Keywords

Introduction

Scientific knowledge consists in the search for truth, but it is not the search for certainty. Karl Popper (a philosopher of science)

Although it is a common experience and knowledge for human beings that bee stings are naturally harmful due to painful and anaphylactic reactions, understandings of the biological, pharmacological, and toxicological effects and the underlying mechanisms of bee venom-induced pain have not been well gained due to lack of scientific investigations until the end of the last century (Chen and Lariviere 2010). Empirically, the bee venom injection, referred to as bee venom therapy (BVT), has been used as a complementary and alternative therapeutic method for thousands of years to treat many diseases, including rheumatoid arthritis, bursitis, tendinitis, shingles (herpes zoster), multiple sclerosis, wounds, gout, burns, and infections (Son et al. 2007; Chen and Lariviere 2010). Recently, attempts have also been made to use bee venom and its chemical components for potential cancer treatment (Son et al. 2007; Chen and Lariviere 2010). Historically, the use of BVT in patients can be traced back to ancient Egypt, Greece, and China. In literature, Philip Terc is believed to be the first user of BVT for treatment of rheumatic patients (American Apitherapy Society Inc. 1989; Chen and Lariviere 2010). The BVT was then introduced to the United States of America by one of Philip Terc’s followers (Beck 1935; Mraz 1981; American Apitherapy Society Inc. 1989). Although BVT is likely to be a promising therapeutic alternative for treatment of chronic pain and other diseases (Son et al. 2007), so far its efficacy and safety have not been approved by food and drug authorities worldwide (Chen and Lariviere 2010).

Interests in understanding of the bee venom-induced pain and its underlying mechanisms have been dramatically increased since the initial characterizations of the altered pain-related behaviors and inflammation in rats (Lariviere and Melzack 1996, 2000; Chen et al. 1999b; Chen and Chen 2000). It has been stably characterized that subcutaneous (s.c.) bee venom injection into one hind paw of rats results in an immediate, long-term spontaneous alterations in behaviors relevant to pain and nociception lasting at least more than 1 h for quantitative observation (Lariviere and Melzack 1996; Chen et al. 1999b). The bee venom-induced spontaneous pain-related behaviors have been later identified as a spinally processed nonvoluntary nociceptive reflex (paw flinches) and a supraspinally processed voluntary self-caring and favoring behaviors (paw lifting, licking, and biting) in rats (Ren et al. 2008). Moreover, it has been further identified that pain hypersensitivity evoked by thermal or mechanical stimulus modality can also occur spatially at the site of bee venom injection (primary thermal and mechanical hyperalgesia ), at an area remote to the bee venom injection site (secondary thermal hyperalgesia), and at symmetrical paw contralateral to the bee venom injection site (mirror-image thermal hyperalgesia) lasting for 48–96 h (Chen et al. 1999b; Chen and Chen 2000). Unlike other algogens (pain-producing substances) (Chen and Lariviere 2010), the nociceptive and inflammatory effects of s.c. bee venom have no species difference in mammals ranging from mice (Lariviere et al. 2002), to rats (Lariviere and Melzack 1996; Chen et al. 1999b; Chen and Chen 2000), to cats (Chen et al. 1998), and to human beings (Koyama et al. 2000, 2003; Sumikura et al. 2003, 2006), suggesting a share of common nociceptive and inflammatory mechanisms among mammals in response to bee stings.

In the past two decades, nociceptive and inflammatory effects of bee venom and its pain-producing components on the somatosensory system have been well studied (Chen 2003, 2007, 2008; Chen and Lariviere 2010). Furthermore, the antinociceptive and anti-inflammatory effects of bee venom have also been reported and reviewed (Son et al. 2007; Chen and Lariviere 2010). In this chapter, the scientific questions of what, how, and why bee venom causes pain and sensory hypersensitivity will be fully addressed, while the mechanisms of the BVT will not be dealt with in the current review because it is beyond the scope of this topic.

Bee Venom and Its Biological Components

As listed in Tables 1 and 2, (honey) bee venom (of Apis mellifera) is a complex composition of polypeptides, enzymes, amines, lipids, and amino acids (Habermann 1972; Gauldie et al. 1976, 1978; Lariviere and Melzack 1996; Chen and Lariviere 2010). Among the various chemical constituents of bee venom, the most unique biologically active substances are some polypeptides, including melittin, apamin, mast-cell degranulating (MCD) peptide, mastocytolytic peptide, minimine, secapin, melittin F, cadiopep, and adolapin (Tables 1 and 2). In general, the bee venom polypeptides are likely to act on the nervous system in both the peripheral nervous system and the central nervous system (CNS) as activators and/or modulators of ion channels and other molecular targets, leading to various biological, pharmacological, and toxicological actions (Tables 1 and 2). However, so far the specifications of these toxic peptides in terms of biological actions, pharmacological and toxicological effects, and structure–function relationships remain mostly unknown. The key effects of some polypeptides and enzymes found in greatest abundance in bee venom are briefly described below.

Table 1 Chemical and physical properties of some major peptides derived from honeybee (Apis mellifera) venoma
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Table 2 Ingredients of honey bee (Apis mellifera) venom and their biological effects
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Melittin

Melittin is a strongly basic 26 amino acid polypeptide which constitutes 40–60% of the whole dry bee venom (Chen and Lariviere 2010). Its physical and chemical properties are listed in Table 1. It has various biological, pharmacological, and toxicological actions including strong surface activity on cell lipid membranes, hemolyzing activity, antibacterial and antifungal activities, and antitumor properties (Table 2; also see Habermann 1972; Son et al. 2007; Chen and Lariviere 2010). Recently, it has become known that melittin is also a strong pain-producing substance able to activate and sensitize nociceptor cells in the periphery via opening of nonselective cation channels − transient receptor potential vanilloid receptor 1 (TRPV1) mediated by the activation of phospholipase A2 (PLA2) cascade pathways (Du et al. 2011). In this process, the activation of ATP P2X and P2Y receptors and phosphorylation of protein kinase A (PKA) and mitogen-activated protein kinases (MAPKs) are also believed to be involved (Hao et al. 2008; Lu et al. 2008; Yu et al. 2009; Du et al. 2011; for details, see below). Moreover, tetrodotoxin-resistant subunit Nav1.9 of voltage-gated sodium channel (VGSC) is upregulated in small cells of the dorsal root ganglia (DRG) by s.c. injection of melittin (Yu et al. 2013). Contrarily, melittin is also believed to be the active substance of bee venom in producing antinociceptive effects when applied to the acupoint of a subject (“apipuncture”) (Son et al. 2007). However, the molecular and cellular mechanisms underlying the antinociceptive effects of melittin remain unclear.

Apamin

Apamin is another important bee venom neurotoxic polypeptide of 18 amino acids comprising 2–3% of whole dry bee venom (Chen and Lariviere 2010). Its physical and chemical properties are listed in Table 1. It possesses a selective inhibitory action on calcium-dependent potassium channels (CDPC) that are involved in regulation of the after-hyperpolarization period that affects the frequency of action potentials in the nervous system (Table 2). Based upon its selective action, apamin has been widely used as a tool drug for studying the characteristics of CDPC.

Mast-Cell Degranulating (MCD) Peptide

MCD peptide, also known as peptide 401, is a bee venom polypeptide with 22 amino acids and constitutes 2–3% of whole dry bee venom (Chen and Lariviere 2010). Its physical and chemical properties are listed in Table 1. It was originally named due to its biological action of causing release of histamine from mast cells (Chen and Lariviere 2010). MCD peptide has specific binding sites in the hippocampus, and application of this peptide onto hippocampal slices was shown to result in the production of long-term potentiation (LTP) in the CA1 area that is distinct from the LTP evoked by conditioning electrical stimulation (Table 2). MCD peptide may also be involved in the pathogenesis of epilepsy (Table 2). The molecular and cellular mechanisms underlying MCD peptide-evoked LTP in the hippocampus are not clear, but some reports showed that it has selective binding sites and actions on voltage-dependent potassium channels (Table 2).

Adolapin

Adolapin is a basic polypeptide with 103 amino acid residues and comprises 1% of whole dry bee venom (Chen and Lariviere 2010). Its physical and chemical properties remain unclear. It is the only one that has been shown to have antinociceptive, anti-inflammatory, and antipyretic effects primarily (Table 2). Adolapin can inhibit prostaglandin synthesis via inhibition of cyclooxygenase (COX) activity (Table 2).

Enzymes

There are two major enzymes in the bee venom. One is PLA2 that constitutes 10–12% of whole dry bee venom and has various pharmacological and toxicological effects (Chen and Lariviere 2010). Cellular PLA2 is a membrane-associated phospholipid-converting enzyme that is important in the production of arachidonic acid (AA), which is further metabolized to prostaglandins by COX and to leukotrienes and eicosanoids by lipoxygenase (LOX). PLA2 exhibits complex interactions with melittin that can result in potentiation or in inhibition of secretory PLA2 effects depending on the peptide/phospholipid ratio (Table 2). PLA2 has effects in a range of cells related to nociception including astrocytes and neurons and possibly microglial cells. PLA2 is also involved in pronociceptive glutaminergic neurotransmission in the substantia gelatinosa of the dorsal horn of the spinal cord. The other major enzyme in the bee venom is hyaluronidase that constitutes 1.5–2% of whole dry bee venom (Chen and Lariviere 2010). Bee venom hyaluronidase may share the same property as the endogenous hyaluronidase that breaks down hyaluronic acid in tissues (Table 2).

