693, p = 0.0076) with a significant reduction at its 10 mg/kg dose (p = 0.0072), but no significant decrease (χ² (2, 15) = 3.877; p = 0.1439) in the percentage of animals vomiting was observed.

Furthermore, the antiemetic effect of U73122 (1, 2.5 and 5 mg/kg) was also assessed against vomiting caused by the dopamine D_2/3R agonist, quinpirole (2 mg/kg, i. p.) (Figures 8E,F). The mean frequency of quinpirole-induced emesis (KW (3, 16) = 12.21, p = 0.0067) was significantly reduced with complete suppression at its 5 mg/kg dose (p = 0.0060). Significant decrease (χ² (3, 16) = 11.92; p = 0.0077) in the percentage of animals vomiting were noted at its 2.5 (60%; p = 0.0261) and complete protection at 5 mg/kg dose (100%; p = 0.0009).

Next, the antiemetic effect of varying doses of U73122 was tested against vomiting caused by the muscarinic M₁R agonist, McN-A-343 (2 mg/kg, i. p.) (Figures 8G,H). U73122 pretreatment (2.5, 5 and 10 mg/kg) also significantly reduced the frequency of McN-A-343-induced emesis (KW (3, 20) = 12.97, p = 0.0047) in a dose-dependent manner with significant reduction at 10 mg/kg dose (p = 0.0015). The percentage of shrews vomiting was also reduced in a dose-dependent fashion (χ² (3, 20) = 14.4; p = 0.0024) with significant reductions at 5 (50%; p = 0.0455) and complete protection at its 10 mg/kg dose (100%; p = 0.0005).

We then investigated the antiemetic potential of U73122 against vomiting evoked by Ca²⁺ channel regulators, such as the selective LTCC agonist FPL64176 (10 mg/kg, i. p.) and the SERCA inhibitor thapsigargin (0.5 mg/kg, i. p.) (Figure 9). U73122 (5 and 10 mg/kg) significantly attenuated the mean frequency of FPL64176-induced vomiting in a dose-dependent manner (KW (2, 15) = 6.195, p = 0.0384) with a significant reduction at 10 mg/kg dose (p = 0.0257) (Figures 9A,B). In addition, the percentage of shrews vomiting in response to FPL64176 was also suppressed by U73122 (χ² (2, 15) = 6; p = 0.0498) with a 66.7% reduction at its 10 mg/kg (p = 0.0143). Figures 9C,D show that U73122 (2.5, 5 and 10 mg/kg) dose-dependently suppressed vomiting caused by thapsigargin (0.5 mg/kg, i. p.). The frequency of thapsigargin-induced emesis (KW (3, 20) = 18.09, p = 0.0004) was significantly reduced at 5 (p = 0.0023) and complete reduction occurred at its 10 mg/kg dose (p = 0.0007). Likewise, significant decreases (χ² (3, 20) = 17.33; p = 0.0006) in the percentage of animals vomiting were also noted at its 5 (83.3%; p = 0.0034) and complete protection at the 10 mg/kg dose (100%; p = 0.0005).

Phospholipase C (PLC) activation is a crucial component in cellular signaling arising from the activation of diverse GPCRs, the largest family of cell membrane-bound receptors, and plays critical roles in signal transduction (Dorsam and Gutkind, 2007). It has been established that a wide variety of GPCRs mediate vomiting when stimulated by endogenous stimuli such as neuropeptides (e.g., substance P) (Carpenter et al., 1984), biogenic amines (e.g., histamine) (Bhargava and Dixit, 1968), lipids (e.g. prostaglandins) (Kan et al., 2006) as well as diverse pharmaceutical agents (Darmani et al., 2014; Zhong et al., 2018). However, to date, scant evidence exists to support a role for PLC in the emetic processes. In this study we demonstrated that a PLC activator, m-3M3FBS, induces vomiting in the least shrew model of emesis, with a mean maximal frequency (6.25 vomits ±2.6) of vomits occurring at its 50 mg/kg (i.p.) dose in up to 90% tested shrews. Because of solubility issues, we could not test a larger dose of m-3M3FBS. In addition, the PLC inhibitor U73122 not only suppressed m-3M3FBS-evoked vomiting, but also vomits induced by selective agonists of diverse emetic receptors including serotonin 5-HT₃-, neurokinin NK₁-, dopamine D_2/3-, and muscarinic M₁-receptors. Thus, the present study provides direct evidence for PLC participation in emesis. The major limitation of this study is that the molecular mechanisms by which PLC activation-mediates vomiting remain to be deciphered. In the following sections, we attempt to discuss the potential mechanisms by which downstream signaling molecules following administration of the PLC activator m-3M3FBS may contribute to the evoked emesis.

