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P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in... Background: The purinergic P2X3 receptor (P2X3R) expressed in the dorsal root ganglion (DRG) sensory neuron and the P2X7 receptor (P2X7R) expressed in the surrounding satellite glial cell (SGC) are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats. Findings: We immunostained DRGs of immature rats and found that P2X3Rs were expressed only in neurons and P2X7Rs were expressed only in SGCs. Western blot analyses indicated that P2X3R expression decreased while P2X7R expression increased with the age of rats. Electrophysiological studies showed that the number of DRG neurons responding to the stimulation of the P2XR agonist, a,b-meATP, was higher and the amplitudes of a,b- meATP-induced depolarizations were larger in immature DRG neurons. As a result, P2X3R-mediated flinching responses were much more pronounced in immature rats than those found in adult rats. When we reduced P2X7R expression with P2X7R-siRNA in postnatal and adult rats, P2X3R-mediated flinch responses were greatly enhanced in both rat populations. Conclusions: These results show that the P2X7R expression increases as rats age. In addition, P2X7Rs in SGCs exert inhibitory control on the P2X3R expression and function in sensory neurons of immature rats, just as observed in adult rats. Regulation of P2X7R expression is likely an effective way to control P2X3R activity and manage pain relief in infants. Keywords: Purinergic signaling, P2X3 receptors, P2X7 receptors, Dorsal root ganglion, P2X7R-P2X3R inhibitory con- trol, Neuron-glia interactions, Nociception, Abnormal pain, Postnatal immature rats, Development Background [1,2]. A neuronal soma with its surrounding SGCs is fre- The DRG neuron is the first (primary) neuron in the quently enclosed by a connective tissue sheath and somatosensory pathway relaying nociceptive (pain), itch forms a distinct morphological unit [3]. There is no evi- dence that classical synaptic contacts exist between neu- and other sensory information from the skin or internal organs to the brain. The cell bodies (somata) of DRG ronal somata in DRGs [2]. We and others have shown neurons are densely packed in a DRG. Each neuronal that neuronal somata communicate bidirectionally with soma is tightly wrapped by a layer of SGCs, which are their surrounding SGCs in DRGs [4,5] and trigeminal often coupled with one another through gap junctions ganglia [6]. The communication modulates the activity of somata thus affecting the afferent inputs into the spinal cord [4]. * Correspondence: lmhuang@utmb.edu Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1069, USA © 2012 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chen et al. Molecular Pain 2012, 8:9 Page 2 of 8 http://www.molecularpain.com/content/8/1/9 Purinergic ionotropic P2X3Rs and P2X7Rs are ligand- Western blots gated ion channels closely associated with the transmis- For Western Blot experiments, DRGs were lysed in 100 sion of nociceptive signals in DRGs [5,7-10]. P2X3Rs are μl of RIPA buffer (1% NP-40, 0.5% Na dexycholate, 0.1% expressed mainly in small- and medium-sized adult SDS, PMSF 10 μl/ml and aprotinin 30 μl/ml). The cell DRG neurons [5,11] and P2X7Rs are expressed only in lysates were centrifuged at 10,000× g for 12 min at 4°C. The concentration of protein in homogenate was deter- SGCs [5,12,13]. We have shown that the P2X3R and mined using a BCA reagent (Pierce). A 50 μgoftotal P2X7R are the two major purinergic receptors involved protein sample from each group was loaded onto a in the bidirectional communication between neurons SDS-PAGE, transferred to a PVDF membrane, incubated and SGCs [4,5,14]. P2X3Rs have been shown to partici- pate in peripheral pain signaling [15-18] and P2X7Rs with a blocking buffer (1 × TBS with 5% w/v fat free dry are involved in the maturation and release of cytokines milk) for 2 hrs and then with rabbit anti-P2X3 (1:1000; [19]. In addition, P2X7R knock-out mice fail to develop Alomone), or anti-P2X7 (1:1000, Alomone) for another hyperalgesia or allodynic pain under inflammation or 2 hours at room temperature. The PVDF membrane nerve injury conditions [20]. The evidence has led to the was then washed 5 times with TBST (1 × TBS and 1% suggestion that P2X7R activation is related to proinflam- Tween 20), incubated with anti-rabbit peroxidase-conju- matory actions in the nociceptive system [21]. It was gated secondary antibody (1:1000; Santa Cruz) for 1 hr therefore unexpected when we found that P2X7R activa- at room temperature and washed 5 times with TBST. tion in SGCs of adult DRGs tonically inhibits the The immunoreactive proteins were detected by expression of P2X3Rs in neurons [5]. This inhibitory enhanced chemiluminescence (ECL kit; Amersham Bios- P2X7R-P2X3R control attenuates the activity of P2X3Rs, ciences) and visualized by exposing the PVDF mem- thus reducing signaling by P2X3R-expressing neurons, brane onto an x-ray film. Loading controls were most of which are nociceptors, under normal and determined by stripping the blots with a buffer contain- injured conditions [5,14]. ing 100 mM 2-mercaptoethanol, 62.5 mM Tris-HCl, In addition to signaling in adults, purinergic receptors and 2% SDS for 20 min and re-probing with mouse and ATP are found to actively participate in embryonic anti-Actin (1:1000, Chemicon) for 2 hr. After washing, the membrane was incubated with anti-mouse peroxi- development in mice and rats [22-25]. P2X3Rs can be dase-conjugated secondary antibody (1:1000, Chemica) seen in day 9.5 mouse embryos (E9.5) [26] and 12.5 day for 1 h. The intensity of protein bands was determined rat embryos [22]. From E14 to postnatal day 1 (P1), using the NIH Image 1.63 software. P2X3Rs are present in nearly all DRG neurons. The expression of P2X3Rs begins to decline after birth and is expressed in ~73% of P7 and ~45% of P14 to adult Immunohistochemistry mouse DRG neurons. At the developmental stage (E14- To immunostain DRG cells, rats were deeply anesthe- P7), most DRG neurons are small in size [23]. After P7, tized with pentobarbital (80 mg/kg, i.p.) and fixed with some neurons start to mature into large cells. From P14 4% paraformaldehyde plus 0.2% picric acid. The L4 and to adults, P2X3Rs are found mainly in small diameter L5 DRGs were removed, postfixed for 1 hr and put in a DRG neurons which account for ~50% of total adult 30% sucrose solution overnight. Tissue was then mouse DRG neurons [23]. P2X3Rs are found in ~ 40% embedded in an OCT compound and cut into 10 μm of adult rat DRG neurons [11]. Although P2X3R expres- transverse DRG sections on a cryostat and washed with sion has been found to decrease with age, it is not normal goat serum (10%) and Triton X (0.2%). Primary known if P2X7R expression increases with age and if antibodies