Phospholipase Cb 3 Mediates the Scratching Response Activated by the Histamine H1 Receptor on C-Fiber Nociceptive Neurons

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provided by Elsevier - Publisher Connector Neuron 52, 691–703, November 22, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.neuron.2006.09.036 Phospholipase Cb 3 Mediates the Scratching Response Activated by the Histamine H1 Receptor on C-Fiber Nociceptive Neurons

Sang-Kyou Han,1 Valeria Mancino,1 mucosal transitional surfaces, or certain mucosal sur- and Melvin I. Simon1,* faces such as the roof of the mouth. Itching is reliably 1 Division of Biology, 147-75 induced by a variety of chemical stimuli, the best exam- California Institute of Technology ple of which is histamine acting on histamine receptors. Pasadena, California 91125 Histamine or other possible pruritic mediators are mainly released from cutaneous mast cells, which are located close to sensory fibers in the skin (Botchkarev Summary et al., 1997; Hagforsen et al., 2000). Histamine can excite a subpopulation of polymodal C-fiber nociceptors Phospholipase Cb (PLCb) isozymes represent a family (Handwerker et al., 1991). A subset of itch-selective C- of molecules that link G protein-coupled receptors fibers has been identified that is insensitive to mechan- (GPCRs) to an intracellular signaling network. Here, ical and heat stimuli, but responds to histamine with very we investigated the function of PLCb isozymes in sen- slow conduction velocities (Schmelz et al., 1997). How- sory neurons by using mutant mice deficient for ever, the precise cellular and molecular mechanisms specific PLCb family members. Expression analysis that transduce the histamine response in sensory fibers indicated that PLCb3, one of the four isoforms, is pre- are still obscure. Molecular analysis has so far identified dominantly expressed in a subpopulation of C-fiber four subtypes of histamine receptors, H1–H4 (Bachert, nociceptors. A subset of these neurons expressed 2002; Haaksma et al., 1990; Oda et al., 2000; Zhu et al., the histamine H1 receptor. Ca2+ imaging studies re- 2001). Pharmacological experiments have suggested vealed that PLCb3 specifically mediates histamine- that histamine-induced itching involves the H1, and pos- induced calcium responses through the histamine H1 sibly the H3 or H4, receptors (Bell et al., 2004; Hager- receptor in cultured sensory neurons. In line with mark et al., 1979). In guinea pig sensory neurons, hista- this, we found that PLCb32/2 mice showed significant mine H1 receptors appear to be expressed in a subset of defects in scratching behavior induced by histamine; isolectin B4+ (IB4+) neurons (Kashiba et al., 1999). There histamine-trifluoromethyl-toluidine(HTMT),aselective is also evidence that histamine H3 receptors are located H1 agonist; and compound 48/80, a mast cell activator. on peripheral endings of peptidergic nerve fibers in the These results demonstrate that PLCb3 is required to rat (Ohkubo et al., 1995). mediate ‘‘itch’’ sensation in response to histamine act- Numerous G protein-coupled receptors (GPCRs) are ing on the histamine H1 receptor in C-fiber nociceptive expressed in nociceptive sensory neurons. Phospholi- neurons. pase Cb (PLCb) is one of the key players in the signaling cascade that links GPCRs to an intracellular signaling Introduction network. Four major G protein-coupled PLCb isoforms have been characterized: PLCb1, PLCb2, PLCb3, and Sensory neurons innervating the skin detect a variety of PLCb4. GPCR-mediated hydrolysis of phosphatidylino- stimuli, including touch, pain, pressure, temperature, sitol 4,5-biphosphate (PIP2) by PLCb generates two and chemicals. They report the presence of these stimuli primary intracellular messengers, diacylglycerol (DAG) to the spinal cord, and subsequently to the brain. Noci- and inositol 1,4,5-triphosphate (IP3), which mediate the ceptors represent a subpopulation of sensory neurons activation of protein kinase C (PKC) and intracellular 2+ that are activated by noxious stimuli. They can be Ca release, respectively (Exton, 1996). Moreover, sev- broadly divided into two classes: one group has small- eral physiological and functional studies have shown diameter cell bodies and slowly conducting, unmyelin- that PLCb signaling is engaged in a diverse range of ated axons (known as C-fibers), whereas the other has sensory functions by specifically or selectively coupling medium-diameter cell bodies and faster conducting, to GPCRs. For example, PLCb4 is involved in mediating lightly myelinated axons (known as Ad fibers). In addi- inflammatory pain in the ventral posterolateral thalamic tion, C-fiber nociceptors can be subdivided on the basis nucleus (VPL) by coupling to metabotropic glutamate of histological markers or requirements for neurotropic receptor type 1 (mGluR1) (Miyata et al., 2003). Previously, factors (Molliver et al., 1997; Nagy and Hunt, 1982; PLCb3-deficient mice were shown to have enhanced Snider and McMahon, 1998). Although C-fiber nocicep- morphine responsiveness (Xie et al., 1999). PLCb2 sig- tive neurons are mainly involved in pain perception, it naling is also required to mediate bitter, sweet, and has become evident that a specific subset of C-fibers umami taste signal transduction (Zhang et al., 2003). is specialized to report the sensation of ‘‘itch’’ (Magerl However, despite indications of an important role for et al., 1990; Rees and Laidlaw, 1999; Schmelz et al., PLC-mediated pathways in sensory signal transduction, 1997). There is also evidence that A-fibers contribute there is only indirect evidence supporting their specific to the itch sensation (Graham et al., 1951; Handwerker functions in primary sensory neurons (Xie et al., 1999). et al., 1987; Magerl, 1991). In order to understand how this protein family with Itch is defined as a sensation that evokes scratching multiple isoforms plays a role in sensory neurons, (1) behavior. It can be elicited only in the skin, skin-to- we determined the cell type-specific distribution and localization of PLCb isoforms in sensory neurons. (2) We then identified histamine as a PLCb3-dependent agonist *Correspondence: [email protected] by screening agonists using the dorsal root ganglia Neuron 692