As we discussed in the introduction section, Ca²⁺, ERK1/2 and PKC are important downstream effectors in PLC-dependent signal transduction. In the current study, vomiting induced by the PLC activator m-3M3FBS was followed by increased expression of c-Fos and ERK1/2 phosphorylation in the brainstem dorsal vagal complex, indicating central activation of emetic nuclei (the area postrema, the NTS and DMNV) following systemic administration of m-3M3FBS. The c-Fos expression in the enteric nervous system of the jejunum showed a weak response upon m-3M3FBS-induced vomiting, whereas the statistics analysis showed no significant difference (p = 0.077) between vehicle controls and m-3M3FBS-administed shrews. Since the brainstem area postrema lacks a complete blood-brain barrier (Wickham, 2020), circulating drugs may directly act on this emetic region, which would then sends output to the NTS for integration, and ultimately sensory signals are conveyed to the DMNV which mediates the emetic motor signals to gastrointestinal tract during the vomiting process (Hornby, 2001; Darmani and Ray, 2009; Bashashati and McCallum, 2014). According to published literature, m-3M3FBS can directly activate different PLC isoforms in various cell types, which can lead to generation of both IP₃ as well as subsequent increase in intracellular Ca²⁺ (Bae et al., 2003). Moreover, different PLC isoforms (PLC_β, PLC_γ, PLC_δ, and PLC_ε) can also regulate the ERK1/2 signaling cascade (Owusu Obeng et al., 2020).

In this study the emetic action of m-3M3FBS appears to be sensitive to inhibitors of ERK1/2 (U0126), PKC (GF109203X), and IP₃R (2-APB), which are in line with current understanding that DAG, IP₃, and Ca²⁺ mobilization, are involved in PLC-dependent cell signaling. In addition, our published findings implicate ERK1/2 phosphorylation in the brainstem DVC as a common emetic signal elicited by systemic administration (i.p.) of diverse emetogens including the: 1) NK₁ receptor agonist GR73632 (5 mg/kg), 2) LTCC activator FPL64176 (10 mg/kg), 3) the SERCA inhibitor and thus an intracellular Ca²⁺ signaling amplifier, thapsigargin (0.5 mg/kg), 4) the 5-HT₃ receptor agonist 2-Methyl-5-HT (5 mg/kg) (Zhong et al., 2014; Zhong et al., 2016; Zhong et al., 2018; Zhong et al., 2019), 5) chemotherapeutic agent cisplatin (10 mg/kg, i. p.) (Darmani et al., 2015), and 6) Akt inhibitor MK-2206 (10 mg/kg, i. p.) (Zhong et al., 2021). Moreover, ERK1/2 inhibitors such as PD98059 or U0126, exert substantial or partial antiemetic effects against vomiting-evoked by the above discussed emetogens except cisplatin.

PKC phosphorylation in least shrew brainstem has also been found to be associated with vomiting caused by several emetogens including cisplatin (Darmani et al., 2013; Darmani et al., 2015), FPL64176 and GR73632 (Zhong et al., 2018; Zhong et al., 2019). Furthermore, the PKC inhibitor GF109203X can suppress the vomiting evoked by either FPL64176 or GR73632 (Zhong et al., 2018; Zhong et al., 2019).

Both IP₃R and RyR contribute to increased intracellular Ca²⁺ concentration following PLC activation (Kim et al., 2015). In this study, inhibitors of both IP₃R (2-APB) and RyR (dantrolene) displayed antiemetic efficacy against m-3M3FBS-induced vomiting, suggesting the involvement of these intracellular endoplasmic reticulum Ca²⁺ release channels in emesis. Whether the antiemetic role of 2-APB and dantrolene contribute to inhibition of intracellular Ca²⁺ release needs further Ca²⁺ imaging studies on cells or brain slices of an emetic species. Previously we have found that dantrolene and 2-APB exhibit differential antiemetic efficacy against vomiting elicited by several emetogens, such as the 5-HT₃R agonist 2-Methyl-5HT (Zhong et al., 2014), FPL64176 (Zhong et al., 2018), thapsigargin (Zhong et al., 2016), and NK₁R agonist GR73632 (Zhong et al., 2014; Zhong et al., 2016; Zhong et al., 2018; Zhong et al., 2019). Therefore, RyRs and IP₃Rs ion-channels may be differentially regulated by different emetogens and may be potential targets for emesis prevention. 2-APB has often been used as an IP₃R inhibitor, but other studies suggest it also acts as a store-operated Ca²⁺ entry inhibitor (Hofer et al., 2013). This non-selectivity of 2-APB may help explain our current findings in that the reduction in frequency of m-3M3FBS-induced vomiting by 2-APB is significant at its 2.5 mg/kg dosage, but not at the10 mg/kg dosage.