used included rabbit anti-P2X7R (Alomone, the regulation between P2X7 and P2X3 exists in devel- 1:200) or guinea pig anti-P2X3R (Chemicon, 1:10,000). oping DRGs. Secondary antibodies included FITC or Texas Red anti- rabbit, or anti-guinea pig IgG (Vector Lab, 1:200). To Methods compare the fluorescence intensity of labeled cells in Animals control and test groups, tissue samples from different Neonatal (3-16 day old) and adult (~250 g) Sprague Daw- groups were processed simultaneously and analyzed ley rats were used in this study. For in vitro experiments, using an identical fluorescence intensity threshold. To rats were anesthetized with pentobarbital (50 mg/kg, i.p.) avoid the possibility of double-counting, labeled cells on and L4 and L5 DRGs were then removed. All experimen- every fifth DRG section were analyzed. tal procedures were approved by the Institutional Animal Electrophysiology Care and Use Committee at University of Texas medical To record the electrical activity of DRG neurons, one L4 Branch and were in accordance with the guidelines of the or L5 DRG with an attached sciatic nerve was isolated National Institutes of Health and of the International from a rat. The preparation was superfused with the Association for the Study of Pain (IASP). Chen et al. Molecular Pain 2012, 8:9 Page 3 of 8 http://www.molecularpain.com/content/8/1/9 artificial cerebral spinal fluid (ACSF), which contained Behavioral experiments (in mM) 115 NaCl, 5.6 KCl, 1 NaH PO , 2.0 CaCl ,1 Nocifensive, i.e., flinching, behavioral studies were con- 2 4 2 MgCl ,11glucose,and 25 NaHCO ,atroomtempera- ducted on P15-16 and adult rats, which were treated 2 3 ture. Sharp electrodes filled with 3 M potassium acet- with either con-siRNA or P2X7R-siRNA eight to nine ate with a resistance ranging from 50 MΩ to 90 MΩ days before. The siRNA was applied intrathecally using a modified direct transcutaneous intrathecal method were used for recordings. DRG neurons were stimu- [31]. Rats were placed under individual plexiglass domes lated either by square pulses (0.1-1 ms) applied to the and acclimated for 1 hr before experiments. Following sciatic nerve through a suction electrode or by 40 ms an injection of a,b-meATP (1 mM, 50 μl), rats were current steps applied to the soma through an intracel- lular recording electrode. Only data obtained from immediately put back under the dome. The flinching cells with a resting potential ≤ - 40 mV and overshoot- behavior was assessed by determining paw withdrawal ing spikes were accepted for analyses. In adult DRG, duration (PW duration), i.e., the total time that the cells were classified as Ab,Aδ or C cells by axonal hindpaw was lifted in the air in a 1 min time bin conduction velocity (CV) with the criteria of CV > 8.5 [32,33]. m/sasAb cells, CV = 1-8.5 m/s as Aδ cells, and CV < 1 m/s as C cells. In cases where CVs could not be Data analyses established, the action potential duration (APD), i.e., Data were expressed as means ± SE or as percentages. duration measured at the half peak AP amplitude, and Student’s t, Fisher Exact or Mann-Whitney tests were the existence of inflection on the falling phase of used to access the significance of changes. Comparisons action potential (AP) were used as the criteria to sepa- between multiple means were done with one-way analy- rate cell groups [27]. Cells with an APD > 3.8 ms and sis of variance (ANOVA) followed by the Holm-Sidak AP inflection were considered as C cells; cells with an post hoc test. A P<0.05 was considered significant. APD ≤ 3.8 ms and AP inflection were categorized as Aδ cells; cells with an APD < 3.8 ms and no AP inflec- Results tion were considered Ab cells. The same APD and AP A high percentage of DRG neurons express P2X3Rs in inflection criteria were used to categorize cell types of immature rats DRGs in early postnatal ages, during which most of We first determined if P2X3Rs are expressed only in DRG neurons and P2X7Rs are expressed only in SGCs the axons were unmyelinated [28]. Only C and Aδ in immature rats as observed in adult cells [5]. DRG DRG neurons were included in our analyses. Chemi- neurons and SGCs were differentiated by their morpho- cals, e.g., a,b-meATP (100 μM) and TNP-ATP (1 μM) were bath applied at a flow rate of ~ 4 ml/min. A logical characteristics. The somata of DRG neurons had membrane potential change of ≥ 2 mV, which was lar- a rounded appearance with diameter ≥ 10 μm [34]. Each ger than 2 times the standard deviation of the baseline soma had a prominent nucleus with relative clear level, was set as a positive response, similar to the cri- nucleoplasm and a low nucleus/cytoplasm ratio [35]. On terion used by Stebbing et al. [29]. the other hand, SGCs were elongated (width ≤ 2 μm) flattened cells that enveloped each neuronal soma siRNAs [35,36]. The SGC had a flattened nucleus which con- Small interfering RNA directed against P2X7 mRNA tained dense chromatins and a high nucleus/cytoplasm (P2X7R-siRNA) or a non-targeting control siRNA (con- ratio. In P6 rats, P2X3R immunostain was found only in siRNA) (20 μM, 10.5 μl) (Thermo Scientific) was used DRG neurons, not in SGCs (Figure 1A). On the other in our experiments. The sense sequence of con-siRNA hand, P2X7Rs were found only in SGCs (Figure 1A). An was 5’-UAGCGACUAAACACAUCAA-3’.The P2X7R- obvious difference in P2X3R distribution between imma- siRNA consisted of four pooled 21-necleotide duplexes. ture and adult DRGs is that a high percentage of DRG The sequences were as follows: (1) Sense GAAACUGC- neurons expressed P2X3Rs in immature rats. In P6 CUCCCGUCUCAUU; Antisense 5’-P. UGAGACGGGA DRGs, 74.83 ± 2.03% (n = 3) of DRG neurons expressed GGCAGUUUCUU; (2) Sense GGAUCCAGAGCGUG P2X3Rs whereas only 43.45 ± 5.03% (n = 3) neurons AAUUAUU; Antisense 5’-P UAAUUCACGCUCUGGA expressed P2X3Rs in adult DRGs neurons (Figure 1B). UCCUU; (3) Sense CCAAGCCGACGUUAAAGUAUU; Antisense 5’-P UACUUUAACGUCGGCUUGGUU; (4) The activity of P2X3Rs in DRG neurons decreases with rat age Sense GAUACGCCAAGUACUAUAAUU; Antisense 5’- We then determined the function of P2X3Rs in DRG P UUAUAGUACUUGGCGUAUCUU. SiRNA-polyethy- neurons by measuring their responses to the P2XR ago- leneimine (PEI) complex (0.18 μl100mM PEIper μg nist, a,b-meATP. DRG neurons were categorized in C, RNA) was prepared according to the method described Aδ and Ab cell groups using the criteria described in by Tan et al. [30] before use. Chen et al. Molecular Pain 2012, 8:9 Page 4 of 8 http://www.molecularpain.com/content/8/1/9 Figure 2 High P2X3R-mediated activity in DRG neurons of immature rats.