Nociceptive and Inflammatory Effects of Subcutaneous Bee Venom and Its Pain-Producing Constituents

Experimental Human Studies

Pain sensation produced by a bee sting is a common unpleasant experience of human beings. However, due to the risk of anaphylaxis and systemic reactions to its allergens, so far there has been no experimental report of whole bee venom injection into humans for the primary purpose of studying pain. At the beginning of the twenty-first century, due to increased interest in animal studies on bee venom-induced pain, the first experimental study on pain and inflammatory responses of healthy adults (two women and five men) to intradermal (i.d.) injection of melittin was carried out (Koyama et al. 2000). The pain intensity was rated using a 0–10 visual analog scale (VAS) in which scores of 0, 5, and 10 indicated “no pain,” “moderate pain,” and “intolerable pain,” respectively. A sharp pain sensation (score > 8.0) was reported in all 7 subjects immediately after i.d. injection of melittin (5 μg in 50 μl saline) into the volar aspect of one forearm. The pain sensation declined gradually and totally disappeared at 3 min after melittin injection. It was clearly described that there was no itch sensation following melittin injection, suggesting that significant histamine might not be released. Meanwhile, melittin produced a visual flare surrounding a wheal near the injection site that disappeared within 2 h. This local inflammatory response was also characterized by an increase in skin temperature monitored by a computer-assisted infrared thermograph. The melittin-induced peak increase in skin temperature was at least 10 min delayed compared to the peak pain sensation, and this process was sustained for at least 1 h. Topical lidocaine gel administration markedly blocked the melittin-induced visual flare and the increased skin temperature but not the pain sensation, suggesting that the local inflammatory response induced by melittin is neurogenic and mediated by dorsal root reflex and/or axonal reflex and sympathetic regulation (Koyama et al. 2000, 2002). The painful effects of two higher doses of melittin (10 and 50 μg in 50 μl saline) have been also observed in two other experimental studies on healthy human volunteers (Sumikura et al. 2003, 2006). The peak pain intensity was similar to the abovementioned report; however, the duration of the pain sensation was much longer with the higher doses used and demonstrated a dose-related increase in the duration of the pain. A similar relationship has been reported in rats following whole bee venom injection (Lariviere and Melzack 1996; Chen and Lariviere 2010; see below). The doses of melittin used in the human experiments are far less than the amount delivered by one bee sting, which contains about 140 μg of dried bee venom per sting and 40–60% melittin (Schumacher et al. 1992, 1994). Moreover, i.d. injection of melittin results in primary heat and mechanical hyperalgesia as well as a secondary heat hyperalgesia identified in an area remote from the injection site (Sumikura et al. 2003, 2006). There is no anaphylaxis and systemic reactions in the human subjects receiving i.d. melittin injection.

Experimental Animal Studies

Nociceptive and Inflammatory Effects of Bee Venom Injection

The primary purpose of the first experimental animal study on the nociceptive and inflammatory effects of bee venom was to establish a new rat model for the study of pain (Lariviere and Melzack 1996). Subsequently, the bee venom-induced pain-related behavioral responses have been well studied in different species including cats, rats, and many strains of mice (Chen et al. 1998, 1999b; Chen and Chen 2000, 2001; Lariviere et al. 2002; for details see Chen and Lariviere 2010). A comparative study of the effects of six venoms from honeybee, bumblebee, yellow jacket and paper wasps, and yellow- and white-faced hornets on rats showed that honeybee venom was the most potent to produce pain in animals (Lariviere and Melzack 2000). Generally, animals respond behaviorally to a given dose of s.c. bee venom injection in a similar pattern and time course. They display painful spontaneous behaviors by a bout of self-caring and favoring actions such as flinching, licking, and biting the injected paw robustly in the first 5–10 min, followed by a slow decline in pain scores over 1 h of observation. Dose–effect investigation shows that the intensity and time course of the bee venom-induced spontaneous pain-related behaviors are dose dependent (Fig. 1). The bee venom-induced pain-related behaviors are shown to be sensitive to pharmacological intervention by morphine and nonsteroidal anti-inflammatory drugs (NSAIDs), demonstrating that the bee venom-induced behaviors are nociceptive or painful. The lower mammalian response pattern and time course are similar to the human response, and thus, there is no evidence for species differences. The consistency of behavioral responses to bee venom among different species reflects common, natural biological processes in mammals in response to bee stings.

Fig. 1
figure 1

Dose effect of subcutaneous injection of bee venom (BV) on spontaneous pain-related behaviors in rats. (a) Time courses of spontaneous paw flinches induced by subcutaneous injection of six doses of BV. (b) A fitted curve of dose effect following BV injection

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Hyperalgesia is referred to as an enhanced painful sensation evoked by painful stimulation (mechanically or thermally nociceptive), while allodynia is referred to as a painful sensation evoked by non-painful stimulation (mechanically or thermally non-nociceptive) under the conditions of tissue and/or nerve injury (McMahon and Koltzenburg 2006; Chen et al. 2013). In animals, pain hypersensitivity (hyperalgesia and allodynia) can be assessed and quantified by measuring changes in paw withdrawal thermal latency (PWTL) or paw withdrawal mechanical threshold (PWMT) between pre- and posttreatment of bee venom. The phenomena of pain hypersensitivity in rats following s.c. bee venom injection were first identified by Chen and his colleagues (Chen et al. 1999b; Chen and Chen 2000, 2001). Following at least 1 h after bee venom injection when spontaneous pain-related behaviors disappear, radiant thermal or mechanical von Frey filament stimuli can be applied to the injection site and its remote surrounding area. Dramatic reductions in PWTL or PWMT can be consistently identified during the period between 2 and 96 h after bee venom injection, indicating the occurrence of primary heat and mechanical hyperalgesia in the injured area of the paw (Chen et al. 1999b, 2000, 2001, 2003; Chen and Chen 2000). This result was consistent with what was seen in experimental human subjects in response to melittin injection (Sumikura et al. 2003). Moreover, a secondary and a mirror-image heat, but not mechanical, hyperalgesia can also be identified in rats, which can also be seen in the same human subjects (Sumikura et al. 2006). This is an interesting clinical phenomenon referred to as mirror-image pain that occurs on the side of the body contralateral to the injured side (Chen et al. 2013; Chen and Lariviere 2010). It is also intriguing to note that the time course of hyperalgesia to thermal and mechanical stimulus modalities in the primary injury area is different. Primary mechanical hyperalgesia lasts more than 96 h, while primary heat hyperalgesia lasts 48 h (Chen 2003, 2007, 2008; Chen and Lariviere 2010). Dose–effect investigation also reflects difference in percent maximal possible effect (% MPE) between thermal and mechanical pain hypersensitivities induced by s.c. bee venom in rats (Fig. 2). These results implicate distinct underlying mechanisms of the bee venom-induced pain hypersensitivity evoked by different stimulus modalities.

Fig. 2
figure 2

Dose effect of subcutaneous injection of bee venom (BV) on thermal or mechanical pain hypersensitivity in rats. (a) Percent maximal possible effect (% MPE) of six doses of BV injections on thermal pain hypersensitivity. Dose-dependent increase in % MPE can be clearly established between 0.001 and 0.05 mg of BV; however, dose effect disappears when the dose is higher than 0.1 mg. (b) Percent MPE of six doses of BV injections on mechanical pain hypersensitivity. Dose-dependent increase in % MPE can be clearly established between 0.001 and 0.01 mg of BV; however, dose effect disappears when the dose is higher than 0.05 mg. PWTL paw withdrawal thermal latency, PWMT paw withdrawal mechanical threshold. Primary injury indicates s.c. BV injection site; mirror image indicates symmetrical site on the contralateral paw to the BV injection

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The bee venom-induced local inflammatory responses have also been scored by measurement of increased volume of the injected paw (Lariviere and Melzack 1996). It has been shown that edema develops immediately and reaches a maximal plateau at about 15–20 min after injection. The time course of edema outlasts the spontaneous pain-related behaviors and gradually disappears by 48 h after bee venom injection. The dose-dependent effect of bee venom on paw edema is well established between the doses ranging from 0.001 to 0.1 mg (in 50 μl saline), while good dose-dependent effect cannot be seen for plasma extravasation (Fig. 3). Indomethacin, an NSAID, could effectively inhibit the bee venom-induced paw edema. There is no overt necrosis developed in response to bee venom injection, and no rat shows signs of allergic reaction, even when retested within 1 month after the first testing (Lariviere and Melzack 1996).

Fig. 3
figure 3

Dose effect of subcutaneous injection of bee venom (BV) on inflammatory responses in rats. (a) Percent maximal possible effect (% MPE) of six doses of BV injections on paw volume (edema). (b) Percent MPE of six doses of BV injections on plasma extravasation. Paw edema reflected by paw volume induced by s.c. BV injection has a great dose-dependent effect; however, plasma extravasation does not

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Nociceptive and Inflammatory Effects of Bee Venom Peptide Constituents

As listed in Tables 1 and 2, bee venom is a complex composition of more than 20 constituents or ingredients. Four major polypeptides including melittin, apamin, MCD peptide, and a novel PLA2-related peptide have been separated, purified, and structurally identified from whole dried bee venom so as to search for active long-term pain-producing components (Chen et al. 2006). In animal studies, it has been revealed that all four of the polypeptides could produce marked local inflammatory responses (edema) when measured 1 h after s.c. injection. However, the nociceptive and hyperalgesic effects differ from substance to substance. Among the four polypeptides tested subcutaneously, melittin, MCD peptide, and PLA2-related peptide are able to produce distinct nociceptive paw flinches; however, only melittin causes pain-related behaviors lasting for nearly 1 h. Because all three polypeptides induce nociception that peaks shortly after injection, it has been proposed that they activate nociceptors more quickly (Chen et al. 2006). Upon examining the effects of the four polypeptides, only melittin and apamin are shown to result in heat as well as mechanical hypersensitivity at the primary injury site, and only melittin-induced primary mechanical hypersensitivity lasts over 48 h. Moreover, the melittin-induced number of rat paw flinches is surprisingly and particularly similar to the number induced by bee venom during the late period (20–60 min after injection), but less than the bee venom-induced paw flinches during the early period (0–19 min after injection). Thus, among the components of bee venom, melittin has been demonstrated to be the major polypeptide responsible for the prolonged painful stimulation of bee venom injection, leading to both tonic nociception and hypersensitivity, while the other polypeptides contribute only to the early nociceptive responses within 10–20 min after injection (Chen and Lariviere 2010).