(DRG) neurons derived from mutant mice and their wild- (28/284) of neurons expressed both isoforms. On the type littermates. (3) Finally, histamine receptor agonists other hand, PLCb3 or PLCb4 mRNA were not found in were intradermally (i.d.) injected into the rostral part of PLCb32/2 TG or PLCb42/2 DRG (Figures 2G–2I and the skin on the back of the mouse, and the itch response 2J–2L), confirming the specificity of the probes. The was assessed by quantifying subsequent scratching in PLCb1 probe gave insufficiently strong signals to be wild-type and mutant mice. Our data demonstrate that used in such double-labeling experiments. PLCb3 is required to mediate the itch signal generated via histamine H1 receptor activation on peripheral C- PLCb3 Expression Is Restricted to C-Fibers, whereas fiber nociceptive neurons. PLCb4 Is Mainly Expressed in A-Fibers Since DRG contains diverse subpopulations of primary Results sensory neurons, we next determined whether PLCb3 and PLCb4 are expressed in more restricted subsets Distribution of Gaq Family Members and PLCb of these neurons. Nociceptive sensory neurons can be Isoforms in Adult Mouse DRG and Spinal Cord divided into three main populations based on conduc- All phospholipase Cb family members (b1–b4) can be tion velocity (nonpeptidergic C-fiber, peptidergic C- activated by interaction with heterotrimeric G protein fiber, and Ad fiber nociceptors). Nonpeptidergic C-fiber a q(Gaq) family members (Gaq, Ga11, Ga14, and Ga15) nociceptors can be identified by binding of Griffonia and are thus functionally linked to a given set of GPCRs. simplicifolia isolectin B4 on the cell surface. In the In order to understand how these protein families with mouse, combined fluorescent labeling for isolectin B4 multiple isoforms are organized, the expression of with in situ hybridization with the PLCb3 probe indicated Gaq/PLCb mRNAs was comparatively examined in adult that 83% (386/465) of the cells expressing PLCb3 coin- mouse DRG and spinal cord by in situ hybridization cided with 85% (386/455) of IB4+ neurons (Figures 3A– using digoxygenin-labeled riboprobes. This analysis re- 3C). Consistent with this result, double in situ analysis vealed that three Gaq members (Gaq, Ga11, and Ga14) indicated that PLCb3 is coexpressed in 85% (495/582) (Figures 1A–1D) and three PLCbs(b1, b3, and b4) (Fig- of the neurons expressing the purinergic receptor ures 1I–1L) are expressed in DRG neurons. In spinal P2X3, which is also primarily expressed in IB4+ neurons cord, two Gaq members (Gaq and Ga11) and, as previ- (Figures 3D–3F). Furthermore, 70% (343/490) of PLCb3+ ously reported (Miyata et al., 2003), two PLCb isoforms neurons coincided with 100% (343/343) of the neurons (b1 and b4), are expressed (Figures 1E–1H and 1M– expressing the MrgD (Mrgprd) receptor (Figures 3G–3I), 1P). Gaq and Ga11 are expressed in every neuron of which is known to be exclusively expressed in IB4+ neu- the DRG and spinal cord as shown (Figures 1A–1B and rons whose peripheral fibers innervate the epidermis in 1E–1F). Interestingly, Ga14 is highly expressed in a sub- the skin (Zylka et al., 2003, 2005). Since PLCb3 is ex- set of DRG neurons (Figure 1C) and is also expressed in pressed in some non-IB4+ neurons, additional double- a few motor neurons in the ventral horn of the spinal cord labeling experiments with PLCb3 versus Calcitonin (Figure 1G). Ga15 expression was virtually absent in Gene-Related Peptide (CGRP) were carried out. We both DRG and spinal cord (Figures 1D and 1H). Similarly, found that 28% (113/401) of PLCb3-expressing neurons three PLCb isoforms, PLCb1, b3, and b4, are expressed coincided with 34% (113/326) of CGRP+ neurons (Fig- at different levels in subpopulations of the DRG neurons ures 3J–3L). DRG neurons can be classified into C-fiber (Figures 1I–1L). PLCb2 mRNA was not detected (Fig- and A-fiber based on the degree of myelination. These ure 1J). Amongst the three PLCbs expressed in DRG two subgroups can be distinguished by NF-200 immu- neurons, PLCb3 showed the highest expression (Fig- noreactivity, which is considered a marker of myelinated ure 1K), whereas no PLCb3-positive signals were de- fibers (Lawson and Waddell, 1991). Double immunolab- tected in the spinal cord (Figure 1O). PLCb4 mRNA is eling using anti-PLCb3 and anti-NF-200 showed that also expressed in a subset of DRG neurons, but at PLCb3 does not overlap with NF-200+ neurons (less much lower levels compared with PLCb3(Figure 1L). than 2%) (Figures 3M–3O). Therefore, these expression However, PLCb4 expression was broadly detected in data demonstrate that PLCb3 is primarily expressed the spinal cord (Figure 1P). Faint signals of PLCb1 in IB4+, MrgD+, and P2X3+ C-fiber nociceptors in the mRNA were detected in limited regions in both DRG mouse. and spinal cord (Figures 1I and 1M). In contrast to PLCb3, PLCb4 was not expressed in IB4+ neurons (Figures 4A–4C), but rather in a subset of PLCb3 and PLCb4 Are Expressed in Largely CGRP+ neurons (Figures 4D–4F). Notably, most PLCb4 Nonoverlapping Subsets of Sensory Neurons was expressed in NF-200+ neurons (Figures 4G–4I). The expression of PLCb1, PLCb3, and PLCb4 in subsets Therefore, PLCb4 is primarily expressed in myelinated of sensory neurons raises the question of whether these A-fibers, whereas expression of PLCb3 is restricted to molecules are expressed in distinct or overlapping sub- neurons with histological markers indicating unmyelin- sets. We applied double-labeled in situ hybridization ated, C-fiber nociceptors. with combined probes of PLCb3 and PLCb4 using di- goxigenin and fluorescein riboprobes. These double in PLCb3 Protein Is Localized Both Peripherally situ probes were hybridized in parallel on DRG or tri- and Centrally geminal ganglia (TG) sections derived from wild-type, In situ analysis does not provide information as to the lo- PLCb32/2, or PLCb42/2 mice. These experiments indi- calization of the PLCb3 protein. Moreover, it is important cated that PLCb3 and PLCb4 are primarily expressed to know whether PLCb3 can act peripherally and that in nonoverlapping subsets of both adult DRG (Figures PLCb3+ neurons project to a specific lamina layer known 2A–2C) and TG neurons (Figures 2D–2F). Only 10% to relay the information to the central nervous system. The Role of PLCb3 in Nociceptive Sensory Neurons 693

Figure 1. Distribution of Gaq Family Mem- bers and PLCb Isoforms in Adult Mouse DRG and Spinal Cord In situ hybridization with the indicated probes was performed on sections through the tho- racic spinal cord. The upper panels show expression of Gaq and PLCb family members. They are high- (203 objective) magniﬁcation views, and the corresponding bottom panels represent low- (53 objective) magniﬁcation views. (A–H) In situ hybridization showing expression of Gaq family members (Gaq, Ga11, Ga14, and Ga15) in adult mouse DRG and spinal cord. (I–P) In situ hybridization showing expression of four PLCb isoform mRNAs in DRG and spinal cord. Note that PLCb3 expression is predominant in a subpopulation of DRG neurons, but is not detected in the spinal cord.