(A) a,b-meATP induced significant depolarizations in DRG neurons of immature rats. In this example, a,b-meATP evoked 8.6 mV depolarization in a P4 DRG neuron. The depolarization was reversibly blocked by the P2X antagonist, TNP- ATP (1 μM). (B) a,b-meATP induced a much reduced, i.e., 2 mV, depolarization in an adult DRG neuron. (C) The percentage of DRG neurons responded to a,b-meATP was high in immature rats and greatly reduced in adult rats (P3-P14 rats: 55.0%, adult rats: 15.4%. Fisher exact test, *P < 0.05). The amplitude of depolarizations induced by a,b-meATP was reduced as rats matured (P3-P14 rats: 7.0 ± 0.8 mV, n = 11; adult rats: 2.0 ± 0 mV, n = 2. Mann-Whitney test, *P < 0.05) Figure 1 P2X3Rs are expressed in a higher percentage of DRG smaller than that obtained in immature DRGs (Figure neurons in immature rats.(A) Immunocytochemical staining of 2C). P2X3Rs and P2X7Rs in DRGs of P6 and adult rats. P2X3R labels (red) were found only in neurons; P2X7R labels (green) were found only in SGCs. (Scale bars: P2X3R, P6 = 40 μm, Adult = 50 μm; P2X7R, P6 P2X7R-P2X3R inhibitory control exists in immature DRGs =25 μm, Adult = 30 μm) (B) Percentage of DRG neurons In a previous study [5], we showed that reducing P2X7R expressing P2X3Rs decreases significantly during postnatal expression or blocking P2X7R activity in SGC cells development (P6 rats: 74.83 ± 2.03%, n = 3; adult rats: 43.45 ± upregulates P2X3R expressions in adult DRG neurons. 5.03%, n = 3. Student t-test, *P < 0.05) This P2X7R-P2X3R inhibitory control is important in maintaining proper functions of P2X3Rs. To determine the METHODS section. Responses to a,b-meATP were if such P2X7R-P2X3R control is also present in imma- studied only in C and Aδ cells. Sharp intracellular ture DRGs and if it contributes to the enhanced P2X3R microelectrodes were used to measure the depolariza- expression in immature rats, we first compared the tion induced by bath applied a,b-meATP (100 μM). P2X3R and P2X7R protein expressions in DRGs isolated Under current clamp conditions, 11 out of 20 (55.0%) from P6, P15 and adult rats. As rat aged, the P2X7R recorded DRG neurons from P3-P14 rats responded to expression increased (P6/Adult: 0.42 ± 0.05, n = 5; P15/ a,b-meATP with ≥ 2 mV depolarization (Figure 2). The adult 0.64 ± 0.06; n = 3). At the same time, the P2X3R average amplitude of a,b-meATP induced depolariza- expression was found to decrease (P6/adult: 1.71 ± 0.28; tions was 7.0 ± 0.8 mV (n = 11). In four immature neu- P15/adult: 1.42 ± 0.11; n = 5) (Figure 3). Thus, P2X7R rons, the effect of the P2X1R and P2X3R antagonist, and P2X3R expressions are negatively correlated, similar TNP-ATP, was also studied. TNP-ATP completely to those observed in adult rats. blocked the a,b-meATP-induced depolarization (Figure To determine the functional consequence of P2X7R- 2A). Only 2 out of 13 (15.4%) neurons tested responded P2X3R expression control in immature rats, nocifensive, to a,b-meATP in adult DRGs (Figure 2B). The average i.e., flinching, behavioral studies were conducted on amplitude of depolarizations induced by a,b-meATP in P15-16 and adult rats, which were treated with either adult DRG neurons (2.0 ± 0.0 mV, n = 2) was much con-siRNA or P2X7R-siRNA. We applied P2X7R-siRNA Chen et al. Molecular Pain 2012, 8:9 Page 5 of 8 http://www.molecularpain.com/content/8/1/9 Immature Con- P2X7- Con- P2X7- siRNA siRNA siRNA siRNA P2X7 P2X3 β-Actin β-Actin P2X7 P2X3 1.2 1.2 0.8 0.6 0.4 0 0 Con-siRNA P2X7-siRNA Con-siRNA P2X7-siRNA Figure 3 P2X3R expression decreases and P2X7R expression increases with rat age. Western blot analyses of P2X3Rs and P2X7Rs were conducted on the DRG protein samples isolated from * Immature Adult P6, P15 and adult rats. P2X3R and P2X7R expressions were expressed in relative intensity with respect to those of adult rats, P2X7-siRNA which was set at 1.0. During postnatal development, the P2X7R Con-siRNA expression increased (P6/Adult: 0.42 ± 0.05; P15/adult 0.64 ± 0.06; n 10 * * = 3-5), while the P2X3R expression decreased (P6/adult: 1.71 ± 0.28; P15/adult: 1.42 ± 0.11; n = 5) (One-way ANOVA, *P < 0.05) 0 5 15 10 20 0 5 15 10 20 Time (min) Time (min) intrathecally to P7 rats to down regulate P2X7R expres- Figure 4 P2X7R-P2X3R inhibitory control exists in immature sion. Eight to nine days later, the effect of P2X7R- rats.(A) P2X7R-siRNA treatment down-regulated P2X7R expression siRNA on P2X3R-mediated flinch behaviors induced by while increased P2X3R expression in P15-16 rats. P2X3R and P2X7R a,b-meATP application to the rat left hindpaw was expressions in P2X7R-siRNA rats were expressed in relative intensity determined. We first determined if P2X7R-siRNA is with respect to those in Con-siRNA rats, which was set at 1.0. (B) effective in knocking down P2X7R expression and if The a,b-meATP induced much more pronounced P2X3R-mediated flinching responses in con-siRNA immature rats than in adult rats. In P2X3Rs are upregulated. Following P2X7R-siRNA treat- both immature and adult rats, a,b-meATP evoked significantly larger ment, P2X7R expression was reduced (P2X7R-siRNA/ flinching responses in P2X7R-siRNA rats than in con-siRNA rats. Con-siRNA = 0.52 ± 0.14, n = 4, Student t-test, P < 0.05), while the P2X3R expression in immature P2X7R- siRNA rats was increased (P2X7R-siRNA/Con-siRNA = 1.32 ± 0.02, n = 3, P < 0.05) (Figure 4A). In presence of from 74.8% in P6 rats to 43.5% in adult rats (Figure 1), con-siRNA, a,b-meATP-induced flinching responses a result similar to those obtained in mice [23]. During were much more pronounced in immature rats than postnatal development, P2X3R switches from being uni- those found in adults. This result is consistent with the versally expressed in DRG neurons to limitedly observations that both P2X3R expression and function expressed in IB4-positive neurons [23]. In adult DRG are elevated in immature rats (Figures 1 and 2). Follow- neurons, P2X3Rs are localized in small and medium- ing P2X7R-siRNA treatment, the a,b-meATP-induced sized DRG neurons that process nociceptive signals flinch behaviors in immature rats were significantly [17,37,38]. To determine if the change in P2X3R expres- enhanced, similar to that observed in adult rats (n = 4- sion corresponds to a change in P2X3R-mediated 7, One-wayANOVA,P<0.05) (Figure4B).Thus,a responses, we studied the depolarizations evoked by the lower expression of P2X7Rs in immature DRGs resulted P2X1R and P2X3R selective agonists, a,b-meATP, in in a higher expression and hence an enhanced function intact DRG neurons. Since P2X1 expression in DRGs is of P2X3Rs. The observation suggests that P2X7R-P2X3R low [38], the a,b-meATP-induced depolarization is inhibitory control plays an important role in regulating likely to be mediated mainly by P2X3R-containing the expression and function of P2X3Rs in immature receptors in DRGs. We chose to determine P2X3R func- rats, as shown in adult rats (Figure 4B) [5]. tion in intact DRG neurons rather than in dissociated DRG neurons because the cell dissociation procedure Discussion appears to alter the percentage of cells responding to Our immunocytochemical