It has been also surprisingly revealed that the melittin-induced nociceptive responses can be partially inhibited by both pre- and posttreatment with capsazepine (CPZ), a potent antagonist of the thermal nociceptor TRPV1, suggesting the involvement of this molecular target in the melittin-induced nociception (Chen et al. 2006). Since CPZ can reverse the melittin-induced primary heat hypersensitivity, but has no effect on the melittin-induced primary mechanical hypersensitivity, it highly supports the notion that the activation of TRPV1 by melittin specifically contributes to persistent nociception and primary heat hyperalgesia (Chen et al. 2006). Altogether, it becomes clear that during the symphony of bee venom-produced inflammatory pain and hypersensitivity, different components of bee venom play different roles in the entire process; however, melittin is likely to play a central role in the production of the long-lasting pain, hyperalgesia, and local inflammation following bee sting in mammals.

Neuronal Activities in Response to Bee Venom and Melittin Along the Pain Pathways

Activation and Sensitization of Primary Nociceptive Neurons

To understand how primary nociceptive neurons respond to bee venom, direct effects of melittin, the major pain-producing polypeptide of bee venom, were examined using in vitro whole-cell patch-clamp recordings of primary sensory neurons acutely dissociated from rat’s DRG (Du et al. 2011). Topical application of melittin resulted in [Ca2+]i rise in 69% of the cells tested (20–35 μm in diameter) (Fig. 4a, b). In current clamp mode, 55% of the recorded DRG neurons were shown to respond to melittin with tonic discharge of action potentials (APs) following a slow membrane depolarization (Fig. 4c). The duration of melittin-induced firing ranged from 70 to 1200 s and was most intense during the 200–800 s time period (Fig. 4d, e). The melittin-responsive DRG cells were defined as nociceptors because the APs exhibited typical electrophysiological characteristics of nociceptive cells: (1) a long AP duration and a prolonged after-hyperpolarization; (2) an inflection on its falling phase, which is generally considered a characteristic of nociceptors (Fig. 4c′); (3) capsaicin sensitive; and (4) IB4-positive cell sensitive. Accordingly, the majority of the melittin-sensitive cells recorded are likely to be primary nociceptive neurons.

Fig. 4
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Melittin (Mel)-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and action potential firing in primary sensory (DRG) neurons of rats. (a) Graph shows that a subpopulation of acutely isolated rat DRG neurons was able to respond to Mel (2.0 μM) with a [Ca2+]i rise (red vs. black). Typical traces of [Ca2+]i changes were simultaneously recorded during the application of Mel for 30 s. (b) Distribution histogram of the cell body sizes for Mel-sensitive (n = 93) and Mel-insensitive (n = 41) DRG neurons. (c) Application of Mel (2.0 μM, for 50 s) to a DRG cell evoked a slow membrane depolarization (resting membrane potential, RMP = −56 mV before Mel application; dotted line) and a period of tonic action potential (AP) discharge. Inset c′ shows the waveform of the AP recorded at the indicated time, 133 s after the Mel application. (d) Histogram showing time course of firing frequency averaged from 45 Mel-sensitive DRG neurons. (e) Distribution histogram of cell body diameters for Mel-sensitive (n = 56) and Mel-insensitive (n = 46) DRG neurons as measured in current clamp experiments (From Fig. 1 of Du et al., Melittin activates TRPV1 receptors in primary nociceptive sensory neurons via the phospholipase A2 cascade pathways. Biochem Biophys Res Commun 2011; 408:32–37. With permission from Elsevier)

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Using voltage clamp recordings at a holding potential of −70 mV, it was found that melittin could evoke large inward currents in 42% of the recorded DRG neurons in a concentration-dependent manner (Fig. 5a, b; Du et al. 2011). Moreover, repeated application of a given concentration of melittin (2 μM for 200–300 s duration, 20 s intervals) had sensitizing effects on the inward currents, as evidenced by increased current amplitude following the second and the third melittin application compared to the first response (Fig. 5c, d). TRPV1, also known as the capsaicin receptor and proton-activated ion channel, is well known to be a thermal nociceptor transducing nociceptive heat (>42 °C) stimuli into a series of pain “signals” (action potentials) at the peripheral terminals of a population of primary nociceptive neurons (Chen et al. 2013). Co-application of CPZ, a selective TRPV1 antagonist, could completely block the occurrence of the melittin-induced inward currents (Fig. 5e, f) and [Ca2+]i rise (Fig. 5g–i) in a reversible manner (Du et al. 2011), suggesting a selective action rather than nonselective pore-forming effects of melittin on the primary sensory neurons (Chen and Lariviere 2010). In this study, further experiments showed that: (1) inhibitions of PLA2, but not phospholipase C (PLC), could suppress the melittin-induced inward currents; (2) inhibitors of COX and LOX, two key components of the AA metabolism pathway, each partially suppressed the inward current evoked by melittin; (3) inhibitor of protein kinase A (PKA), but not of PKC, also abolished the melittin-induced inward currents. These results indicate that melittin can excite primary nociceptive neurons at least in part by activating TRPV1 receptors via PLA2-COXs/LOXs cascade pathways (Fig. 5j; Du et al. 2011). In another two experiments, roles of TRPC in mediation of the melittin-induced activation of primary nociceptive neurons and pain-related behaviors were also studied (Ding et al. 2011, 2012). It was revealed that direct application of SKF-96365, a potent antagonist of TRPC3/6/7, could block the melittin-induced inward current and [Ca2+]i rise (Ding et al. 2011) and the melittin-induced spontaneous pain-related behaviors as well as thermal pain hypersensitivity (Ding et al. 2012). Similar to TRPV1, TRPC family is also a nonselective cation channel that is highly permeable to Ca2+ when activated. Because it is known that TRPC1, TRPC3, and TRPC6 are the major subunits localized in the rat DRG and intracellular diacylglycerol (DAG) is an endogenous activator of TRPC3/6/7 channels that are sensitive to SKF-96365, it can be proposed that melittin, which can insert itself into the lipid membrane (Chen and Lariviere 2010), causes pore formation that leads to the release of ATP and the activation of P2X channels and P2Y G-protein-coupled receptors (GPCRs) (Lu et al. 2008). The activation of GPCRs by melittin results in the production of DAG and inositol 1,4,5-triphosphate (IP3), and this may serve as another route for melittin to activate the subpopulation of nociceptor cells containing IB4, but not TRPV1 (Ding et al. 2011, 2012).

Fig. 5
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Effects of topical application of melittin (Mel) on the current responses of primary sensory (DRG) neurons. (a) Amplitude of current evoked from a DRG cell following successive applications of Mel (10 μM, 5 μM, 1 μM) showed dose-dependent increase in evoked current. (b) Plot of mean inward current amplitude evoked by different doses of melittin (2.5 μM, 5 μM, 10 μM) in DRG cells (n = 17, 15, 7, respectively). *# P < 0.05 compared to lowest dose (Kruskal–Wallis ANOVA on ranks with Dunn’s post test). For this experiment each cell was exposed to only one dose. (c) Sample trace of a current recording from a DRG cell following three repeated applications of 2 μM Mel (Mel1, Mel2, and Mel3). There was a clear sensitization of the Mel-evoked inward current following repeated applications. (d) Average responses to successive applications of 2 μM Mel, normalized to the amplitude of the first response. **## P < 0.01 vs. Mel1 (Wilcoxon signed rank test with Bonferroni correction, n = 20). Cells with no response to the first application are excluded from the analysis. (e) Co-application of capsazepine (CPZ, 5 μM), a TRPV1 antagonist, resulted in a significant blockade of the 2.0 μM Mel-evoked inward currents from a holding potential of −70 mV (n = 5). (f) Statistical analysis of inhibitory effects of CPZ on the Mel-induced inward current. ** P < 0.01, Mel + CPZ vs. Mel (first application) (Student’s paired t-test). (g) Repeated application of Mel-evoked rises in intracellular Ca2+ concentration ([Ca2+]i) measured by calcium imaging. (h) Blocking effects of CPZ on melittin-induced rise in [Ca2+]i measured by calcium imaging. (i) Statistical analysis of inhibitory effects of CPZ on the Mel-induced rises in [Ca2+]i. ** P < 0.01, Mel + CPZ vs. Mel (paired t-test). (j) A proposed scheme showing direct action of Mel on the DRG cells where it activates and sensitizes TRPV1 by PLA2-LOX/COX metabolites (a–i merged from Figs. 2 and 3 of Du et al. Melittin activates TRPV1 receptors in primary nociceptive sensory neurons via the phospholipase A2 cascade pathways. Biochem Biophys Res Commun 2011; 408:32–37. With permission from Elsevier)

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More recently, it was revealed that s.c. injection of melittin caused upregulation of tetrodotoxin-resistant VGSC subunits Nav1.9 and Nav1.8 in the DRG cells (Yu et al. 2013). However, antisense-mediated knockdown of Nav1.9, but not Nav1.8, in the DRG resulted in inhibition of melittin-induced pain-related behaviors (Yu et al. 2013). Contrarily, Nav1.8, but not Nav1.9, was shown to be involved in complete Freund’s adjuvant (CFA)-induced inflammatory pain (Yu et al. 2011). Patch-clamp recordings of DRG cells dissociated from rats 2 h after s.c. melittin injection revealed that the melittin-responsive cells were tonic type, but not phasic type, in terms of electrophysiological characteristics (Yu et al. 2014). This suggests that the tonic subpopulation of the DRG cells studied is likely to be a key primary nociceptor cellular type in production and conduction of spike firing induced by melittin and bee venom.

Activation and Sensitization of Spinal Dorsal Horn Neurons

The dorsal horn of the spinal cord is structurally and functionally involved as the first synaptic relay in the mediation of nociceptive information from the periphery to the CNS (McMahon and Koltzenburg 2006; Chen et al. 2013). To see how the spinal dorsal horn neurons respond to s.c. bee venom and melittin injection is of particular importance for understanding of the spinal mechanisms of the bee venom-induced spontaneous pain and hypersensitivity.