The complete blockade of m-3M3FBS-induced vomiting by LTCC inhibitor nifedipine is not surprising. In the least shrew emesis model, nifedipine exhibits broad-spectrum antiemetic efficacy against vomiting evoked by a myriad of emetogens including agonists of the LTCC (FPL64176), 5-HT₃- (e.g., 5-HT or 2-Me-5-HT), NK₁- (GR73632), dopamine D_2/3- (apomorphine or quinpirole), muscarinic M₁- (McN-A-343) receptors (Darmani et al., 2014). Ca²⁺ mobilization is involved in both triggering neurotransmitter release coupled with receptor activation, as well as post-receptor excitation-contraction coupling (Zuccotti et al., 2011), which can be an important aspect of vomit induction, and has been further discussed in one of our reviews (Zhong et al., 2017). The mechanism underlying the antiemetic potential of Ca²⁺ signaling inhibitors may be closely related to inhibition of Ca²⁺ mobilization and emetic receptor-mediated downstream signaling pathways involving key molecules, such as PKA, PKC, ERK1/2, and Ca²⁺/calmodulin-dependent protein kinase II (Zhong et al., 2017).

m-3M3FB-evoked emesis is also sensitive to antagonists of both 5-HT₃R and NK₁R (palonosetron and netupitant, respectively). It remains to be determined whether the PLC activator m-3M3FBS stimulates release of 5-HT or substance P, or both, which would subsequently activate corresponding serotonin 5-HT₃R and/or neurokinin NK₁R to evoke vomiting, which our accompanying antagonist studies imply. To the best of our knowledge, there is no published literature to indicate PLC activation by m-3M3FBS would cause release of such emetic mediators. However, it is known that the PLC inhibitor U73122 inhibits lipopolysaccharide-induced prostaglandin E₂ production in mice, a process attributed to the inhibition of PLC pathway (Hou et al., 2004). Moreover, lipopolysaccharide is a potent emetogen in pigs (Girod et al., 2000).

U73122 can selectively inhibit the PLC-dependent signaling process, and thus has proven useful in evaluating PLC-dependent cell activation both in vitro and in vivo when administered i. p. or intravenously (Hou et al., 2004). In the present study, U73122 suppressed vomiting evoked by the PLC activator m-3M3FBS, or via the activation of key emetic receptors (serotonin 5-HT₃, neurokinin NK₁, dopamine D_2/3, muscarinic M₁) induced by their corresponding specific agonists (2-Methyl-5-HT, GR73632, quinpirole, McN-A-343, respectively) and Ca²⁺ signaling amplifiers (FPL64176 and thapsigargin) (Darmani et al., 2014; Zhong et al., 2016). U73122 is a PLC isoform-nonspecific inhibitor which also attenuates stimulus-evoked intracellular Ca²⁺ accumulation as well as ERK1/2 phosphorylation in cellular studies (Shen et al., 2013). Therefore, the mechanism underlying the antiemetic potential of U73122 could be interpreted via inhibition of Ca²⁺ mobilization and ERK1/2 signaling, both of which play important roles in the vomiting-mediated by various emetic receptors as demonstrated in our previous studies (Zhong et al., 2017). Another potential explanation could be stimulation of emetic receptors (e.g., 5-HT₃R, LTCC) may activate PLC through Ca²⁺ mobilization (Thore et al., 2005).

The tested antiemetic doses of U73122 may nonspecifically attenuate vomiting via a general decrease in locomotor activity. Thus, we investigated the effect of U73122 on locomotor activity parameters of least shrews using a computerized video tracking, motion analysis and behavior recognition system (EthoVision) as described in our published studies (Darmani, 2002). The tested antiemetic doses of U73122 in this study did not affect either the velocity or distance travelled by the shrews (Supplementary Figure S1). Thus, inhibition of motor activity per se does not contribute to the antiemetic potential of U73122.

Per our introduction section, in addition to the described limitation of the current study concerning molecular mechanisms by which PLC activation-mediates vomiting, the potential anti-nauseous activity of U73122 in the least shrew remains to be established. Indeed, published anatomical and pharmacological data imply several higher brain structures (e.g., amygdala, cortex), as well as neurotransmitters (e.g., cholinergic system, histaminergic), their corresponding receptors, and cellular processes (e.g., expression of immediate early genes, kinase signaling pathway) are probably involved in nausea (Welzl, 2001). As suggested by our current emesis/antiemesis findings, PLC activators and inhibitors would probably exert similar nauseous/anti-nauseous outcomes which would need to be investigated.