study showed that the per- the agonist [29]. We found that 55% of recorded DRG centage of DRG neurons expressing P2X3Rs decreases neuronsofP3-P14ratsresponded to a,b-meATP PW duration (s) Rel. intensity Chen et al. Molecular Pain 2012, 8:9 Page 6 of 8 http://www.molecularpain.com/content/8/1/9 (Figure 2). In contrast, a,b-meATP evoked depolariza- [25]. Different from DRGs, in addition to expressing in tions in only 15.4% of adult DRG neurons, a percentage microglia cells, P2X7Rs are also expressed in brain neu- similar to that obtained by Stebbing et al. [29]. Thus, an rons, e.g., Purkinje cells in the cerebellum [40]. increased number of DRG neurons expressed P2X3Rs As animals mature, the P2X7R expression increases as obtained from immunostain analyses is consistent with P2X3R expression decreases in DRGs (Figure 3). More an increased number of neurons responding to a,b- important, we showed that knocking down P2X7R expression with P2X7R-siRNA significantly increases the meATP. P2X3R-mediated flinch responses in P15-16 rats (Figure We also analyzed the a,b-meATP-induced depolariza- 4). Thus, P2X7Rs negatively control the expression and tions in individual DRG neurons. The slow kinetics of the a,b-meATP-induced depolarizations is likely a result function of P2X3Rs in immature rats just as those of bath application of the agonist [29]. Because of the observed in adult rats [5]. Since P2X7R expression is small percentage of adult DRG neurons responding to low in P6-15 day rats (Figure 3), a reduced P2X7R- a,b-meATP, the kinetic difference in the depolarizing P2X3R inhibitory control is likely a mechanism contri- responses of immature and adult DRG neurons was not buting to the high expression and function of P2X3Rs in studied. We found that the amplitudes of a,b-meATP- DRGs of immature rats. induced depolarizations in individual adult DRG neu- Many challenges exist in pain management in infants. rons is ~3.5 fold smaller than those recorded in imma- The expressions and functions of transmitters and ture neurons (Figure 2). receptors undergo changes during development [41]. We further studied the P2X3R expression using Wes- The descending control pathways from the brainstem to tern analyses and P2X3R functions through behavioral the spinal cord in infants are not completely developed. analyses. In immature rats, the P2X3R protein expres- Sensory inputs are integrated, both spatially and tempo- sion was high (Figure 3) and the a,b-meATP application rally [41]. In addition, verbal communication with to the rat paw evoked much larger flinching behavioral infants is limited. Compared to adults, infants are more responses in immature rats (Figure 4B). An increase in sensitive to repeated noxious stimuli and chronic tissue receptor expression in Western analyses and an increase inflammation [42]. On the other hand, injury to periph- in behavioral responses could result from a larger num- eral nerves in infants gives rise to much less allodynia ber of cells expressing receptors and responding to a,b- and neuropathic pain than those observed in adults meATP respectively. They also can result from an [43,44]. Therefore, the information obtained from stu- dies of pain treatment in adults is not directly applicable increase in the receptor expression and function in indi- to infants. We showed before that, in adult rats, total vidual neurons. Thus, an increase in the amplitude of a, b-meATP evoked depolarizations in individual neurons P2X3R protein expression is upregulated in DRG neu- (Figure 2) is consistent with the results of Western and rons after inflammation [45] and the membrane traffick- behavioral analyses. Combining immunocytochemical, ing of P2X3Rs increases following spared nerve injury electrophysiological and behavioral studies, we conclude [32]. It is of interest to determine if purinergic receptors that P2X3R expression and P2X3R-mediated cellular respond to tissue and nerve injury in immature DRGs in activity are high in immature DRG neurons and are a similar manner as in adult DRGs. Our observations reduced as rats mature. The high level of P2X3R expres- that P2X3R and P2X7R expression are negatively corre- sion in the somata of immature DRG neurons is likely lated (Figure 3) and P2X7R-P2X3R inhibitory control to give rise to an enhanced transport of P2X3R receptor also exists in developing rats (Figure 4) suggest that reg- to their nerve terminals and thus increased a,b-meATP- ulation of P2X7R expression in SGCs can be a useful induced behavioral responses. Nevertheless, the results strategy for controlling P2X3R activity in sensory neu- do not exclude the possibility that developmental rons and relieving pain in infant patients. changes in the spinal circuitry and descending modula- tion could also contribute to the observed changes in Acknowledgements the behavioral responses in immature rats. The work is supported by grants from National Institutes of Health (NS30045 We found that P2X3Rs are expressed only in DRG and DE17813). neurons and P2X7Rs are expressed only in SGCs (Figure Authors’ contributions 1) in P6 rats, similar to those found in adult DRGs [5]. YC designed the study, performed Western blotting, immunocytochemical There are similarities and differences between the distri- and electrophysiological experiments and prepared the manuscript. GL carried out the behavioral experiments. LMH designed and coordinated the bution of P2X3Rs and P2X7Rs in DRGs and those in study and prepared the manuscript. All authors read and approved the final the developing rat brain. Similarities include that manuscript. P2X3Rs are not found in glial cells [39] and P2X3Rs Competing interests mRNA expression decreases while P2X7R expression The authors declare that they have no competing interests. increases and/or persists in the developing rat brain Chen et al. Molecular Pain 2012, 8:9 Page 7 of 8 http://www.molecularpain.com/content/8/1/9 Received: 30 June 2011 Accepted: 7 February 2012 21. McGaraughty S, Chu KL, Namovic MT, Donnelly-Roberts DL, Harris RR, Published: 7 February 2012 Zhang XF, Shieh CC, Wismer CT, Zhu CZ, Gauvin DM, Fabiyi AC, Honore P, Gregg RJ, Kort ME, Nelson DW, Carroll WA, Marsh K, Faltynek CR, Jarvis MF: P2X7-related modulation of pathological nociception in rats. References Neuroscience 2007, 146:1817-1828. 1. Huang TY, Cherkas PS, Rosenthal DW, Hanani M: Dye coupling among 22. 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Pain 2005, 114:386-396. neurobiological approach. Nat Clin Pract Neurol 2009, 5:35-50. Chen et al. Molecular Pain 2012, 8:9 Page 8 of 8 http://www.molecularpain.com/content/8/1/9 45. Xu GY, Huang LY: Peripheral inflammation sensitizes P2X receptor- mediated responses in rat dorsal root ganglion neurons. J Neurosci 2002, 22:93-102. doi:10.1186/1744-8069-8-9 Cite this article as: Chen et al.: P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons. Molecular Pain 2012 8:9. 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P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