To determine the presence of the PLCb3 protein in pe- be transported anterogradely in peripheral axons from ripheral nerve fibers, we initially carried out immunos- the DRG soma. taining of PLCb3 in the mouse skin, sciatic nerve fibers, To ascertain whether PLCb3 is expressed in central and DRG culture. However, PLCb3 immunoreactivity nerve fibers projecting to the spinal cord, immunohisto- was difficult to detect in peripheral nerve fibers, al- chemical staining was performed in perfused mouse though anti-PLCb3 antibody specifically detected spinal cord sections. Since PLCb3 is not expressed in PLCb3 protein in the soma area of the DRG neurons spinal neurons, as shown in Figure 1 O, PLCb3 immuno- (see Figure S1 in the Supplemental Data). To convince reactivity in the dorsal horn reflects the projection of ourselves that PLCb3 is distributed to the peripheral the central nerve fiber from DRG neurons (Figure 5B). nerve fibers, we examined PLCb3 transport in the rat, To define the laminar localization of PLCb3, we stained which provides a better experimental model for this pur- perfused spinal cord sections with Alexa Fluor-conju- pose. Following double ligation of the sciatic nerve for gated lectin IB4 and PKCg antibody (Figure 5B). The for-

18 hr, a western blot was performed on nerve segments mer is a marker for lamina II; the latter, for laminae IIi and (w3 mm) cut on various sites, including the proximal and lamina III interneurons. Double immunostaining experi- distal sides of the double ligature. The PLCb3 rabbit ments revealed that PLCb3 immunoreactivity was local- polyclonal antibody specifically identified PLCb3 protein ized in the superficial region of the dorsal horn that at the expected molecular weight (w130 kDa) in DRG includes the IB4+ laminae, and that it was dorsal to the lysates derived from wild-type mice (Figure 5A: lane 1), PKCg+ lamina (Figure 5B). This result is consistent with but did not detect PLCb3 protein in the DRG lysates of the finding that PLCb3 is mainly expressed in IB4+ neu- the PLCb3-deficient mice (Figure 5A: lane 2). The bands rons and expressed in some peptidergic C-fiber DRG corresponding to PLCb3 were present in each of the rat neurons. We further sought to determine if PLCb3is sciatic nerve segments with much weaker intensity present in the synaptic compartment of the mouse spinal compared with that of the DRG lysates (Figure 5A: lane cord. Spinal cord synaptosomes were prepared using 3–6), explaining why PLCb3 immunostaining did work the methods described by Guo et al. (2001). Western well in DRG soma but wasn’t observed in peripheral blot analysis of PLCb3 clearly shows that PLCb3 colocal- nerve fibers. The blot was reprobed using an anti b-actin izes with the synaptic vesicle marker synaptophysin, a 38 monoclonal antibody to confirm that equal amounts of kDa synaptic vesicle protein, indicating that PLCb3is PLCb3 were present in each lane (Figure 5A, bottom presynaptically localized at the central terminals of the panel). As shown, PLCb3 significantly accumulated DRG neurons (Figure 5C). only on the proximal side of the first ligature (Figure 5A: lane 4) compared with the other nerve segments, includ- PLCb3 Mediates Histamine-Induced Calcium ing the distal side of the second ligature. This eliminates Responses by Specifically Coupling to the Histamine the possibility that the presence of PLCb3 resulted from H1 Receptor on Sensory Neurons sources extrinsic to the nerve cell and demonstrates Based on the finding that expression patterns of PLCb3 that PLCb3 present in the adult rat sciatic nerve must and PLCb4 are mutually exclusive in sensory neurons, Neuron 694

Figure 2. Nonoverlapping Expression of PLCb3 and PLCb4 in Adult Mouse Sensory Neurons Shown are 1 mm confocal microscope optical sections of adult DRG or trigeminal ganglia (TG) derived from wild-type and PLCb3or PLCb4 knockout mice and processed in parallel for double-label in situ hybridization with combined probes for PLCb3 and PLCb4. PLCb4-positive cells were counted as cells having a fluorescence level greater than the background fluorescence resulting from iden- Figure 3. PLCb3 Is Predominantly Expressed in C-Fiber Nociceptors tically processed sections derived from PLCb42/2 mice. By alternat- ing the light source or adjusting the contrast of confocal micros- One micrometer confocal microscope optical sections of adult copy, we observed that the PLCb4-positive signals were restricted mouse DRG are shown. They were processed for double-label in to the cell soma. In all panels, PLCb3 is in red, and PLCb4 in green. situ hybridization (A–L) and double immunofluorescence (M–O) (A–F) Double-label in situ with PLCb3 and PLCb4 probes in DRG and with the indicated markers. In all panels, the PLCb3 probe is in TG sections. PLCb3 staining mostly does not overlap with PLCb4 red, and the counter markers are in green. For double labeling with staining. (G–I) Double-label in situ on trigeminal sections derived Griffonia simplicifolia IB4 lectin, sections were stained with Alexa Fluor 488-conjugated IB4 lectin following in situ hybridization with from PLCb3-deficient mice. (J–L) Double-label in situ on DRG sec- + tions derived from PLCb4-deficient mice. These controls illustrate the PLCb3 cRNA probe. (A–I) PLCb3 is expressed in most IB4 and P2X3+ neurons and all MrgD+ neurons. (J–L) PLCb3 is partially the signal specificity of each probe. Scale, bottom right, is the + same for all panels (80 mm) unless indicated otherwise. expressed in CGRP neurons. (M–O) Anti-rabbit PLCb3 antibody costained with mouse anti-NF-200 antibody, indicating that PLCb3 expression is not found in NF-200+ neurons. Scale, bottom right we tested the possibility that they may be specifically (80 mm), is the same for all panels. coupled to receptors in these different subsets of cells 2+ and thus may mediate distinctive sensory signaling. In (10 mM) resulted in robust [Ca ]i release in the majority order to test this hypothesis, we prepared acutely disso- of cells. Compared with wild-type, PLCb3orPLCb4- ciated DRG neurons derived from both adult mice that deficient cells were not different with respect to the per- were deleted for PLCb3 or PLCb4 and from their wild- centage of responsive cells (Table 1) or the mean of peak type littermates and examined the effects of various ag- amplitude (mean F340/F380 ratio) when treated with 2+ onists on [Ca ]i as a screen for the presence of PLCb3 these two ligands (Figure 6A). In wild-type cells, on aver- or PLCb4-specific ligand effects. Adenosine 50-triphos- age, 15% to w20% of the cells in the population re- phate (ATP), Uridine 50-triphosphate (UTP), capsaicin, sponded to BK (1 mM), histamine (100 mM), or UTP (100 histamine, and bradykinin (BK) have been shown to elicit mM). These three agonists are thought to induce in- 2+ 2+ [Ca ]i increases in dissociated DRG neurons: ATP is creases in [Ca ]i through PLC activation. Nevertheless, a nonselective agonist that acts on the P2X and P2Y re- the percentage of neurons responding to BK or UTP was ceptors; capsaicin is a specific agonist for TRPV1; BK is not different between cells derived from wild-type or a specific agonist for B1R and B2R; histamine is a spe- mutant mice (Table 1), and no significant differences in cific agonist for histamine receptors; and UTP is a selec- the mean F340/F380 ratio were observed between wild- tive agonist for P2Y2/P2Y4 receptors (Caterina et al., type and mutant cells upon application of BK or UTP 1997; Nicolson et al., 2002; Thayer et al., 1988; Ueno (Kruskal-Wallis ANOVA analysis: p > 0.1) (Figure 6A). In et al., 1999). Application of ATP (100 mM) or capsaicin contrast, when histamine was applied, the percentage The Role of PLCb3 in Nociceptive Sensory Neurons 695