Spatiotemporal Properties of Spinal Dorsal Horn Neural Activities

Neural activity biomarker c-Fos, an immediate early proto-oncogene protein, has been widely used for the study of spinal dorsal horn neural activities in response to painful stimuli (Luo et al. 1998; Chen and Lariviere 2010). It has been clearly shown that neuronal activities associated with pain in the spinal dorsal horn can be localized mainly in the superficial layers (laminae I–II) and the deep layers (laminae IV–VI) (Chen et al. 2013). The superficial and deep layers contain pain-related neurons that receive input from primary nociceptive afferents (Chen et al. 2013). Thus, localization of c-Fos-like immunoreactivity can reflect spatiotemporal characteristics of the dorsal horn functional state. Briefly, 30 min after bee venom injection into the ipsilateral hind paw, expression of c-Fos protein became localized within laminae I–II of lumbar spinal cord. The spatial range of neuronal activities extended parallel to the number increase of c-Fos-positive neurons in both superficial and deep layers at 1 h and reached peak level at 2 h after bee venom injection (Luo et al. 1998). The c-Fos-positive neurons began to decline in number in both superficial and deep layers at 4 h and completely disappeared at 96 h after bee venom injection (Luo et al. 1998). In comparison with the time course of the bee venom-induced behavioral nociceptive responses and hyperalgesia, it is likely that c-Fos expression reflects establishment of a sensitized state at the spinal dorsal horn which requires at least 30 min of persistent primary afferent input to the central site. The sensitized state of the spinal dorsal horn is responsible for the development and maintenance of pain hypersensitivity that disappears with the disappearance of spinal c-Fos expression (Luo et al. 1998; Chen et al. 1999b; Chen and Chen 2000).

The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases found in a variety of cells, transducing a broad range of extracellular stimuli into diverse intracellular responses by producing changes in transcriptional modulations of key genes as well as posttranslational modifications of target proteins. The roles of three MAPK family members, extracellular signal-regulated kinases (ERKs), p38 MAPK, and c-Jun N-terminal kinase (JNK), in mediation of the bee venom and melittin-induced neural activities and pain-related behaviors have been well studied (Yu and Chen 2005; Cao et al. 2007; Guo et al. 2007; Cui et al. 2008; Hao et al. 2008; Liu et al. 2007, 2011; Li et al. 2008; Yu et al. 2009). Temporal and spatial features of activated ERK and p38 MAPK in the spinal dorsal horn in response to s.c. bee venom injection were examined using immunocytochemical staining in rats (Cui et al. 2008). Subcutaneous injection of bee venom resulted in very quick phosphorylation of ERKs in superficial layer neuronal cell bodies within 2 min that gradually declined within 1 day; however, the phosphorylation of p38 MAPK in the superficial layer neurons began 1 h later than ERKs, reached peak at about 2 h, and was maintained active until the end of 7 days of observation. There were very few ERK- and p38-labeled neurons observed in the deep layers of the dorsal horn, suggesting that only neurons in the superficial layers use these two types of MAPKs in response to bee venom injection. A dramatic phenomenon was that phosphorylated p38 MAPKs began to be localized in microglia across laminae III–IV 1 day after bee venom challenge and reached peak on the 3rd day when bee venom-induced pain hypersensitivity almost disappeared in behavioral observations, implicating that appearance of microglia with p38 MAPK phosphorylation in the dorsal horn is not likely to be involved in the maintenance of pain and hyperalgesia, instead, probably as a scavenger to remove “dead” cells and repair injury. Throughout the observation period, there was neither ERKs nor p38 MAPK detected in astrocytic cells, suggesting that astrocytes may use other protein kinases in the dorsal horn in response to peripheral injury. Actually, there are region-related differences in distribution between different isoforms of a given subtype of MAPKs along the somatosensory system (Guo et al. 2007; Liu et al. 2007). For example, under normal state ERK1 and JNK54 were highly expressed in the spinal dorsal horn with very low level of ERK2 and JNK46 signal; however, in the primary somatosensory cortex (S1 area) or the hippocampal formation, ERK2 and JNK46 were highly expressed. However, under the bee venom-induced inflammatory pain state, ERK2 phosphorylation and JNK46 phosphorylation were distinctly increased in the dorsal horn, suggesting that spinal ERK1 and JNK54 are constitutive isoforms, whereas ERK2 and JNK46 are inducible by peripheral nociception.

In summary, s.c. injection of bee venom indeed induces long-term activation of spinal dorsal horn neural activities that expands from superficial layers to the deep layers in a synchronized way. As a response to the peripheral ongoing input induced by bee venom, ERKs are quickly phosphorylated and peaked at 2 min in the superficial neurons, followed by appearance of p38 MAPK with peak timing at 2 h in the superficial neurons as well. In contrast, the activation of microglia is much later than the activation of neurons, and phosphorylation of microglial p38 MAPK peaks on the 3rd day after bee venom injection. The modulation of the bee venom-induced nociceptive processes at the spinal dorsal horn is likely more complex in nature than previously thought and needs to be systematically studied further.

In Vivo Electrophysiological Recordings of Spinal Dorsal Horn Neuronal Activities

There are at least three classes of neurons, low-threshold mechanoreceptive (LTM) neurons, wide-dynamic-range (WDR) neurons, and nociceptive-specific (NS) neurons in the spinal dorsal horn serving as transducer, encoder, and probably filter of various somatosensory information (Chen et al. 2013). The functional classification of dorsal horn neurons is based upon their neuronal response characteristics to natural mechanical stimuli applied to their cutaneous receptive fields (cRF). LTM neurons are driven mostly by innocuous stimulation and located mainly in laminae III–IV; WDR neurons are driven by noxious as well as non-noxious stimulation and located mainly within lamina V; NS neurons are driven only by noxious stimulation and located mainly in lamina I (Chen et al. 2013). Among the three classes of dorsal horn neurons, there are reliable lines of evidence showing that the majority of WDR neurons, but not NS neurons, are intercalated in the circuitry responsible for the nociceptive withdrawal reflex (Chen et al. 2013). Thus, spinal WDR neurons may play very important roles in the mediation of nociceptive responses and hypersensitivity observed in pain-related behaviors following s.c. bee venom injection. The first electrophysiological recording of the response to bee venom injection was made by Chen and his colleagues on the WDR neurons located mainly within laminae IV–VI of the spinal dorsal horn in anesthetized cats (Chen et al. 1998, 1999a). Similar to what was observed in the behavioral assays, s.c. injection of bee venom into the center of the cRF resulted in an immediate sustained increase in neuronal firing that reached peak within 5 s and declined until the end of the 60-min observation period. The bee venom-induced spike discharges of WDR neurons could be suppressed by systemic morphine and lidocaine locally applied onto the sciatic nerve that resulted in blockade of primary afferent input. In these studies, responsiveness of spinal WDR neurons to both non-nociceptive and nociceptive mechanical stimuli was also significantly enhanced 1–2 h after bee venom injection, suggesting that spinal WDR neurons that mediate and encode spinal nociceptive reflex are sensitized by peripheral bee venom injection. Because the response pattern and time course of spinal WDR neuronal activities are quite similar to those of behavioral manifestation induced by s.c. bee venom injection and because blockade of altered spinal neuronal activities results in parallel disappearance of the bee venom-induced pain-related behaviors (see Chen and Lariviere 2010), it is rational to believe that the sensitized dorsal horn WDR neurons play a key role in the mediation of paw withdrawal reflex facilitation, displayed as persistent paw flinches and hyperalgesia. Recordings made of the same class of neurons in the anesthetized rats show similar altered neuronal responses in the dorsal horn in response to bee venom treatment (You and Chen 1999; Zheng et al. 2002, 2004).

In an excellent electrophysiological study, it was found that injection of either bee venom or melittin into the cRF of spinal WDR neurons resulted in a dose-dependent increase in spontaneous spike discharges in terms of both frequency and duration in anesthetized rats (Li and Chen 2004). The melittin-induced spike firing of spinal dorsal horn WDR neurons can be completely blocked by local peripheral injection of CPZ into the ipsilateral cRF, but not the contralateral paw, suggesting that melittin has local nociceptive effects rather than systemic effects (Fig. 6). Similar to the bee venom effects, local peripheral injection of melittin into the same WDR neuronal cRF resulted in increase in responsiveness to both thermal and mechanical stimuli compared to the baseline controls (Fig. 7a, b). This functionally sensitized state of the spinal nociceptive neurons following melittin injection can be reflected by a clear leftward shift of the functional curves of the stimulus intensity−responsiveness relationship for both thermal and mechanical hypersensitivities (Fig. 7c, d). Intriguingly, the leftward shift of the thermal functional curves was completely reversed by local peripheral injection of CPZ into the ipsilateral cRF of spinal WDR neurons, whereas that of the mechanical functional curves was not affected by the same treatment, strongly suggesting existence of different underlying mechanisms between thermal and mechanical hypersensitivities (Fig. 7a–d). To demonstrate the roles of spinal dorsal horn WDR neurons in the mediation of bee venom-induced withdrawal reflex facilitation, simultaneous pairwise recordings were made in both WDR neurons and single motor units (SMU) along the spinally organized nociceptive reflex circuitry (You et al. 2003a). The result showed an immediate parallel increase in spike discharges of the paired WDR neuron and SMU that lasted for 1 h in response to injection of bee venom in their common cRFs (Chen 2003; You and Arendt-Nielsen 2005). These paired recordings also showed a parallel enhancement of mechanically nociceptive responsiveness as well as wind-up responses 1–2 h after bee venom treatment (Chen 2003; Chen and Lariviere 2010). These data reliably demonstrate the neuronal basis underlying thermal and mechanical hypersensitivity observed in behavioral studies. In another study, responses of lamina VII multi-receptive nociceptive neurons were studied in anesthetized, spinalized rats (You et al. 2008). In contrast to the long-term response patterns of dorsal horn WDR neurons, s.c. bee venom injection evoked a short-term (<10 min) firing of this population of neurons. However, the neurons were switched to give a long-term biphasic firing with an early phase (4–13 min) that was followed by a late tonic firing phase (28–74 min) after a quiescent period (4–11 min) in the spinalized rats. The data is interesting and suggests the existence of two separate modulatory systems in the spinal dorsal horn. Spinal WDR neurons that serve as encoders of spinal nociceptive withdrawal reflex are not likely tonically controlled by descending antinociceptive systems, while the deeper-layer nociceptive neurons that send nociceptive information along the spinoreticulothalamic tract to the medial thalamus are likely tonically controlled by a descending antinociceptive system. The response characteristic of lamina I NS neurons to s.c. bee venom injection remains uninvestigated, and thus the precise role of NS neurons needs to be determined.