Molecular Pain , Volume 8 (1) – Feb 7, 2012

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Springer Journals
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Copyright © 2012 by Chen et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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1744-8069
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1744-8069
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10.1186/1744-8069-8-9
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22314033
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Abstract

Background: The purinergic P2X3 receptor (P2X3R) expressed in the dorsal root ganglion (DRG) sensory neuron and the P2X7 receptor (P2X7R) expressed in the surrounding satellite glial cell (SGC) are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats. Findings: We immunostained DRGs of immature rats and found that P2X3Rs were expressed only in neurons and P2X7Rs were expressed only in SGCs. Western blot analyses indicated that P2X3R expression decreased while P2X7R expression increased with the age of rats. Electrophysiological studies showed that the number of DRG neurons responding to the stimulation of the P2XR agonist, a,b-meATP, was higher and the amplitudes of a,b- meATP-induced depolarizations were larger in immature DRG neurons. As a result, P2X3R-mediated flinching responses were much more pronounced in immature rats than those found in adult rats. When we reduced P2X7R expression with P2X7R-siRNA in postnatal and adult rats, P2X3R-mediated flinch responses were greatly enhanced in both rat populations. Conclusions: These results show that the P2X7R expression increases as rats age. In addition, P2X7Rs in SGCs exert inhibitory control on the P2X3R expression and function in sensory neurons of immature rats, just as observed in adult rats. Regulation of P2X7R expression is likely an effective way to control P2X3R activity and manage pain relief in infants. Keywords: Purinergic signaling, P2X3 receptors, P2X7 receptors, Dorsal root ganglion, P2X7R-P2X3R inhibitory con- trol, Neuron-glia interactions, Nociception, Abnormal pain, Postnatal immature rats, Development Background [1,2]. A neuronal soma with its surrounding SGCs is fre- The DRG neuron is the first (primary) neuron in the quently enclosed by a connective tissue sheath and somatosensory pathway relaying nociceptive (pain), itch forms a distinct morphological unit [3]. There is no evi- dence that classical synaptic contacts exist between neu- and other sensory information from the skin or internal organs to the brain. The cell bodies (somata) of DRG ronal somata in DRGs [2]. We and others have shown neurons are densely packed in a DRG. Each neuronal that neuronal somata communicate bidirectionally with soma is tightly wrapped by a layer of SGCs, which are their surrounding SGCs in DRGs [4,5] and trigeminal often coupled with one another through gap junctions ganglia [6]. The communication modulates the activity of somata thus affecting the afferent inputs into the spinal cord [4]. * Correspondence: lmhuang@utmb.edu Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1069, USA © 2012 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chen et al. Molecular Pain 2012, 8:9 Page 2 of 8 http://www.molecularpain.com/content/8/1/9 Purinergic ionotropic P2X3Rs and P2X7Rs are ligand- Western blots gated ion channels closely associated with the transmis- For Western Blot experiments, DRGs were lysed in 100 sion of nociceptive signals in DRGs [5,7-10]. P2X3Rs are μl of RIPA buffer (1% NP-40, 0.5% Na dexycholate, 0.1% expressed mainly in small- and medium-sized adult SDS, PMSF 10 μl/ml and aprotinin 30 μl/ml). The cell DRG neurons [5,11] and P2X7Rs are expressed only in lysates were centrifuged at 10,000× g for 12 min at 4°C. The concentration of protein in homogenate was deter- SGCs [5,12,13]. We have shown that the P2X3R and mined using a BCA reagent (Pierce). A 50 μgoftotal P2X7R are the two major purinergic receptors involved protein sample from each group was loaded onto a in the bidirectional communication between neurons SDS-PAGE, transferred to a PVDF membrane, incubated and SGCs [4,5,14]. P2X3Rs have been shown to partici- pate in peripheral pain signaling [15-18] and P2X7Rs with a blocking buffer (1 × TBS with 5% w/v fat free dry are involved in the maturation and release of cytokines milk) for 2 hrs and then with rabbit anti-P2X3 (1:1000; [19]. In addition, P2X7R knock-out mice fail to develop Alomone), or anti-P2X7 (1:1000, Alomone) for another hyperalgesia or allodynic pain under inflammation or 2 hours at room temperature. The PVDF membrane nerve injury conditions [20]. The evidence has led to the was then washed 5 times with TBST (1 × TBS and 1% suggestion that P2X7R activation is related to proinflam- Tween 20), incubated with anti-rabbit peroxidase-conju- matory actions in the nociceptive system [21]. It was gated secondary antibody (1:1000; Santa Cruz) for 1 hr therefore unexpected when we found that P2X7R activa- at room temperature and washed 5 times with TBST. tion in SGCs of adult DRGs tonically inhibits the The immunoreactive proteins were detected by expression of P2X3Rs in neurons [5]. This inhibitory enhanced chemiluminescence (ECL kit; Amersham Bios- P2X7R-P2X3R control attenuates the activity of P2X3Rs, ciences) and visualized by exposing the PVDF mem- thus reducing signaling by P2X3R-expressing neurons, brane onto an x-ray film. Loading controls were most of which are nociceptors, under normal and determined by stripping the blots with a buffer contain- injured conditions [5,14]. ing 100 mM 2-mercaptoethanol, 62.5 mM Tris-HCl, In addition to signaling in adults, purinergic receptors and 2% SDS for 20 min and re-probing with mouse and ATP are found to actively participate in embryonic anti-Actin (1:1000, Chemicon) for 2 hr. After washing, the membrane was incubated with anti-mouse peroxi- development in mice and rats [22-25]. P2X3Rs can be dase-conjugated secondary antibody (1:1000, Chemica) seen in day 9.5 mouse embryos (E9.5) [26] and 12.5 day for 1 h. The intensity of protein bands was determined rat embryos [22]. From E14 to postnatal day 1 (P1), using the NIH Image 1.63 software. P2X3Rs are present in nearly all DRG neurons. The expression of P2X3Rs begins to decline after birth and is expressed in ~73% of P7 and ~45% of P14 to adult Immunohistochemistry mouse DRG neurons. At the developmental stage (E14- To immunostain DRG cells, rats were deeply anesthe- P7), most DRG neurons are small in size [23]. After P7, tized with pentobarbital (80 mg/kg, i.p.) and fixed with some neurons start to mature into