Figure 4. PLCb4 Is Mainly Expressed in A-Fiber Sensory Neurons (A–C) One micrometer confocal microscope optical sections of adult mouse DRG hybridized in situ with the indicated PLCb4 probe (green), stained with Alexa Fluor 594-conjugated lectin IB4 (red). (D–F) Double-label in situ with PLCb4 (green) and CGRP probes (red). (G–I) Double labeling of PLCb4 probes with NF-200 (red), a marker for myelinated A-fibers in DRG. Anti-NF-200 antibody was applied to DRG section after in situ hybridization with the PLCb4 antisense probe. Note that PLCb4 is expressed in IB42 and NF-200+ neurons, in contrast to the expression pattern of PLCb3. Scale, bottom right (80 mm), is the same for all panels. of responding neurons showing calcium responses was significantly decreased only in PLCb3-deficient cells (Table 1). The mean F340/F380 ratio induced by histamine appeared to be at basal level (1.01 6 0.01, n = 40) in PLCb32/2 cells compared with wild-type (1.43 6 Figure 5. PLCb3 Protein Is Distributed Peripherally and Centrally 2/2 0.05, n = 23) and PLCb4 cells (1.60 6 0.13, n = 15). (A) A representative western blot showing that PLCb3 protein accu- To further characterize the histamine-sensitive neu- mulates only on the anterograde side (lane 4) of a proximal nerve rons, we examined the responses to 100 mM histamine segment. The sciatic nerves of a three-week-old rat were ligated in followed by a mixture of 1 mM BK and 100 mM UTP. All his- two places for 18 hr, after which the ligatures were removed and 3 2/2 mm segments homogenized. The tissue extracts were then sub- tamine-sensitive neurons in wild-type and in PLCb4 jected to a 4%–12% gradient SDS-PAGE, the protein was trans- cells also responded to a mixture of BK and UTP (Fig- ferred onto nitrocellulose, and a western blot was performed using ure 6B). A mixture of BK and UTP elicited increases in anti-PLCb3 antibody (1:800). After that, the blot was reprobed by 2+ [Ca ]i in w30% of wild-type and mutant cells, which is b-actin antibody (1:20,000), which was used as a control to compare comparable to the combined percentage of neurons levels of protein loading in each lane. To show PLCb3 antibody spec- responding when BK or UTP were applied individually. ificity, the same amounts of DRG lysate derived from wild-type and PLCb3-deficient mice were loaded in lane 1 and 2, respectively, and These results indicate that histamine-sensitive neurons subjected to immunoblot analysis. Each lane corresponds to the consist of two different subsets: BK-sensitive or UTP- DRG segments illustrated. sensitive cells. (B) Confocal (203) images of dorsal horn of perfused spinal cord Next, we examined the activation and desensitization showing that PLCb3 immunoreactivity was detected dorsal to + kinetics in response to histamine. Application of 100 mM PKCg interneurons located in lamina IIi and III. Scale, bottom right histamine to wild-type neurons elicited a rapid rise in (80 mm), is the same for all panels. 2+ (C) Representative western blot showing PLCb3 expression in [Ca ]i, with peak responses achieved within 10–20 s. synaptosomes prepared from spinal cord of wild-type and PLCb3- The successive application of histamine led to a reduced deficient mice. Solubilized DRG lysates (0.8 mg/ml) and synapto- response compared with the peak height elicited by the somal proteins (0.1 mg/ml) were separated by SDS-PAGE, trans- first application (Figure 6C), indicating desensitization. ferred to a nitrocellulose sheet, and immunoreacted with PLCb3 Since histamine-induced calcium responses may antibody or anti-synaptophysin antibody (1:2000). Note that PLCb3 result from the activation of four isotypes, H1, H2, H3, colocalizes with synaptophysin in the spinal synaptosome preparation. Similar results were obtained in at least three separate and H4, we tried to determine which receptor mediates experiments. 2+ the histamine-induced increase in [Ca ]i. We used histamine receptor antagonists relatively specific for each receptor isotype: mepyramine (H1 receptor antagonist), Neuron 696