Fig. 6
figure 6

Blocking effect by local peripheral TRPV1 antagonism on the spike responses of spinal dorsal horn pain-signaling neurons to subcutaneous injection of melittin in anesthetized rat. (a) The melittin-induced sustained period of spike responses of a spinal dorsal horn wide-dynamic-range (WDR) neuron cannot be influenced by local peripheral application of saline (vehicle). (b) Local peripheral application of capsazepine (0.3 mg CPZ in 50 μl 50% dimethysulphoxide), a selective TRPV1 antagonist, into the melittin injection site (Ipsil, ipsilateral) resulted in a dramatic reduction in spike responses of a spinal WDR neuron. (c) Contralateral (Contl) administration of CPZ had no effect on the melittin-induced spike firing of a spinal WDR neuron. (d) Averaged blocking effects of CPZ on the melittin-induced spike discharges of spinal WDR neurons. *** P < 0.001 CPZ (Ipsil) vs. vehicle or CPZ (Contl) (n = 11 for each group). Arrows indicate the timing for injection of CPZ or vehicle 10 min after subcutaneous injection of melittin (Partially from the original data of Li and Chen. Altered pain-related behaviors and spinal neuronal responses produced by s.c. injection of melittin in rats. Neuroscience 2004; 126:753–762. With permission from Elsevier)

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Fig. 7
figure 7

Blocking effect by local peripheral TRPV1 antagonism on the melittin (Mel)-induced thermal hypersensitivity of spinal dorsal horn pain-signaling neurons in anesthetized rat. (a) Subcutaneous Mel injection enhanced the responsiveness of a spinal WDR neuron to suprathreshold nociceptive thermal stimuli (>42 °C) applied to its cutaneous receptive field (cRF). Local peripheral application of CPZ (0.3 mg in 50 μl 50% dimethysulphoxide) into the Mel injection site [Mel + CPZ (Ipsil)] resulted in a dramatic inhibition of spinal WDR neuronal responsiveness to thermal stimuli, whereas contralateral administration of CPZ [Mel + CPZ (Contl)] had no effect. (b) Local application of CPZ did not influence the Mel-induced mechanical hypersensitivity of a spinal WDR neuron regardless of either route of administrations. (c) Intensity-responsiveness functional curves of spinal WDR neurons plotted from rats (n = 11 for each group). † P < 0.05; **†† P < 0.01; ***††† P < 0.001 Mel + Vel vs. Baseline; Mel + CPZ (Ipsil) vs. Mel + Vel or Mel + CPZ (Contl). Br brush, Pr pressure, pin pinch, n.s. no significance (Partially from the original data of Li and Chen. Altered pain-related behaviors and spinal neuronal responses produced by s.c. injection of melittin in rats. Neuroscience 2004; 126:753–762. With permission from Elsevier)

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In summary, in vivo electrophysiological recordings of spinal dorsal horn neurons in both cats and rats reveal similar results of a centrally sensitized state following s.c. bee venom or melittin injection which is responsible for animal paw withdrawal reflex facilitation and altered pain sensation such as hyperalgesia and allodynia.

Spinal Release of Excitatory and Inhibitory Amino Acids in Response to Bee Venom Injection

Given that melittin and other algogenic components of bee venom are able to activate nociceptors upon diffusion to peripheral free nerve endings when injected subcutaneously, pain “signals” (APs) would be generated and conducted anterogradely to the central terminals of primary nociceptive neurons, leading to release of neurotransmitters at the dorsal horn of the spinal cord (Chen et al. 2013). This hypothesis was tested by measuring the release of both excitatory amino acids (EAAs) and inhibitory amino acids (IAAs) and other amino acids involved in recycling of metabolites of those neurotransmitters (Yan et al. 2009). It is well known that glutamate and aspartate are important excitatory neurotransmitters used by the primary afferent to affect the spinal dorsal horn in addition to neuropeptides such as substance P and CGRP (Chen and Lariviere 2010; Chen et al. 2013). Correspondingly, glycine and γ-aminobutyric acid (GABA) are important inhibitory neurotransmitters used by local inhibitory interneurons in the dorsal horn (Chen and Lariviere 2010; Chen et al. 2013). Thus, simultaneous monitoring of both EAAs and IAAs at the spinal level in awake animals experiencing painful stimulation is of particular importance for the understanding of the basic neurotransmitters mediating long-lasting pain.

Cerebrospinal fluid (CSF) samples were collected by microdialysis in conscious rats every 20 min for a period of 120 min when spontaneous nociceptive responses were induced by s.c. bee venom injection (Yan et al. 2009). At least nine amino acids including excitatory (e.g., glutamate, aspartate), inhibitory (glycine, GABA, taurine), and other metabolites were analyzed by high-pressure liquid chromatography. The results showed an immediate increase in the concentration of all the nine amino acids at 20 min; however, in the remaining period of 100 min, EAAs remained above the baseline levels, while IAAs soon decreased to the baseline levels and thereafter to a level much lower than the baseline for the remaining time. The bee venom-induced imbalance between EAAs and IAAs at the spinal cord could be reversed by pre-blockade with bupivacaine at the injection site during which the bee venom-induced pain-related behaviors disappeared completely. These results suggest that the imbalance between EAAs and IAAs at the spinal cord may be associated with the maintenance of persistent pain-related behaviors induced by bee venom (Yan et al. 2009). These results directly support the idea that pain-related behaviors are caused by persistent activation of postsynaptic pain-related neurons driven by ongoing activation of primary nociceptors that use glutamate and aspartate as neurotransmitters in the central terminals projecting to the dorsal horn of the spinal cord (Yan et al. 2009). The results that intrathecal pre- and post-blockade of glutamate NMDA and non-NMDA receptors can inhibit bee venom-induced spontaneous pain-related behaviors provide more strong supporting evidence for the roles of EAAs in mediation of the bee venom-induced pain (You et al. 2002, 2003b; Yan et al. 2009).

Cortical Activation and Synaptic Metaplasticity in “Pain Matrix” Induced by Bee Venom Injection

There are tremendous amounts of brain imaging studies in both human and animal subjects supporting that pain is a complex experience consisting of sensory-discriminative, affective-motivational, and cognitive-evaluative dimensions (McMahon and Koltzenburg 2006; Liu and Chen 2009, 2014; Zhao et al. 2009; Chen et al. 2013). Now it has been gradually known that noxious information caused by tissue or nerve damage is processed by a widely distributed, hierarchically interconnected neural network, referred to as neuromatrix, in the brain (“pain matrix”) (Melzack 2005). To study the effects of s.c. bee venom on the cortical responses, three areas, including the primary somatosensory cortex (S1), the anterior cingulate cortex (ACC), and the hippocampal formation, have been investigated using multiple experimental approaches (Guo et al. 2007; Liu et al. 2007, 2011, 2012; Chang et al. 2008; Ren et al. 2008; Zhao et al. 2009; Gong et al. 2010; Lyu et al. 2013; Lu et al. 2014; for details, see review by Liu and Chen 2014).

The Primary Somatosensory Cortex

The S1 area is believed to be involved in perceiving pain sensation at the cortical level (McMahon and Koltzenburg 2006; Chen et al. 2013). To investigate how the S1 area responds to bee venom injection spatiotemporally, c-Fos immunoreactive neuronal activities in the hindlimb representative of the S1 area were observed 1 h, 2 h, 3 h, and 4 h after s.c. bee venom injection into one hind paw (Chang et al. 2008). Compared to naïve and saline-treated rats, c-Fos-labeled neurons became densely increased in superficial layers (II–III), but less increased in deep layers (IV–VI), following s.c. bee venom injection. The mean number of c-Fos-positive neurons double labeled with selective neuronal biomarker NeuN began to increase at 1 h and reached peak at 2 h within the layers II–III after bee venom treatment that was followed by gradual decrease afterward. The time course of c-Fos expression in the layers IV–VI was in parallel with that of the superficial layers, but with a much lower density and magnitude. This result demonstrates that bee venom in the periphery can evoke increased neuronal activities in the S1 area with predominant localization in layers II–III.

In other two studies, subtypes of two isoforms of MAPKs including ERK1/ERK2 and JNK46/JNK54 were also examined following s.c. bee venom injection (Guo et al. 2007; Liu et al. 2007). In the S1 area of the cortex, ERK2 was expressed more abundantly than ERK1, while there was a larger amount of ERK1 than ERK2 in the spinal cord, suggesting existence of a region-specific expression of different subtypes of ERKs along the pain pathway . Moreover, phosphorylated ERK2 (pERK2), not phosphorylated ERK1 (pERK1), was normally expressed with a high level in the S1 area, but neither pERK1 nor pERK2 was detectable in normal spinal cord, suggesting that under normal condition the S1 cortices use pERK2 in processing non-nociceptive information from dorsal column–medial lemniscus system. However, under condition following bee venom injection, ERK1 was more remarkably phosphorylated than ERK2 in the S1 area, while pERK2 exhibited stronger response than pERK1 in the spinal cord, implicating that the S1 uses pERK2 while the spinal dorsal horn uses pERK1 as a mechanism in processing nociceptive information from the dorsal horn–spinothalamocortical tract (Guo et al. 2007). Furthermore, pJNK46 was shown to be normally expressed in the S1 area, but not in the spinal cord, while neither of the two structures contained pJNK54 (Liu et al. 2007). Subcutaneous bee venom resulted in a significant increase in the phosphorylation of both JNK isoforms in the S1 area for a long period (lasting at least 48 h). Nevertheless, JNK46 exhibited a much higher activation than JNK54 in the spinal cord, whereas the same noxious stimulation elicited evident activation of JNK54 in the S1 area, leaving JNK46 less affected (Liu et al. 2007).