large cells. From P14 4% paraformaldehyde plus 0.2% picric acid. The L4 and to adults, P2X3Rs are found mainly in small diameter L5 DRGs were removed, postfixed for 1 hr and put in a DRG neurons which account for ~50% of total adult 30% sucrose solution overnight. Tissue was then mouse DRG neurons [23]. P2X3Rs are found in ~ 40% embedded in an OCT compound and cut into 10 μm of adult rat DRG neurons [11]. Although P2X3R expres- transverse DRG sections on a cryostat and washed with sion has been found to decrease with age, it is not normal goat serum (10%) and Triton X (0.2%). Primary known if P2X7R expression increases with age and if antibodies used included rabbit anti-P2X7R (Alomone, the regulation between P2X7 and P2X3 exists in devel- 1:200) or guinea pig anti-P2X3R (Chemicon, 1:10,000). oping DRGs. Secondary antibodies included FITC or Texas Red anti- rabbit, or anti-guinea pig IgG (Vector Lab, 1:200). To Methods compare the fluorescence intensity of labeled cells in Animals control and test groups, tissue samples from different Neonatal (3-16 day old) and adult (~250 g) Sprague Daw- groups were processed simultaneously and analyzed ley rats were used in this study. For in vitro experiments, using an identical fluorescence intensity threshold. To rats were anesthetized with pentobarbital (50 mg/kg, i.p.) avoid the possibility of double-counting, labeled cells on and L4 and L5 DRGs were then removed. All experimen- every fifth DRG section were analyzed. tal procedures were approved by the Institutional Animal Electrophysiology Care and Use Committee at University of Texas medical To record the electrical activity of DRG neurons, one L4 Branch and were in accordance with the guidelines of the or L5 DRG with an attached sciatic nerve was isolated National Institutes of Health and of the International from a rat. The preparation was superfused with the Association for the Study of Pain (IASP). Chen et al. Molecular Pain 2012, 8:9 Page 3 of 8 http://www.molecularpain.com/content/8/1/9 artificial cerebral spinal fluid (ACSF), which contained Behavioral experiments (in mM) 115 NaCl, 5.6 KCl, 1 NaH PO , 2.0 CaCl ,1 Nocifensive, i.e., flinching, behavioral studies were con- 2 4 2 MgCl ,11glucose,and 25 NaHCO ,atroomtempera- ducted on P15-16 and adult rats, which were treated 2 3 ture. Sharp electrodes filled with 3 M potassium acet- with either con-siRNA or P2X7R-siRNA eight to nine ate with a resistance ranging from 50 MΩ to 90 MΩ days before. The siRNA was applied intrathecally using a modified direct transcutaneous intrathecal method were used for recordings. DRG neurons were stimu- [31]. Rats were placed under individual plexiglass domes lated either by square pulses (0.1-1 ms) applied to the and acclimated for 1 hr before experiments. Following sciatic nerve through a suction electrode or by 40 ms an injection of a,b-meATP (1 mM, 50 μl), rats were current steps applied to the soma through an intracel- lular recording electrode. Only data obtained from immediately put back under the dome. The flinching cells with a resting potential ≤ - 40 mV and overshoot- behavior was assessed by determining paw withdrawal ing spikes were accepted for analyses. In adult DRG, duration (PW duration), i.e., the total time that the cells were classified as Ab,Aδ or C cells by axonal hindpaw was lifted in the air in a 1 min time bin conduction velocity (CV) with the criteria of CV > 8.5 [32,33]. m/sasAb cells, CV = 1-8.5 m/s as Aδ cells, and CV < 1 m/s as C cells. In cases where CVs could not be Data analyses established, the action potential duration (APD), i.e., Data were expressed as means ± SE or as percentages. duration measured at the half peak AP amplitude, and Student’s t, Fisher Exact or Mann-Whitney tests were the existence of inflection on the falling phase of used to access the significance of changes. Comparisons action potential (AP) were used as the criteria to sepa- between multiple means were done with one-way analy- rate cell groups [27]. Cells with an APD > 3.8 ms and sis of variance (ANOVA) followed by the Holm-Sidak AP inflection were considered as C cells; cells with an post hoc test. A P<0.05 was considered significant. APD ≤ 3.8 ms and AP inflection were categorized as Aδ cells; cells with an APD < 3.8 ms and no AP inflec- Results tion were considered Ab cells. The same APD and AP A high percentage of DRG neurons express P2X3Rs in inflection criteria were used to categorize cell types of immature rats DRGs in early postnatal ages, during which most of We first determined if P2X3Rs are expressed only in DRG neurons and P2X7Rs are expressed only in SGCs the axons were unmyelinated [28]. Only C and Aδ in immature rats as observed in adult cells [5]. DRG DRG neurons were included in our analyses. Chemi- neurons and SGCs were differentiated by their morpho- cals, e.g., a,b-meATP (100 μM) and TNP-ATP (1 μM) were bath applied at a flow rate of ~ 4 ml/min. A logical characteristics. The somata of DRG neurons had membrane potential change of ≥ 2 mV, which was lar- a rounded appearance with diameter ≥ 10 μm [34]. Each ger than 2 times the standard deviation of the baseline soma had a prominent nucleus with relative clear level, was set as a positive response, similar to the cri- nucleoplasm and a low nucleus/cytoplasm ratio [35]. On terion used by Stebbing et al. [29]. the other hand, SGCs were elongated (width ≤ 2 μm) flattened cells that enveloped each neuronal soma siRNAs [35,36]. The SGC had a flattened nucleus which con- Small interfering RNA directed against P2X7 mRNA tained dense chromatins and a high nucleus/cytoplasm (P2X7R-siRNA) or a non-targeting control siRNA (con- ratio. In P6 rats, P2X3R immunostain was found only in siRNA) (20 μM, 10.5 μl) (Thermo Scientific) was used DRG neurons, not in SGCs (Figure 1A). On the other in our experiments. The sense sequence of con-siRNA hand, P2X7Rs were found only in SGCs (Figure 1A). An was 5’-UAGCGACUAAACACAUCAA-3’.The P2X7R- obvious difference in P2X3R distribution between imma- siRNA consisted of four pooled 21-necleotide duplexes. ture and adult DRGs is that a high percentage of DRG The sequences were as follows: (1) Sense GAAACUGC- neurons expressed P2X3Rs in immature rats. In P6 CUCCCGUCUCAUU; Antisense 5’-P. UGAGACGGGA DRGs, 74.83 ± 2.03% (n = 3) of DRG neurons expressed GGCAGUUUCUU; (2) Sense GGAUCCAGAGCGUG P2X3Rs whereas only 43.45 ± 5.03% (n = 3) neurons AAUUAUU; Antisense 5’-P UAAUUCACGCUCUGGA expressed P2X3Rs in adult DRGs neurons (Figure 1B). UCCUU; (3) Sense CCAAGCCGACGUUAAAGUAUU; Antisense 5’-P UACUUUAACGUCGGCUUGGUU; (4) The activity of P2X3Rs in DRG neurons decreases with rat age Sense GAUACGCCAAGUACUAUAAUU; Antisense 5’- We then determined the function of P2X3Rs in DRG P UUAUAGUACUUGGCGUAUCUU. SiRNA-polyethy- neurons by measuring their responses to the P2XR ago- leneimine (PEI) complex (0.18 μl100mM PEIper μg nist, a,b-meATP. DRG neurons were categorized in C, RNA) was prepared according to the method described Aδ and Ab cell groups using the criteria described in by Tan et al. [30] before use. Chen et al. Molecular Pain 2012, 8:9 Page 4 of 8 http://www.molecularpain.com/content/8/1/9 Figure 2 High P2X3R-mediated activity in DRG neurons of immature rats.