Table 1. Percentage of DRG Neurons Showing Calcium Responses to Each Agonist

ATP BK Capsaicin Histamine UTP WT 73.1% (79/108) 19.9% (32/161) 71.4% (70/198) 15.9% (71/447) 14.6% (18/123) PLCb32/2 70.1% (43/61) 17.7% (44/249) 76.8% (43/56) 2.5% (5/200) 16.9% (23/136) PLCb42/2 73.0% (27/37) 23.2% (59/254) 72.3% (47/65) 18.1% (36/199) 22.5% (25/111) Ratio (F340/F380) baselines of all individual neurons measured for 40 s prior to agonist application. Responses were defined by the peak value in the change of the ratio that was greater than 20% of the baseline value for each neuron, when each drug was applied; the sample size represents the fraction of responding neurons per total number of cells tested. cimetidine (H2 receptor antagonist), and thioperamide mice suffer from locomotor ataxia of their hindlimbs, (H3 receptor antagonist). As previously reported (Nicol- as previously reported (Kano et al., 1998). son et al., 2002), only the H1 receptor antagonist, mepyr- Figures 7A–7D show the time course of scratching amine (10 mM), abolished the histamine-induced calcium bouts for 30 min at 5 min intervals after the injection of responses in wild-type neurons. BK and UTP respon- each compound. Typically, the first scratching was ob- siveness, however, were not changed in the presence served within 2 to w3 min following injection, and of mepyramine (Figure 6D). peaked within 10 min in wild-type mice. Comparison of Single in situ analysis revealed that the H1 receptor is wild-type and PLCb32/2 mice revealed significant differ- expressed in 14.5% (58/399) of the sensory neurons, ences in scratching initiation time (Ti), peak, and total and all of the H1-positive cells were observed to be bouts of scratching against compounds tested (Figure 7). amongst the small-diameter neurons (Figure 6E). More- When histamine was applied, PLCb32/2 mice showed over, double in situ analysis confirmed that 90% (26/29) delays in the first scratching response (WT: Ti = 143 6 of the H1 receptor-expressing neurons coexpressed 10 s, versus KO: Ti = 227 6 19 s; p = 0.002), with a signif- with PLCb3(Figure 6F). Western blotting detected the icant reduction in mean total bouts of scratching over H1 receptor in mouse whole DRG, and the level of ex- a period of 30 min (WT: 152 6 17, versus KO: 46 6 9; pression of histamine H1 receptor was not changed in p = 0.001), but with the same peak at 5 to w10 min as mutant mice (Figure S2B), indicating that absence of in wild-type mice. When the selective histamine H1 re- PLCb3orPLCb4 did not effect the expression levels of ceptor agonist HTMT was applied, the effect of PLCb3 the H1 receptor in the DRG. deficiency was the most pronounced. The total mean in- In order to identify the types of neurons that show cidence of scratching in wild-type mice was 117 6 22, histamine responses, acutely dissociated mouse DRG whereas in PLCb3-deficient mice it was only 7 6 4 bouts neurons were labeled with IB4 before recording. In our (Mann-Whitney Test; p < 0.001), which is almost compa- preparation, IB4-labeled cells on the average repre- rable to the response elicited by saline injection sented approximately 50% of the wild-type or mutant (Figure 7E). The total mean number of scratching bouts DRG neurons in culture. PLCb3 immunoreactive cells elicited by saline over the 30 min was 5 6 2 bouts represented 70% (319/485) of the total neuron culture, (mean 6 SEM., n = 6). In wild-type mice, injection of and most IB4+ neurons were PLCb3+, which is consistent clobenpropit, an H3 antagonist and H4 agonist, evoked with the results of the in situ analysis. In wild-type cells, the highest total incidence of scratching (362 6 47) and histamine evoked calcium responses in 23.4% (56/239) also showed the fastest response (Ti =836 8 s) among of IB4+ and 7.2% (15/208) of the IB42 neurons. In PLCb3- the compounds tested. The most significant difference deficient IB4+ cells, the responses were almost com- was observed in scratching at peak time and first pletely abolished (only 1% [1/103] cells responded), scratching response (KO: Ti = 272 6 65 s; p = 0.016), while while in PLCb3-deficient IB42 neurons, 4.1% (4/97) the incidences of scratching after 10 min were not signif- showed calcium responses. Taken together, these re- icantly different between wild-type and PLCb32/2 mice sults demonstrate that PLCb3 is specifically coupled to (Figure 7C). Among the tested drugs, the response to the histamine H1 receptor and mediates histamine- clobenpropit shown in PLCb32/2 mice was the least induced calcium responses in a sensory neuron popula- affected (Figure 7E). Compared with wild-type mice, tion that consists almost entirely of IB4+ neurons. only a 45% decrease in the total number of scratching bouts (WT: 362 6 47, versus KO: 196 6 52; p = 0.033) was observed, suggesting that a PLCb3-independent PLCb3 Is Required for Scratching Induced mechanism exists in clobenpropit-induced scratching by Histamine Receptor Agonists responses. The identification of histamine as a PLCb3-specific ago- Itch in humans and rodents can be triggered by hista- nist led to speculation that PLCb3 might mediate itching mine release from skin mast cells upon mast cell de- in vivo. Pruritic compounds, including histamine, were granulation. Among mast cell degranulators, compound injected i.d. into the rostral part of the back skin of 48/80 has been reported as a potent mast cell histamine wild-type, PLCb3-, or PLCb4-deficient mice, and itch releaser in both in vitro and in vivo studies (Kuraishi et al., was assessed by quantifying the scratching. Mice used 1995; Nakayama et al., 2002). Thus, we next examined in the scratching behavioral assay were littermates or the potential role of PLCb3 in compound 48/80-induced age-matched, but not sex matched because in prelimi- scratching responses. When compound 48/80 was nary experiments we couldn’t find any significant sex injected, significant scratching was observed in wild- differences in scratching reactions to the tested drugs type mice, which peaked within 10 min and extended (Figure S3). In the case of PLCb42/2 mice, we were not for 30 min, whereas in PLCb32/2 mice, the peak was able to determine the scratching responses since these significantly delayed with significant reduction at all The Role of PLCb3 in Nociceptive Sensory Neurons 697