Multielectrode array (MEA) recordings in vivo enable experimenters to identify ensemble WDR and NS neurons at the S1 area in freely moving rats (Wang et al. 2011; Chen’s unpublished data). Subcutaneous injection of bee venom into the cRFs of both WDR and NS neurons resulted in a parallel increase in spike discharges and pain-related behaviors lasting for at least 1 h in a similar time course pattern to the spinal dorsal horn responses (Chen and Lariviere 2010; Liu and Chen 2014). Moreover, slice recordings using MEA (8×8 channels) probe and patch-clamp technique in vitro enable experimenters to evaluate the effects of s.c. bee venom on the synaptic plasticity at the cortical level (Zhao et al. 2009; Liu et al. 2011, 2012; Lyu et al. 2013; Lu et al. 2014; for review see Liu and Chen 2014). The results showed that s.c. bee venom caused a dramatic switch in synaptic efficacy from the LTP-resistant state to the LTP-inducible state (Chen’s unpublished data), implicating a great impact of s.c. bee venom on the neural plasticity in the S1 area.

The Anterior Cingulate Cortex

The medial prefrontal cortex including the prelimbic, sublimbic, and the ACC is believed to be a critical brain structure involved in mediation of direct pain-related emotion and vicarious empathy for pain (Ren et al. 2008; Li et al. 2014; Liu and Chen 2014). The synaptic mechanisms of the ACC underlying different types of pain have been well studied in several animal models of pain (Liu and Chen 2014). Briefly, s.c. bee venom injection resulted in dramatic spatial enlargement of synaptic connections as well as leftward shift of input–output (I-O) functional curves of synaptic transmission in comparison with naïve and saline controls (Lu et al. 2014); however, the induction of LTP could not be altered in the same experimental preparation (Lu et al. 2014). Patch-clamp recordings of the slices containing the ACC harvested 2 hours after s.c. bee venom injection also showed a hyperexcitable state under which enhanced firing rate can be seen (Gong et al. 2010). The bee venom-induced hyperexcitable state of the ACC neurons is largely due to disinhibition of tonic control of excitatory synaptic transmission by inhibitory synaptic input, because enhanced excitatory postsynaptic currents (EPSCs) and reduced inhibitory postsynaptic currents (IPSCs) could be seen following s.c. bee venom injection, implicating that the balance between the excitatory and inhibitory synaptic functions is disrupted by s.c. bee venom injection (Gong et al. 2010).

The Hippocampal Formation

The hippocampal formation, traditionally believed to be a key brain structure in learning and memory, has also been demonstrated to be involved in processing of pain information (Liu and Chen 2009; 2014). Similar to the S1 area, in the hippocampus (Guo et al. 2007), (1) there was a larger amount of ERK2 than ERK1 in naïve state; (2) pERK2, not pERK1, was normally expressed with a high level in the hippocampus; (3) following bee venom injection, however, ERK1 was more remarkably activated than ERK2 in the hippocampus. These results implicate that, similar to the S1 area, the hippocampus uses ERK2 to process information under the naïve condition; however, it uses ERK1 to process nociceptive information under the peripheral inflammatory pain state.

Using 8×8 MEA recordings of brain slices containing the hippocampal formation, entorhinal–dentate gyrus (DG)/CA1 synaptic responses can be observed following electrical stimulation of the perforant path (PP) through which direct axonal projections from the entorhinal cortical (EC) neurons are sent to the DG or the CA1 (Zhao et al. 2009). The EC-DG/CA1 pathways are believed to be major projections mediating information from other cortical areas to the hippocampus via relays in the EC (Liu and Chen 2009, 2014). Following s.c. bee venom injection, there were great changes in both synaptic connections spatially and synaptic efficacy temporally. For example, the EC-DG/CA1 synaptic connections were significantly increased in number when MEA recordings were made on the hippocampal slices from rats with bee venom injection (Zhao et al. 2009). Meanwhile, LTP, an electrical phenomenon reflecting temporal synaptic plasticity (enhancement of synaptic efficacy) induced by theta-burst stimulation of the PP, can also be significantly enhanced by preconditioning with s.c. bee venom injection (Zhao et al. 2009). This suggests that metaplasticity, a concept coined by Abraham and Bear in 1996 indicating a plasticity of synaptic plasticity (Abraham and Bear 1996; Abraham 2008), occurs in the hippocampus of rats following preconditioning with bee venom-induced peripheral inflammatory pain.

Pharmacological inhibition of activations of either ERK by U0126 or of JNK by SP600125 significantly reduced the bee venom-enhanced LTP in both EC-DG and EC-CA1 synaptic transmissions, while bath application of the p38 MAPK inhibitor SB239063 resulted in a further increase in the LTP magnitude in the same samples (Liu et al. 2011). In a similar pattern, the bee venom-enhanced LTP could be reversed by antagonism of metabotropic glutamate receptor 5 (mGluR5) with 2-methyl-6-(phenylethynyl)-pyridine; however, the bee venom-enhanced LTP could be further augmented by antagonism of mGluR1 by a selective antagonist 7-hydroxyiminocyclopropan [b] chromen-1α-carboxylic acid ethyl ester perfusion (Liu et al. 2012). These data implicate that mGluR5 and ERK/JNK signaling link is likely to be involved in facilitating the hippocampal metaplasticity (enhancement of LTP) induced by bee venom injection, whereas mGluR1 and p38 MAPK signaling link is likely to be involved in counteracting the processes of hippocampal metaplasticity. More interestingly, s.c. injection of bee venom resulted in sustained (>8 h) phosphorylation of mammalian target of rapamycin (mTOR) and its downstream target p70 S6 kinase (S6K) in the hippocampus (Lyu et al. 2013). The bee venom-induced metaplasticity (enhanced LTP) and the increased phosphorylation of mTOR-S6K signaling pathway in the hippocampus were able to be suppressed by systemic administration of rapamycin, an mTOR inhibitor (Lyu et al. 2013). These data suggest that the bee venom-induced mTOR activation is involved in hippocampal metaplasticity that may affect behaviors of rats. As a supporting line of evidence, rats injected with bee venom exhibited markedly reduced ambulation and exploratory activity in the open field due to over self-caring and favoring behaviors in a corner (signs of anxiety). Systemic administration of rapamycin could completely reverse the bee venom-induced anxiety-like behaviors (Lyu et al. 2013).

Since spatial reorganization of synaptic connections is likely a consequence of metaplasticity, it can be deduced that pharmacological modulation of metaplasticity would result in a parallel reversal of the bee venom-induced spatial enlargement of synaptic connections in the hippocampus. As lines of supporting evidence, antagonisms or blockades of mGluR5, ERK/JNK, and mTOR-S6K signaling links result in the reversal of the bee venom-induced spatial synaptic reorganization; however, antagonism or blockade of mGluR1 and p38 MAPK signaling link results in enhancing effects of the bee venom-induced spatial synaptic reorganization.

In summary, peripheral inflammatory pain state induced by s.c. bee venom can cause a form of the LTP-enhanced metaplasticity in the hippocampal formation that may result in spatial reorganization of synaptic connections at network level, leading to anxiety-like behaviors seen in the bee venom test. Moreover, there exists an inter-counteracting system required to maintain the balance or homeostasis of neural network in the hippocampal formation that can be interrupted by peripheral persistent pain. The signaling link between mGluR1 and p38 MAPK may exist as a counteracting factor against the LTP-enhanced form of metaplasticity; however, the signaling links between mGluR5 and ERK/JNK and mTOR-S6K may act as facilitating factors mediating the LTP-enhanced form of metaplasticity.

Proposed Mechanisms of Bee Venom-Induced Pain

Peripheral Mechanisms

The underlying peripheral mechanisms of bee venom-induced nociception and pain hypersensitivity have been proposed according to many experimental data (for review see Chen and Lariviere 2010). In general, two sets of mechanisms are likely to be involved: (1) direct actions of the bee venomous pain-producing constituents on the primary nociceptors and (2) indirect actions of the bee venomous constituents on the primary nociceptors through algogens and proinflammatory and inflammatory mediators released from mast-cell degranulation, tissue damage, and immune responses. As a consequence, pain-producing substances directly activate cation channel nociceptors leading to pain sensation, while pain-enhancing substances regulate or modify cation channel nociceptors via intracellular cascades leading to hyperalgesia and/or allodynia (pain hypersensitivity).

As shown in Fig. 8, following s.c. injection of bee venom, melittin, a major pain-producing substance, activates TRPV1 receptors by hydroxyeicosatetraenoic acids (HETEs), endogenous ligands of TRPV1 receptor, through catalyzing phospholipids by PLA2 and then AA by LOX that finally produce 5-, 12-, or 15-HETEs, leading to depolarization of primary nociceptor cells (Du et al. 2011). Meanwhile, melittin, MCD peptide, bee venom PLA2, and hyaluronidase may cause tissue damage, leading to releases of adenosine triphosphate (ATP) and photon (H+) that may activate P2-purinoceptor X3 (P2X3) and P2Y, TRPV1, and acid-sensing ionic channel (ASIC) (Chen and Lariviere 2010). Indirect actions of melittin, MCD peptide, and bee venom PLA2 cause degranulation of mast cells, leading to releases of histamine, bradykinin (BK), and 5-hydroxytryptamine (5-HT) that may activate H1 receptor, 5-HT3 receptor, and BK1/2 receptors. Because TRPV1, P2X3, 5-HT3, and ASIC are selective or nonselective cation channels and opening of these cation channels may lead to depolarization of primary nociceptor cells, the activators of these cation channels such as melittin, ATP, 5-HT, and H+ are believed to be pain-producing substances which are responsible for the production of spontaneous pain sensation in humans and pain-related behaviors in animals (Chen and Lariviere 2010). Melittin can also cause production of pain-enhancing substances such as prostaglandin E2 through catalyzing phospholipids by PLA2 and then AA by COX that finally produce various prostanoids, leading to sensitization of primary nociceptors via sensitizing TRPV1 receptors (Chen and Lariviere 2010; Du et al. 2011). Other G-protein-coupled receptors (GPCRs) such as P2Y, H1, and BK1/2 are also likely to be involved in the sensitization of primary nociceptors through PLC- or adenylyl cyclase (AC)-signaling pathways (McMahon and Koltzenburg 2006; Chen et al. 2013). Because MCD peptide, apamin, and tertiapin are blockers of VGPC, calcium-dependent potassium channel (Ca2+–K+), and inward-rectifier potassium channel (Kir), respectively, inactivation of VGPC, Ca2 +–K+, and Kir channels may facilitate depolarization and increase firing rate of primary nociceptor cells (Chen and Lariviere 2010). Further findings support the important roles of TTX-resistant VGSC subunit (Nav1.8 and Nav1.9) in mediation of the bee venom-induced long-term spontaneous pain-related behaviors and pain hypersensitivity because s.c. injection of melittin can induce sustained upregulation of both Nav1.8 and Nav1.9, but not TTX-sensitive, subunits in the small DRG cells (Yu et al. 2013). However, it is intriguing to see that only antisense-mediated knockdown of Nav1.9, but not Nav1.8, in the DRG cells can reverse the melittin-induced pain hypersensitivity. The roles of Nav1.8 cannot be completely excluded in the bee venom-induced nociception because CFA-induced pain hypersensitivity can be suppressed by antisense-mediated knockdown of Nav1.8, but not Nav1.9, suggesting differential roles of TTX-resistant VGSC subunits in different processes of inflammatory pain (Yu et al. 2011).