(A) a,b-meATP induced significant depolarizations in DRG neurons of immature rats. In this example, a,b-meATP evoked 8.6 mV depolarization in a P4 DRG neuron. The depolarization was reversibly blocked by the P2X antagonist, TNP- ATP (1 μM). (B) a,b-meATP induced a much reduced, i.e., 2 mV, depolarization in an adult DRG neuron. (C) The percentage of DRG neurons responded to a,b-meATP was high in immature rats and greatly reduced in adult rats (P3-P14 rats: 55.0%, adult rats: 15.4%. Fisher exact test, *P < 0.05). The amplitude of depolarizations induced by a,b-meATP was reduced as rats matured (P3-P14 rats: 7.0 ± 0.8 mV, n = 11; adult rats: 2.0 ± 0 mV, n = 2. Mann-Whitney test, *P < 0.05) Figure 1 P2X3Rs are expressed in a higher percentage of DRG smaller than that obtained in immature DRGs (Figure neurons in immature rats.(A) Immunocytochemical staining of 2C). P2X3Rs and P2X7Rs in DRGs of P6 and adult rats. P2X3R labels (red) were found only in neurons; P2X7R labels (green) were found only in SGCs. (Scale bars: P2X3R, P6 = 40 μm, Adult = 50 μm; P2X7R, P6 P2X7R-P2X3R inhibitory control exists in immature DRGs =25 μm, Adult = 30 μm) (B) Percentage of DRG neurons In a previous study [5], we showed that reducing P2X7R expressing P2X3Rs decreases significantly during postnatal expression or blocking P2X7R activity in SGC cells development (P6 rats: 74.83 ± 2.03%, n = 3; adult rats: 43.45 ± upregulates P2X3R expressions in adult DRG neurons. 5.03%, n = 3. Student t-test, *P < 0.05) This P2X7R-P2X3R inhibitory control is important in maintaining proper functions of P2X3Rs. To determine the METHODS section. Responses to a,b-meATP were if such P2X7R-P2X3R control is also present in imma- studied only in C and Aδ cells. Sharp intracellular ture DRGs and if it contributes to the enhanced P2X3R microelectrodes were used to measure the depolariza- expression in immature rats, we first compared the tion induced by bath applied a,b-meATP (100 μM). P2X3R and P2X7R protein expressions in DRGs isolated Under current clamp conditions, 11 out of 20 (55.0%) from P6, P15 and adult rats. As rat aged, the P2X7R recorded DRG neurons from P3-P14 rats responded to expression increased (P6/Adult: 0.42 ± 0.05, n = 5; P15/ a,b-meATP with ≥ 2 mV depolarization (Figure 2). The adult 0.64 ± 0.06; n = 3). At the same time, the P2X3R average amplitude of a,b-meATP induced depolariza- expression was found to decrease (P6/adult: 1.71 ± 0.28; tions was 7.0 ± 0.8 mV (n = 11). In four immature neu- P15/adult: 1.42 ± 0.11; n = 5) (Figure 3). Thus, P2X7R rons, the effect of the P2X1R and P2X3R antagonist, and P2X3R expressions are negatively correlated, similar TNP-ATP, was also studied. TNP-ATP completely to those observed in adult rats. blocked the a,b-meATP-induced depolarization (Figure To determine the functional consequence of P2X7R- 2A). Only 2 out of 13 (15.4%) neurons tested responded P2X3R expression control in immature rats, nocifensive, to a,b-meATP in adult DRGs (Figure 2B). The average i.e., flinching, behavioral studies were conducted on amplitude of depolarizations induced by a,b-meATP in P15-16 and adult rats, which were treated with either adult DRG neurons (2.0 ± 0.0 mV, n = 2) was much con-siRNA or P2X7R-siRNA. We applied P2X7R-siRNA Chen et al. Molecular Pain 2012, 8:9 Page 5 of 8 http://www.molecularpain.com/content/8/1/9 Immature Con- P2X7- Con- P2X7- siRNA siRNA siRNA siRNA P2X7 P2X3 β-Actin β-Actin P2X7 P2X3 1.2 1.2 0.8 0.6 0.4 0 0 Con-siRNA P2X7-siRNA Con-siRNA P2X7-siRNA Figure 3 P2X3R expression decreases and P2X7R expression increases with rat age. Western blot analyses of P2X3Rs and P2X7Rs were conducted on the DRG protein samples isolated from * Immature Adult P6, P15 and adult rats. P2X3R and P2X7R expressions were expressed in relative intensity with respect to those of adult rats, P2X7-siRNA which was set at 1.0. During postnatal development, the P2X7R Con-siRNA expression increased (P6/Adult: 0.42 ± 0.05; P15/adult 0.64 ± 0.06; n 10 * * = 3-5), while the P2X3R expression decreased (P6/adult: 1.71 ± 0.28; P15/adult: 1.42 ± 0.11; n = 5) (One-way ANOVA, *P < 0.05) 0 5 15 10 20 0 5 15 10 20 Time (min) Time (min) intrathecally to P7 rats to down regulate P2X7R expres- Figure 4 P2X7R-P2X3R inhibitory control exists in immature sion. Eight to nine days later, the effect of P2X7R- rats.(A) P2X7R-siRNA treatment down-regulated P2X7R expression siRNA on P2X3R-mediated flinch behaviors induced by while increased P2X3R expression in P15-16 rats. P2X3R and P2X7R a,b-meATP application to the rat left hindpaw was expressions in P2X7R-siRNA rats were expressed in relative intensity determined. We first determined if P2X7R-siRNA is with respect to those in Con-siRNA rats, which was set at 1.0. (B) effective in knocking down P2X7R expression and if The a,b-meATP induced much more pronounced P2X3R-mediated flinching responses in con-siRNA immature rats than in adult rats. In P2X3Rs are upregulated. Following P2X7R-siRNA treat- both immature and adult rats, a,b-meATP evoked significantly larger ment, P2X7R expression was reduced (P2X7R-siRNA/ flinching responses in P2X7R-siRNA rats than in con-siRNA rats. Con-siRNA = 0.52 ± 0.14, n = 4, Student t-test, P < 0.05), while the P2X3R expression in immature P2X7R- siRNA rats was increased (P2X7R-siRNA/Con-siRNA = 1.32 ± 0.02, n = 3, P < 0.05) (Figure 4A). In presence of from 74.8% in P6 rats to 43.5% in adult rats (Figure 1), con-siRNA, a,b-meATP-induced flinching responses a result similar to those obtained in mice [23]. During were much more pronounced in immature rats than postnatal development, P2X3R switches from being uni- those found in adults. This result is consistent with the versally expressed in DRG neurons to limitedly observations that both P2X3R expression and function expressed in IB4-positive neurons [23]. In adult DRG are elevated in immature rats (Figures 1 and 2). Follow- neurons, P2X3Rs are localized in small and medium- ing P2X7R-siRNA treatment, the a,b-meATP-induced sized DRG neurons that process nociceptive signals flinch behaviors in immature rats were significantly [17,37,38]. To determine if the change in P2X3R expres- enhanced, similar to that observed in adult rats (n = 4- sion corresponds to a change in P2X3R-mediated 7, One-wayANOVA,P<0.05) (Figure4B).Thus,a responses, we studied the depolarizations evoked by the lower expression of P2X7Rs in immature DRGs resulted P2X1R and P2X3R selective agonists, a,b-meATP, in