Figure 6. Functional and Expression Evidences for Coupling Specificity of PLCb3 with Histamine H1 Receptor in Adult Mouse DRG Neurons (A) Agonist-induced calcium responses in dissociated DRG neurons derived from adult wild-type, PLCb32/2, and PLCb42/2 mice. Treatments with 1 mM bradykinin (BK), 10 mM capsaicin (Cap), 100 mM histamine (HTM), ATP, and UTP were performed. The mean F340/F380 ratio was used 2+ as a measure of peak amplitudes for the indicated agonist-induced increase of [Ca ]i. The magnitudes of histamine-evoked calcium responses were significantly decreased in PLCb32/2 cells compared with wild-type and PLCb42/2 cells (Kruskal-Wallis; for histamine, *p < 0.001, n = 40), but for other agonists, there was no significant difference in increases of mean F340/F380 ratios between any two of these three genotypes (Kruskal-Wallis; for ATP, p = 0.57, n = 23; for BK, p = 0.15, n = 20; for capsaicin, p = 0.38, n = 40; and for UTP, p = 0.10, n = 12). Each point is the mean 6 SEM of at least three independent experiments. (B) In wild-type or PLCb42/2 cells, all neurons responding to histamine also responded to a second challenge with a mixture of 1 mM BK and 100 mM 2+ UTP. InPLCb3-deficient cells, the 100 mM histamine-evoked increase in [Ca ]i issignificantlyimpairedwithoutaffecting the responsesto BK plusUTP. 2+ 2+ (C) Desensitization of Ca mobilization. Histamine (100 mM) was applied to wild-type cells twice for 40 s in a 3.3 min interval. The magnitude of [Ca ]i release induced by the second histamine application was smaller than the first response. (D) Pretreatment of 10 mM mepyramine completely abolished the response to histamine, but left the response to BK and UTP unaltered in the same 2+ neurons. Each line shows [Ca ]i measured in the soma of a representative single neuron. (E) Bright-field photomicrograph showing in situ hybridization with mouse histamine H1 receptor antisense probes in adult mouse DRG section. Note that the H1 receptor mRNA-positive neurons were detected in 14.5% (58/399) of the DRG neurons and were exclusively small-diameter neurons. (F) Shown is a representative confocal image of adult mouse DRG processed for double-label in situ hybridization with PLCb3(green)andH1receptor (red) probes. Arrows indicate double positive cells. Neuron 698

Figure 7. Scratching Behavior Induced by Histamine, HTMT, Compound 48/80, and Clobenpropit in PLCb3+/+ and PLCb32/2 Mice Mice were given an intradermal injection of saline (n = 6 for wild-type), histamine (10 mmol; n = 8 for wild-type; n = 5 for PLCb32/2), HTMT (0.1 mmol; n = 7 for wild-type; n = 14 for PLCb32/2), clobenpropit (0.1 mmol; n = 8 for wild-type; n = 7 for PLCb32/2), and compound 48/80 (30 mg; n = 8 for wild-type; n = 7 for PLCb32/2). Scratch bouts directed to injection site were recorded during the first 30 min after injection. (A–D) Time course of scratching after an intradermal injection of each drug for wild-type (filled rectangle) versus PLCb32/2 (filled circles). The number of bouts of scratching is indicated at each 5 min interval during a 30 min test period. Each value represents the mean 6 SEM. Asterisks indicate the time point at which wild-type mice were significantly different from PLCb32/2 mice (*p < 0.05; two-way ANOVA with Bonferroni correction). (E) The total number of bouts of scratching for wild- type and mutant mice was determined after following the mice for 30 min. As a control, the mice were given an intradermal injection of the same volume of saline. Each value represents the mean 6 SEM of the total count. Compared to wild-type mice, PLCb32/2 mice show significant differences in scratching behavior to the tested drugs (two-way ANOVA: p = 0.001 for histamine, p < 0.001 for HTMT; Mann-Whitney test, p = 0.002 for compound 48/80, p = 0.033 for clobenpropit).

times (Figure 7D). The mean of total bouts of scratching Functional Implications and Distribution of PLCb was decreased by 77% compared with wild-type mice Isozymes in Adult Mouse DRG Neurons (WT: 198 6 33, versus KO: 47 6 13; p = 0.002) as shown PLCb activity is regulated by a number of GPCRs linked in Figure 7E. These results suggest that PLCb3 mediates to the Gaq subfamily (Gaq, Ga11, Ga14, and Ga15) of the itch sensation that results from mast cell activation heterotrimeric GTP binding proteins (G proteins). In in vivo. addition, two of the PLCb isoforms (PLCb2 and PLCb3) can be activated by the Gbg subunit dissociated from Discussion Gai/o subfamily in a pertussis toxin-sensitive manner (Smrcka et al., 1991). PLCb isoforms differ in their sensi- PLCb isozymes represent one of the key families of tivity to stimulation by Gaq subunits or Gbg. These signaling molecules that link GPCRs to intracellular net- biochemical properties may underscore potential PLCb works. Although a large family of GPCRs is expressed in isoform-specific differences in signaling. primary sensory neurons, it is not clear what roles the In adult mouse DRG, PLCb3 is a major isoform with isozymes of a given PLC family play in sensory neurons lower levels of PLCb4 and much lower levels of PLCb1. in vivo, i.e., to what extent they determine the specificity This is analogous to the expression pattern of PLCb of individual signaling pathways. This study demon- isoforms observed in Purkinje cells in the cerebellum strated a high degree of cellular and functional specificity (Kano et al., 1998), but it is essentially different from ex- for different PLC isoforms. We found that PLCb3 is pre- pression patterns in other brain regions, in which PLCb1 dominantly expressed in C-fiber nociceptive peripheral and PLCb4 represent the major neuronal PLCb isoforms. nerves, that it is specifically coupled to the histamine In contrast to PLCb1 and PLCb4, PLCb3 is not H1 receptor, and that it mediates histamine-induced expressed in central nervous tissues (e.g., spinal cord, scratching (itch). thalamus, cortex, etc.) implicated in somatosensory The Role of PLCb3 in Nociceptive Sensory Neurons 699