Fig. 8
figure 8

A schematic drawing of proposed underlying mechanisms of the bee venom-induced peripheral neural plasticity (activation and sensitization). On the left column, venom sac and sting apparatus is shown at the tip of a honeybee abdomen. The major polypeptides and enzymes of the bee venom are listed below (also see Table 1). Subcutaneous injection of bee venom by a syringe is shown on the left top, while a nerve terminal of primary afferent is shown on the bottom right. The direct and indirect actions of each ingredients of the bee venom are proposed. Color symbols representing each ingredient of the bee venom (left) can be clearly seen on the right where melittin (dark red double strand), MCD peptide (red-colored circle), apamin (green-colored circle), and tertiapin (light blue rectangle) bind directly to the membrane of a nociceptor cell leading to the activation of it. Meanwhile, melittin, MCD peptide, bv PLA2 (light green “H”), and hyaluronidase (dark green double balls) cause tissue damage (gray) and release of ATP and H+ that activate P2X3 (thick blue arrow and paired channel)/P2Y (thin red arrow and paired channel), TRPV1 (green arrow and paired channel), and ASIC (purple dashed arrow and paired channel). Indirect actions of melittin, MCD peptide, and bv PLA2 cause degranulation of mast cells (purple) and release histamine, BK, and 5-HT that activate H1 receptor (pink thick arrow and paired receptor), 5-HT3 receptor (blue arrow and paired receptor), and BK1/2 receptors (dark green arrow and paired receptor). The firing of nociceptor terminals will be mediated by voltage-dependent sodium channels (TTXr Nav1.8/1.9), VDCC, VDPC, Kir, and Ca2 + −K+. Dorsal root reflex and axon reflex may cause the release of glutamate and neuropeptides (SP and CGRP) that further activate their autoreceptors on the nociceptor terminals or blood vessels causing inflammatory extravasation (neurogenic) with infiltration of macrophage, immune cells and platelets, and many cytokines (TNF-α, IL1β, PAF, etc.). The syringe indicates transcutaneous injection of bee venom. Abbreviations: 5-HT3 5-hydroxytryptamine receptor 3, 12-HETE 12-hydroxyeicosatetraenoic acids; AA arachidonic acid, ASIC acid-sensing ionic channel, ATP adenosine triphosphate, BK1/2 bradykinin receptors 1/2, bv PLA2 bee venom phospholipase A2, Ca 2 + −K + calcium-dependent potassium channel, CGRP calcitonin-gene-related peptide, COX-1/COX-2 cyclooxygenases 1/2, Glu glutamate, H1 histamine receptor type 1, iGluRs ionotropic glutamate receptors, IL1b interleukin 1b, IL6 interleukin 6, Kir inward-rectifier potassium channel, LOXs lipoxygenases, MAPKs mitogen-activated protein kinases, MCD peptide mast-cell degranulating peptide, MCL peptide mastocytolytic peptide, NK1 neurokinin 1, NOS nitric oxide synthase, P2X3 P2-purinoreceptor X3, P2Y P2-purinoreceptor Y, PAF platelet-activated factor, PGs prostaglandins, PKA protein kinase A, PKC protein kinase C; PKG protein kinase G; SP substance P, TNF-a tumor-necrosis factor-a, TRPV1 transient receptor potential vanilloid receptor 1, TTXr tetrodotoxin resistant, VDCC voltage-dependent calcium channel, VDPC voltage-dependent potassium channel (From Fig. 3 of Chen and Lariviere. The nociceptive and anti-nociceptive effects of bee venom injection and therapy: A double-edged sword. Prog Neurobiol 2010; 92:151–183. With permission from Elsevier)

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Neurogenic inflammation is also proposed to play important roles in mediation of bee venom-induced inflammatory pain processes by releasing glutamate and neuropeptides (SP and CGRP) from the peripheral terminals of primary nociceptor cells due to dorsal root reflex and axon reflex, leading to inflammatory extravasation with infiltration of macrophage, immune cells and platelets, and many cytokines (TNF-α, IL1β, platelet-activated factor, etc.) that exacerbate pain and hypersensitivity (Fig. 8; see Chen and Lariviere 2010; Chen et al. 2013).

Spinal Mechanisms

The spinal dorsal horn is the first relay of synaptic transmission for nociceptive information between primary afferent input and pain-related central neuronal cells (McMahon and Koltzenburg 2006; Chen et al. 2013). The functional state of the spinal dorsal horn has been shown to be activity dependent and is changed by peripheral persistent neural sensitization or plasticity induced by bee venom injection (Chen 2003, 2007, 2008; Chen and Lariviere 2010). The spinal neural mechanisms of bee venom-induced pain and hyperalgesia have also been proposed based upon many experimental data (Chen and Lariviere 2010).

As shown in Fig. 9a, the induction and maintenance of bee venom-induced persistent spontaneous pain-related behaviors are dependent upon the functional state of the synaptic connections between the primary nociceptive nerve terminals (presynaptic component) and dorsal horn pain-signaling neuronal cell bodies (postsynaptic component) (Chen 2007, 2008; Chen and Lariviere 2010). Astrocytes and microglial cells can also be activated by long-term impulse barrages from the periphery induced by s.c. bee venom, forming a dynamic tripartite synaptic composition (Fig. 9a–c; Chen et al. 2013). Following s.c. bee venom injection, the levels of EAAs (glutamate and aspartate) and IAAs (glycine and GABA) at the spinal cord are initially elevated, followed by a sustained increase in EAAs release and decrease in IAAs release (Yan et al. 2009). The sustained release of EAAs from primary afferents plays a key role in coactivation of both ligand-gated ionotropic glutamate receptors (iGluRs), including AMPA/KA and NMDA receptors, and, as a consequence, leads to increase in intracellular Ca2+ concentration (You et al. 2003b; Yan et al. 2009). On the other hand, release of EAAs and SP also activates mGluR group I and neurokinin 1 (NK1) receptors, but not mGluR groups II and III (Zheng and Chen 2001; Yan et al. 2009). This transsynaptic signal transduction via G-protein-mediated signaling results in the phosphorylation of various MAPKs (e.g., ERK and p38 MAPK) (Yu and Chen 2005; Cao et al. 2007; Cui et al. 2008; Li et al. 2008) and protein kinases (PKC, PKA, and PKG) (Li et al. 2000; Li and Chen 2003; Chen 2007, 2008; Chen and Lariviere 2010) as well as enzymes COX-1/COX-2 (Chen and Lariviere 2010). ATP P2X receptors are activated by extracellular ATP leakage in the spinal dorsal horn in response to s.c. bee venom (Zheng and Chen 2000). Meanwhile, at presynaptic component, TRPV1, P2X3, VGCC, Nav1.8, and Nav1.9 are also likely to be activated or upregulated by tonic persistent primary afferent that in turn facilitate neurotransmitter release. Presynaptic localization of NK1, as autoreceptors of SP released from primary afferent terminals, might be further activated and has been demonstrated to enhance both TRPV1- and TTX-resistant Nav1.8 through PKCε (Chen and Lariviere 2010). However, the ongoing spinal dorsal horn neural activities are peripherally dependent, and the establishment of the centrally sensitized state in the spinal cord has been shown to require a certain minimum time window for the accumulation of peripheral input before dorsal horn sensitization can be established (Chen et al. 1998, 1999a, 2000, 2001; You et al. 2002). The maintenance of this centrally sensitized state is also shown to require the activation of peripheral EAAs receptors, P2X and P2Y receptors, protein kinases, and MAPKs (Chen et al. 1999a, 2008; You et al. 2002; Hao et al. 2008; Lu et al. 2008; Yan et al. 2009; Yu et al. 2009).