in a higher expression and hence an enhanced function intact DRG neurons. Since P2X1 expression in DRGs is of P2X3Rs. The observation suggests that P2X7R-P2X3R low [38], the a,b-meATP-induced depolarization is inhibitory control plays an important role in regulating likely to be mediated mainly by P2X3R-containing the expression and function of P2X3Rs in immature receptors in DRGs. We chose to determine P2X3R func- rats, as shown in adult rats (Figure 4B) [5]. tion in intact DRG neurons rather than in dissociated DRG neurons because the cell dissociation procedure Discussion appears to alter the percentage of cells responding to Our immunocytochemical study showed that the per- the agonist [29]. We found that 55% of recorded DRG centage of DRG neurons expressing P2X3Rs decreases neuronsofP3-P14ratsresponded to a,b-meATP PW duration (s) Rel. intensity Chen et al. Molecular Pain 2012, 8:9 Page 6 of 8 http://www.molecularpain.com/content/8/1/9 (Figure 2). In contrast, a,b-meATP evoked depolariza- [25]. Different from DRGs, in addition to expressing in tions in only 15.4% of adult DRG neurons, a percentage microglia cells, P2X7Rs are also expressed in brain neu- similar to that obtained by Stebbing et al. [29]. Thus, an rons, e.g., Purkinje cells in the cerebellum [40]. increased number of DRG neurons expressed P2X3Rs As animals mature, the P2X7R expression increases as obtained from immunostain analyses is consistent with P2X3R expression decreases in DRGs (Figure 3). More an increased number of neurons responding to a,b- important, we showed that knocking down P2X7R expression with P2X7R-siRNA significantly increases the meATP. P2X3R-mediated flinch responses in P15-16 rats (Figure We also analyzed the a,b-meATP-induced depolariza- 4). Thus, P2X7Rs negatively control the expression and tions in individual DRG neurons. The slow kinetics of the a,b-meATP-induced depolarizations is likely a result function of P2X3Rs in immature rats just as those of bath application of the agonist [29]. Because of the observed in adult rats [5]. Since P2X7R expression is small percentage of adult DRG neurons responding to low in P6-15 day rats (Figure 3), a reduced P2X7R- a,b-meATP, the kinetic difference in the depolarizing P2X3R inhibitory control is likely a mechanism contri- responses of immature and adult DRG neurons was not buting to the high expression and function of P2X3Rs in studied. We found that the amplitudes of a,b-meATP- DRGs of immature rats. induced depolarizations in individual adult DRG neu- Many challenges exist in pain management in infants. rons is ~3.5 fold smaller than those recorded in imma- The expressions and functions of transmitters and ture neurons (Figure 2). receptors undergo changes during development [41]. We further studied the P2X3R expression using Wes- The descending control pathways from the brainstem to tern analyses and P2X3R functions through behavioral the spinal cord in infants are not completely developed. analyses. In immature rats, the P2X3R protein expres- Sensory inputs are integrated, both spatially and tempo- sion was high (Figure 3) and the a,b-meATP application rally [41]. In addition, verbal communication with to the rat paw evoked much larger flinching behavioral infants is limited. Compared to adults, infants are more responses in immature rats (Figure 4B). An increase in sensitive to repeated noxious stimuli and chronic tissue receptor expression in Western analyses and an increase inflammation [42]. On the other hand, injury to periph- in behavioral responses could result from a larger num- eral nerves in infants gives rise to much less allodynia ber of cells expressing receptors and responding to a,b- and neuropathic pain than those observed in adults meATP respectively. They also can result from an [43,44]. Therefore, the information obtained from stu- dies of pain treatment in adults is not directly applicable increase in the receptor expression and function in indi- to infants. We showed before that, in adult rats, total vidual neurons. Thus, an increase in the amplitude of a, b-meATP evoked depolarizations in individual neurons P2X3R protein expression is upregulated in DRG neu- (Figure 2) is consistent with the results of Western and rons after inflammation [45] and the membrane traffick- behavioral analyses. Combining immunocytochemical, ing of P2X3Rs increases following spared nerve injury electrophysiological and behavioral studies, we conclude [32]. It is of interest to determine if purinergic receptors that P2X3R expression and P2X3R-mediated cellular respond to tissue and nerve injury in immature DRGs in activity are high in immature DRG neurons and are a similar manner as in adult DRGs. Our observations reduced as rats mature. The high level of P2X3R expres- that P2X3R and P2X7R expression are negatively corre- sion in the somata of immature DRG neurons is likely lated (Figure 3) and P2X7R-P2X3R inhibitory control to give rise to an enhanced transport of P2X3R receptor also exists in developing rats (Figure 4) suggest that reg- to their nerve terminals and thus increased a,b-meATP- ulation of P2X7R expression in SGCs can be a useful induced behavioral responses. Nevertheless, the results strategy for controlling P2X3R activity in sensory neu- do not exclude the possibility that developmental rons and relieving pain in infant patients. changes in the spinal circuitry and descending modula- tion could also contribute to the observed changes in Acknowledgements the behavioral responses in immature rats. The work is supported by grants from National Institutes of Health (NS30045 We found that P2X3Rs are expressed only in DRG and DE17813). neurons and P2X7Rs are expressed only in SGCs (Figure Authors’ contributions 1) in P6 rats, similar to those found in adult DRGs [5]. YC designed the study, performed Western blotting, immunocytochemical There are similarities and differences between the distri- and electrophysiological experiments and prepared the manuscript. GL carried out the behavioral experiments. LMH designed and coordinated the bution of P2X3Rs and P2X7Rs in DRGs and those in study and prepared the manuscript. 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Pain 2005, 114:386-396. neurobiological approach. Nat Clin Pract Neurol 2009, 5:35-50. Chen et al. Molecular Pain 2012, 8:9 Page 8 of 8 http://www.molecularpain.com/content/8/1/9 45. Xu GY, Huang LY: Peripheral inflammation sensitizes P2X receptor- mediated responses in rat dorsal root ganglion neurons. J Neurosci 2002, 22:93-102. doi:10.1186/1744-8069-8-9 Cite this article as: Chen et al.: P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons. Molecular Pain 2012 8:9. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Molecular PainSpringer Journals

Published: Feb 7, 2012

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