circuits (Kano et al., 1998; Rubio et al., 1999; Watanabe found that histamine-induced calcium responses were et al., 1998). significantly impaired in PLCb3-deficient, but not PLCb4- Our data revealed that PLCb3 is highly expressed in deficient, DRG neurons in culture. Moreover, histamine- a subpopulation of the C-fiber nociceptors that includes sensitive cells appear to respond to either BK or UTP. In 85% of the IB4+ neurons and 34% of the peptidergic neu- the majority of tissues, BK and UTP have been reported rons, whereas PLCb4 is mainly expressed in A-fibers. to induce PLC activation through the Gaq/Ga11 G protein Differential expression strongly suggested that PLCb3 isoform. In the DRG, the BK receptors, the B1R and B2R and PLCb4 could play different roles in signaling. In sup- receptors, have been identified in nociceptive neurons port of this argument, PLCb3+ and PLCb4+ neurons in the (Ma et al., 2000; Seabrook et al., 1997), and UTP can excite DRG contain an array of different molecular compo- cutaneous C-fiber nociceptors through the G protein- nents. Some of these differentially expressed compo- mediated receptor P2Y2 (Molliver et al., 2002; Stucky nents include the MrgD receptor, Ga14, and TRPC3 et al., 2004). However, in neither PLCb3- nor PLCb4- (data not shown). Currently, we are investigating whether deficient cultured DRG neurons was the calcium respon- these molecules are directly coupled to PLCb3 and what siveness elicited by BK or UTP affected. Only histamine roles they may play in sensory function. responses required the presence of PLCb3, suggesting C-fiber nociceptors send their central projections to that PLCb3 specifically couples to the histamine H1 the superficial regions of the dorsal horn in the spinal receptor in cultured DRG neurons. These results raise cord. Peptidergic populations target lamina I of the the question of how signaling specificity is achieved in superficial dorsal horn, while the IB4+ population is response to different agonists in the same cell. It is likely focused on lamina II (Hunt and Mantyh, 2001). These that there are multiple intracellular pathways to induce 2+ layers contain projection neurons that transmit various agonist-specific increases in [Ca ]i. However, the nature kinds of sensory information, such as pain or itch, to of these pathways still remains unclear. One possibility is related brain regions, including the midbrain, thalamus, that PLCb1 may be responsible for calcium responses eli- and limbic regions (Braz et al., 2005; Wallengren, 2005). cited by BK or UTP since PLCb1mRNAisweaklydetected As revealed by double immunostaining, PLCb3 is local- in small-diameter neurons. In addition, we cannot rule out ized to lamina I and II dorsal to the PKCg+ layer in the the possibility that other novel PLC isoforms such as PLC3 dorsal horn. Furthermore, we showed that PLCb3is or PLCh may play a role. These two PLC isoforms were colocalized with synaptophysin at the synaptic vesicle also reported to be, in part, coupled to GPCRs through in central terminals, but not in the spinal neurons. There- the release of Gbg (Wing et al., 2003; Zhou et al., 2005). fore, our results indicate that PLCb3 is located in both In either event we are driven to postulate the formation peripheral nerve fibers and central presynaptic regions of isoform-specific circuitry in specifically differentiated and could play a number of roles in signal transduction sensory neurons. and transmission. Neural Mechanisms of Itch The Signaling Specificity of PLCb Isoforms With regard to the peripheral mechanisms of the itch in the DRG Neurons sensation, two distinct models have been proposed Receptor-mediated, PLC isoform-specific or -selective (McMahon and Koltzenburg, 1992). The specificity the- signaling is often found in vivo. Several genetic studies ory proposes that a set of primary afferents exclusively have shown that PLCb1 is involved in signaling in the responds to pruritic stimuli. In contrast, the selectivity cerebral cortex and hippocampus by coupling predomi- hypothesis assumes that some afferents respond to nantly to the muscarinic acetylcholine receptor (Kim both pruritic and painful stimuli, whereas others respond et al., 1997), whereas PLCb4 works through the metabo- only to painful stimuli, and that the two classes have tropic glutamate receptor in the cerebellum or thalamus different central connectivity. Our calcium mobilization (Kano et al., 1998; Miyata et al., 2001, 2003), illustrating assays revealed that at least four distinct subpopula- that a specific combination of G proteins active in signal- tions of chemoreceptor combinations could exist in ing from a given receptor can be involved in the selective DRG neurons in culture: BK+/Histamine2,BK+/Hista- activation of PLCb isozymes. Such specificities can be mine+, UTP+/Histamine2 and UTP+/Histamine+. Since generated in several different ways. For example, BK is mainly algogenic and not pruritogenic, the selectiv- a conserved PDZ binding motif at the C terminus of ity model is the more likely explanation for the sensation a PLCb isoform can interact with a given scaffolding pro- of itch. Unlike the case with BK, it is not clear yet whether tein and form a physically and functionally distinct sig- UTP can act as an algogenic agent or pruritic agent in the naling unit (reviewed in Ranganathan and Ross, 1997). periphery. Thus, it is possible that two different subsets In addition, the signaling specificity of PLCb isoforms of histamine-sensitive neurons may have distinct func- can be achieved through their unique distribution or tions in detecting the itch sensation. In support of this, differential level of expression in a given tissue or cell BK-induced firing patterns (Kasai et al., 1998) appear to (Kano et al., 1998; Kim et al., 1997; Miyata et al., 2003). be different from the pattern evoked by UTP, which Our data also suggest the possibility of PLC isoform- showed a delayed (>1 min) and prolonged burst of action receptor-specific signaling. As previously reported potentials (>2 min), respectively, in nociceptive neurons (Nicolson et al., 2002), we found that the application of (Molliver et al., 2002; Schmelz et al., 2003). Such electro- histamine evoked a calcium response in a fraction physiological properties coincide with the observation (w16%) of DRG neurons in culture by direct activation that the itch sensation starts with a delay of 30 to w60 of the H1 histamine receptor. Most histamine-sensitive s following histamine application by iontophoresis, cells appear to be IB4+ neurons in which the PLCb3 reaching a maximum at 2 to w3 min (Schmelz, 2001). isoform is predominantly expressed. Importantly, we This suggests that the UTP+/histamine+ neuron subset Neuron 700