Fig. 9
figure 9

Schematic drawings of proposed underlying mechanisms of the bee venom (BV)-induced sustained spontaneous pain, primary thermal hyperalgesia, and primary mechanical hyperalgesia in the spinal cord dorsal horn. (a) Shown is a synaptic structure between a presynaptic component (central terminal of primary afferents, left) and a postsynaptic component (membrane of a pain-signaling neuron, right). Astrocytes and microglia are not fully activated at this stage. At the synaptic cleft, in response to the coming of tonic firing action potentials, excitatory amino acids (EAAs, glutamate and aspartate, small colored clear vesicles) are selectively activating ionotropic glutamate receptors (NMDA and AMPA/KA) and metabotropic glutamate receptor group I (mGluR I), while substance P (SP, large dense-cored vesicles) is activating neurokinin 1 (NK1) receptors. At this process, mGluRs II and III are not activated. At presynaptic component, vanilloid receptor TRPV1, voltage-dependent calcium channel (VDCC), and NK1 are activated. ATP (green clear vesicles) released from glial cells or damaged cells due to cytotoxic effects are activating ATP P2X receptors. On the other hand, glycinergic and GABAergic modulations become weak due to lack of inhibitory amino acids. Intracellularly, extracellular signaling-regulated kinase (ERK) and p38 MAPK protein kinases (PKA and PKC and PKG) are phosphorylated and recruited as enhancing modulators of membrane receptors and ion channels. Cyclooxygenases 1/2 (COX-1/COX-2) are also recruited as enzymes catalyzing arachidonic acids to prostaglandins. The inset on the right top shows involvement of descending nociceptive facilitatory pathway from rostral medial medulla (RMM) in the development and maintenance of mirror-image thermal pain hypersensitivity due to the centrally sensitized state. (b) Shown is a tripartite synaptic structure with a presynaptic component (left) and a postsynaptic component (right) and astrocytes or microglia. At the synaptic cleft, in response to thermally nociceptive heat stimuli, EAAs are selectively activating groups I, II, and III of mGluRs but without the activation of ionotropic glutamate receptors. SP is also activating NK1 receptors. At presynaptic component, TRPV1, VDCC, and NK1 are activated. ATP is activating ATP P2X receptors. Intracellularly, ERK and p38 MAPK, PKC, and PKG, but not PKA, are phosphorylated and recruited as enhancing modulators of membrane receptors and ion channels. COX-1/COX-2 is also recruited as enzymes catalyzing arachidonic acids to prostaglandins. (c) Shown is also a tripartite synaptic structure. At the synaptic cleft, in response to mechanically von Frey filament stimuli, EAAs are selectively activating mGluR group I, but without the activation of ionotropic glutamate receptors and mGluR groups II and III. NK1 receptors are not activated. At presynaptic component, TRPV1, VDCC, and NK1 are inactivated. ATP is still activating ATP P2X receptors. Intracellularly, only PKA and PKG, but not PKC, are phosphorylated. COX-1/COX-2 is also involved in this process (Merged from Figs. 4, 5, and 6 of Chen and Lariviere. The nociceptive and anti-nociceptive effects of bee venom injection and therapy: A double-edged sword. Prog Neurobiol 2010; 92:151–183. With permission from Elsevier)

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The spinal mechanisms underlying the bee venom-induced hyperalgesia and/or allodynia (pain hypersensitivity) are much more complex than spontaneous pain due to its different response properties to different stimulus modalities (chemical, thermal, and mechanical) and its spatial location in relation to the primary injury site (primary, secondary, and mirror-image hyperalgesia) (Fig. 9b, c; also see Chen and Lariviere 2010).

Briefly, the iGluRs (NMDA and AMPA/KA) in the dorsal horn are not likely to be involved in the bee venom-induced primary thermal hypersensitivity (Fig. 9b; Chen and Chen 2000; Yan et al. 2009). Instead, GPCRs including mGluR groups I, II, and III and NK1 play important roles in the induction and maintenance of the primary thermal hypersensitivity (Zheng and Chen 2001; Yan et al. 2009). Consequently, intracellular DG-PKC, but not cAMP-PKA, is activated (Li et al. 2000; Li and Chen 2003). Meanwhile, ERK, p38 MAPK, soluble guanylyl cyclase (sGC)-PKG-NOS, and COX-1/COX-2 in the spinal cord are also recruited as important factors in the mediation of the primary thermal hypersensitivity (Yu and Chen 2005; Cao et al. 2007; Cui et al. 2008; Chen 2008; Li et al. 2008).

Through intensive investigations on the spinal mechanisms underlying the bee venom-induced primary mechanical hypersensitivity, it is surprising to note that few pharmacological targets are effectively involved (Fig. 9c; Chen 2007, 2008; Chen and Lariviere 2010). Group I of mGluRs has been demonstrated to be involved in the induction and maintenance of the primary mechanical hypersensitivity, while other membrane receptors including iGluRs (NMDA and AMPA/KA), groups II and III of mGluRs, and NK1 are not likely to be involved (Fig. 9c; Zheng and Chen 2001; Yan et al. 2009). Because inhibition of ATP receptors P2X3/P2X2 and P2Y in the periphery is shown to be effective (Lu et al. 2008), involvement of P2X receptors is highly possible. Intracellular cAMP-PKA and sGC-PKG-NOS, but not DG-PKC, are phosphorylated and responsible for the induction and maintenance of the primary mechanical hypersensitivity (Li et al. 2000; Li and Chen 2003).

The bee venom-induced secondary and mirror-image hypersensitivity share quite similar spinal mechanisms that have been proposed for the bee venom-induced spontaneous nociception (Fig. 9a; Chen 2007, 2008; Chen and Lariviere 2010). However, the mirror-image hypersensitivity has a unique mechanism involving the descending pain facilitatory pathway from the rostral medial medulla to the spinal dorsal horn (see right upper inset of Fig. 9a; Chen et al. 2000, 2001, 2003; You and Arendt-Nielsen 2005).

In summary, the bee venom-induced multiple symptomatic “phenotypes” of nociception and hyperalgesia have separate spinal mechanisms. The spinal mechanisms of these nociception and hyperalgesia are highly associated with the stimulus modalities applied in the periphery.

Cortical Mechanisms

So far, the cortical mechanisms underlying the bee venom-induced pain and hyperalgesia/allodynia (pain hypersensitivity) have not been fully elucidated. However, activations of the S1 area (Guo et al. 2007; Liu et al. 2007; Chang et al. 2008) and the ACC (Ren et al. 2008; Gong et al. 2010; Lu et al. 2014) and the hippocampal formation (Guo et al. 2007; Zhao et al. 2009; Liu et al. 2011, 2012; Lyu et al. 2013) have been consistently induced by s.c. bee venom injection. Moreover, the primary motor cortex has also been shown to be activated by s.c. bee venom (Wang et al. 2011). The activations of the cerebral cortices are likely to be dependent upon the ascending nociceptive input from the peripheral bee venom injection site, because some cortical activations have been demonstrated to be completely eliminated by local peripheral co-injection of bee venom and bupivacaine that can block VGSCs mediating generation and conduction of action potentials (nociceptive impulses) (Zhao et al. 2009; Lu et al. 2014). However, the functional roles of these cortical areas in the production and maintenance of the bee venom-induced pain are not clear due to limited available data from either human subjects or animals. In animals, it has been shown that bilateral complete ACC chemical lesions caused by kainic acid microinjection significantly decrease the bee venom-induced paw lifting and licking behavior (self-caring and favoring behaviors) but produce no influence upon spinally processed spontaneous paw-flinching reflex (involuntary withdrawal reflex) (Ren et al. 2008). Moreover, the bilateral ACC lesions can also relieve the bee venom-evoked primary thermal or mechanical hypersensitivity compared with the sham control group (Ren et al. 2008). These results implicate that the bee venom-induced self-caring and favoring anxiety-like behaviors are mediated in part by the activation of the ACC. The ACC is also likely to be involved in maintaining the bee venom-induced primary thermal or mechanical hypersensitivity through descending facilitatory effects. Furthermore, the bee venom-induced self-caring and favoring anxiety-like behaviors can also be relieved by systemic administration of rapamycin that is shown to inhibit the mTOR-S6K signaling phosphorylation and the LTP-enhancing form of metaplasticity in the hippocampal formation (Lyu et al. 2013; for details also see section “The Hippocampal Formation”).

Based upon the existing human neuroimaging and animal studies associated with the functions of the S1 area, the ACC, and the hippocampal formation (McMahon and Koltzenburg 2006; Chen et al. 2013; Li et al. 2014), it is proposed that the activation of the S1 area is responsible for the spontaneous pain sensation, while the ACC and the hippocampal formation are responsible for the emotional responses and cognitive evaluation in response to s.c. bee venom injection. However, when pain becomes persistent following s.c. bee venom injection, animals would become annoyed and anxious due to LTP-enhancing metaplasticity in the hippocampal formation, and this may reflect the adverse effects of honeybee sting in human beings (Liu and Chen 2014; unpublished data from Chen’s Lab).

Conclusion and Future Directions

Subcutaneous bee venom injection in both humans and animals has been used as a surrogate to study the underlying mechanisms of (honey) bee sting-induced pain and inflammation. Following s.c. bee venom injection, an immediate, sustained period of spontaneous pain and hyperalgesia/allodynia can be induced. Melittin, a major component of bee venom, acts predominantly as an algogen (pain-producing substance) on the primary nociceptor cells to evoke membrane depolarization and firing via the activation of TRPV1 receptors by PLA2-LOX/COX metabolites and activations of P2X3 receptors by ATP or TRPC receptors by GPCRs-PLC-IP3 pathway, leading to peripheral sensitization. The melittin-induced persistent pain signals are conducted to the spinal dorsal horn via mediation of VGSC (Nav1.9), leading to the release of EAAs and SP that activates postsynaptic glutamate and NK1 receptors of spinal WDR neurons, resulting in long-term synaptic plasticity or central sensitization. The melittin-induced peripheral and spinal sensitization of pain-signaling neurons is responsible for the production of spinally processed spontaneous nociceptive reflex and pain hypersensitivity to thermal and mechanical stimuli. Meanwhile, the melittin-induced long-term activation and sensitization of pain-signaling neurons result in synaptic plasticity and metaplasticity of the cerebral cortices (S1 area, ACC, and hippocampus), leading to pain sensation and pain-related emotional responses such as anxiety-like behaviors. During the processes associated with the bee venom-induced plasticity at the levels of the primary nociceptor cells, the spinal dorsal horn, and the cerebral cortical neurons, many extracellular and intracellular molecular events are detected by multiple experimental approaches and are thought to be involved in the bee venom-induced pain and hyperalgesia/allodynia.

The bee venom injection model is one of the most carefully studied animal models of acute inflammatory pain. The use of the bee venom model can be helpful for understanding of the underlying neural mechanisms of naturally occurring pain and hyperalgesia in humans. It can also be used as a tool for screening of novel analgesics through evaluations of both behaviors and neural plasticity and metaplasticity at different levels of the pain pathways. Unraveling of the underlying molecular and cellular mechanisms of the bee venom-induced anxiety-like behaviors at the cortical level would be challenging and of particular importance for understanding of the transition from persistent pain to its comorbidity with emotional disorders and cognitive deficits (see Liu and Chen 2014).