might be more relevant for sensing histamine-evoked via effects on cytokine production. However, this seems pure itch, while BK+/histamine+ neurons may be respon- highly improbable since the time course of histamine-in- sible for grading or regulating the intensity of the itch duced cytokine production in keratinocytes has been sensation. shown to occur in the order of an hour (Matsubara et al., 2005), while the scratching response is rapid and PLCb3 Is a Major Mediator for Sensation occurs on a time scale of minutes. Moreover, PLCb3is of Histamine-Induced Itch In Vivo not likely to be implicated in the neural circuit that Since PLCb3 is highly expressed in C-fiber nociceptive extends beyond the sensory cells since PLCb3 is not sensory neurons, we initially reasoned that PLCb3 might expressed in the spinal neurons or in higher brain areas have a role in nociception. However, mice lacking PLCb3 responsible for somatosensation. Indeed, the scratch- showed normal behavior in various pain behavior tests, ing events appeared to be localized to the site of test such as the hot-plate test run at different noxious tem- agent application. Additionally, when compounds tested peratures, Von-Frey, formalin, and tail immersion tests were injected intraperitoneally, the resultant behaviors (data not shown), precluding a specific role for PLCb3 such as grooming, mouse activity (bearing and locomo- in transducing acute or inflammatory pain. In addition, tion), or some minor scratching were observed to be the we also observed no difference in thermal preference be- same in wild-type and PLCb32/2 mice. These arguments havior at the innocuous temperatures (data not shown). further support the idea that C-fiber nociceptors co- In the skin, histamine is known to be a major mediator expressing both PLCb3 and the H1 receptor are primar- that is released into dermis following mast cell degranu- ily responsible for mediating the itch sensation at the lation. This event appears to be a primary trigger for the level of the skin. itch sensation, which is mediated by C-fiber nociceptors Beside histamine H1 receptors, H3 or H4 receptors located in the uppermost skin layer (Fjallbrant and Iggo, may be involved in scratching responses since a phar- 1961; Schmelz et al., 1997; Tuckett and Wei, 1987). How- macological study showed that the scratching response ever, the signaling mechanisms underlying histamine- in BalbC mice was induced by application of clobenpro- induced itch in the periphery are unclear. The present pit, which is both an H4 receptor agonist and an H3 re- study showed significantly impaired scratching receptor antagonist (Bell et al., 2004). Our study showed actions in PLCb32/2 mice in response to i.d. injection that clobenpropit evoked an unusually high scratching of histamine. This data appear to be very similar to response in C57BL/6. The mean total number of bouts a scratching defect observed in PLCb32/2 mice when of scratching was three times higher than scratching compound 48/80 was injected, which is consistent with induced by HTMT. This may reflect differences in mouse the fact that histamine is a major mediator released strains, which can markedly influence scratching re- from mast cells. Based on our finding that histamine- sponses to drugs (Inagaki et al., 2001). Although cloben- induced calcium responses were mostly eliminated in propit-induced scratching showed a reduction in PLCb3-deficient DRG neurons in culture, it is likely that PLCb32/2 mice, significant scratching responses still re- the scratching response to exogenous histamine results mained. This suggests that in clobenpropit-mediated from direct action on sensory neurons expressing both scratching there are at least two distinct mechanisms histamine receptors and PLCb3. Furthermore, PLCb3- that mediate the scratching behavior, and that they act deficient mice showed almost complete defects in via PLCb3-dependent and -independent pathways. scratching responses evoked by HTMT, a selective H1 However, further studies are required to determine the receptor agonist, which agrees with the finding that his- molecular and cellular mechanisms underlying cloben- tamine-induced calcium responses in DRG neurons propit-dependent scratching in vivo. were completely eliminated by pretreatment with mepyr- Taken together, the available data are consistent with amine, an H1 antagonist. In addition, previous behavioral the notion that the H1 receptor-PLCb3-mediated path- studies have shown that the H1 receptor antagonist way is a major component of the in vivo itch response mepyramine significantly reduced scratching when ap- to histamine. This work clearly defines the signaling plied following the application of histamine or compound events that generate the stereotypic ‘‘itch-scratch’’ 48/80 (Bell et al., 2004; Hagermark et al., 1979; Sugimoto behavior. et al., 1998). Therefore, we conclude that PLCb3 mediates the itch sensation evoked by histamine released from mast cells via the histamine H1 receptor on a subset Experimental Procedures of peripheral C-fiber nociceptors, which are mainly IB4+ neurons. Animals On the other hand, one study reported that compound PLCb3(Xie et al., 1999) and PLCb4(Kano et al., 1998) knockout mice were backcrossed seven and five generations, respectively, onto 48/80 can induce scratching behavior in mast cell- a C57BL/6 background. Animals were obtained by mating heterozy- deficient mice (Inagaki et al., 2002). However, it is not gous animals. Three-week-old wistar rats were used in sciatic nerve clear if a mast cell-independent pathway contributes ligation experiments. All experiments were conducted according to to scratching behavior under the normal physiological protocols approved by The California Institute of Technology Animal condition. Care and Use Committee. Since both the H1 receptor and PLCb3 are known to be broadly expressed in peripheral tissues or cells, the phenotype in PLCb32/2 mice might conceivably result Preparation and Culture of Mouse DRG Neuron The dorsal root ganglia were isolated from adult mice (1 to w2 month from cells other than the primary sensory neuron. One old) and cultures were prepared based on the method described possibility, for example, is that the H1 receptor in kerati- previously (Chopra et al., 2000). For details, see Supplemental nocytes could be involved in mediating itch responses Experimental Procedures. The Role of PLCb3 in Nociceptive Sensory Neurons 701

In Situ Hybridization and Immunofluorescence Acknowledgments Nonisotopic in situ hybridization on frozen 14 mm thick sections was performed as previously described using cRNA probes (Ma et al., We wish to express our deepest gratitude to Xinzhong Dong for 1996; Perez et al., 1999). For immunofluorescence, DRGs were invaluable assistance in our research. We also thank Mark Zylka, collected from perfused mice and processed. Four to eight confocal David Anderson, and Allan Basbaum for valuable comments; Henry images (w1 mm optical thickness) from adult wild-type mice were Lester and Raad Nashimi for gracious assistance with calcium counted to determine the percentage of PLCb3-positive DRG imaging; Iain Fraser for providing PLCb cDNA constructs; Paul C. neurons that coexpressed a given marker. We also checked by Sternweis for providing PLCb3 antibody; and Hee Ju Kim for great counting the percentage of cells expressing a given marker that technical assistance. This work was supported by NIH grant coexpressed PLCb3. Details are available (see Supplemental NS048499. Experimental Procedures). Received: April 19, 2006 Ca2+ Imaging Revised: August 31, 2006 2+ Measurement of [Ca ]i at the individual cell level was performed as Accepted: September 18, 2006 previously described (Dong et al., 2001; Han et al., 2002). Changes in Published: November 21, 2006 the intracellular Ca2+ levels in acutely dissociated neurons derived from wild-type or mutant adult mouse DRG were monitored using the Ca2+-sensitive dye fura-2. For details of calcium imaging, see References Supplemental Experimental Procedures. Bachert, C. (2002). The role of histamine in allergic disease: re- appraisal of its inflammatory potential. Allergy 57, 287–296. Synaptosome Preparation Subcellular fractionation and isolation of synaptosomes from Bell, J.K., McQueen, D.S., and Rees, J.L. (2004). 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