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Participation of satellite glial cells of the dorsal root ganglia in acute nociception
Accepted Manuscript Title: Participation of satellite glial cells of the dorsal root ganglia in acute nociception Authors: J´ulia Borges Paes Lemes, Tais de Campos Lima, D´ebora Oliveira Santos, Amanda Ferreira Neves, Fernando Silva de Oliveira, Carlos Almicar Parada, Celina Monteiro da Cruz Lotufo PII: DOI: Reference:
S0304-3940(18)30258-1 https://doi.org/10.1016/j.neulet.2018.04.003 NSL 33526
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
24-11-2017 29-3-2018 2-4-2018
Please cite this article as: J´ulia Borges Paes Lemes, Tais de Campos Lima, D´ebora Oliveira Santos, Amanda Ferreira Neves, Fernando Silva de Oliveira, Carlos Almicar Parada, Celina Monteiro da Cruz Lotufo, Participation of satellite glial cells of the dorsal root ganglia in acute nociception, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Participation of satellite glial cells of the dorsal root ganglia in acute nociception. Júlia Borges Paes Lemesa,*, Tais de Campos Limaa, Débora Oliveira Santosa, Amanda Ferreira Nevesb, Fernando Silva de Oliveiraa, Carlos Almicar Paradab, Celina Monteiro da Cruz Lotufoa,*
aDepartment b
of Physiological Science, Universidade Federal de Uberlândia, Minas Gerais, Brazil Department of Structural Biology, Universidade Estadual de Campinas, São Paulo, Brazil.
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Corresponding author. Federal University of Uberlândia, Institute of Biomedical Sciences. Pará 1720, Umuarama, Brazil Tel.: +55 34 98855-7679; e-mail address: [email protected] (Lotufo, CM).
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*
Highlights
Nociceptors communicate with satellite glial cells in dorsal root ganglia through ATP release.
Activation of glial P2X7 receptors by ATP participates in acute nociception.
Communication through gap junctions in satellite glial cells is involved in acute pain
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Abstract
At dorsal root ganglia, neurons and satellite glial cells (SGC) can communicate through ATP release and P2X7 receptor activation. SGCs are also interconnected by gap junctions and have been previously
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implicated in modulating inflammatory and chronic pain.We now present evidence that SGCs are also involved in processing acute nociception in rat dorsal root ganglia. Using primary dorsal root ganglia
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cultures we observed that calcium transients induced in neurons by capsaicin administration were followed by satellite glial cells activation. Only satellite glial cells response was reduced by
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administration of the P2X7 receptor antagonist A740003. In vivo, acute nociception induced by intraplantar injection of capsaicin in rats was inhibited by A740003 or by the gap junction blocker carbenoxolone administered at the dorsal root ganglia (L5 level). Both drugs also reduced the second phase of the formalin test. These results suggest that communication between neurons and satellite
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glial cells is not only involved in inflammatory or pathological pain, but also in the transmission of the nociceptive signal, possibly in situations involving C-fiber activation.
Keywords Nociception, satellite glial cells, purinergic P2X7 receptor, Adenosine Triphosphate, Gap Junctions.
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1. Introduction Sensory ganglia are the first station in the somatosensory pathway in which pain can be modulated. Dorsal root ganglia (DRG) are located near the posterior horn of the spinal cord and contain the cell bodies of primary afferent neurons, which transmit the pain signal from the periphery into the central nervous system. Sensory neurons do not form proper synapses at the DRG, but the
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neuronal soma is able to release neurotransmitters, such as glutamate, ATP and Substance P, and it also expresses a variety of receptors for neurotransmitters and inflammatory mediators [1,2,3,4]. At
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DRG, the neuronal soma is enwrapped by satellite glial cells (SGCs) [5,6]. These glial cells play an active role in modulating neuronal activity in painful pathologies. In inflammatory and neuropathic
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conditions, SGCs exhibit morphological changes that are described as SGC activation [7,8]. These
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cells enlarge, proliferate and increase glial fibrillary acidic protein (GFAP) expression. Also, in the
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same pain conditions there is an increase in the expression of connexins that form gap junctions
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between adjacent SGCs [9, 10,11]. Furthermore, activated SGCs in pathological conditions might modulate neuronal sensitivity via production and release of inflammatory mediators, such as IL-1β,
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TNF-α, and prostaglandins [12,1]. It was previously described that sensory neurons are able to communicate with SGCs by releasing neurotransmitters. Electric stimulation of DRG cultures was
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shown to promote neuronal ATP release, which activates P2X7 receptors that are selectively expressed in SGCs [1]. The involvement of SGCs in pain and hyperalgesia has been evaluated using P2X7
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antagonists or gap junction blockers that promotes analgesia and reduces SGCs activation in animal pain models [7,12,13]. In the present study, we describe evidence that P2X7 receptors and gap
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junctions, expressed by SGCs at the DRG, participate in processing acute nociceptive responses in the absence of neuronal sensitization or pathological pain condition.
2. Materials and Methods 2.1. Animals
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Male Wistar rats (200-250g) were housed with food and water ad libitum in a controlled environment of 12/12h light/dark cycle. All experiments were approved by the Committee for Ethical Use of Animals from Universidade Federal de Uberlândia (CEUA/UFU).
2.2. Primary cell culture of Dorsal Root Ganglion
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Rats were euthanized under isoflurane anesthesia and lumbar and thoracic DRGs were harvested and transferred to Hank’s buffered saline solution containing Hepes (10 mM). Ganglia were
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incubated in 0.28 U/mL collagenase (type II; Sigma) for 75 min and in 0.25% trypsin (Sigma) for 12 min. After three washes with DMEM containing 10% of fetal bovine serum, the ganglionic cells were dissociated using a fire-polished glass Pasteur pipette and plated in glass bottom dishes coated with
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Matrigel (BD). Cultures were maintained in DMEM plus 10% of fetal bovine serum and penicillin (50
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U/mL)/streptomycin (50 mg/mL) for 48h at 37 °C with 5% CO2 atmosphere. For intracellular calcium
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detection, cultures were incubated with 5 μM Fluo-3AM (Invitrogen) for 40 minutes and then washed
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with Hanks/HEPES solution. Serial images were acquired using a confocal microscope (LSM 510
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meta, Zeiss). Capsaicin was initially diluted and aliquoted at 10 mM of ethanol and was further diluted in saline for administration at the cell cultures. Capsaicin (1 μM) was administered after vehicle
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(Hanks) or the P2X7 receptor antagonist A740003 (1 nM). Administrations were performed during serial image acquisition using an automatic pipette by adding 10 µl of 10x the final concentration of
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the drug to 90 µl of the Hanks solution bathing the cultured cells. Neuronal and SGCs responses were analyzed by selecting individual cells as ROIs (Region of Interest) and calculating mean gray value
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variations using the ImageJ software (NIH). Each experiment was repeated in 3 different plates, obtained from at least two animals, and individual cells responses were considered for statistics.
2.3.Behavioral tests
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Acute nociception was accessed using the capsaicin and formalin tests. Animals received either intraplantar (i.pl., right hind paw) injections of 50 µl of sterile saline solution (NaCl 0.9%) containing capsaicin (10 µg, Sigma) or formalin (2.5% diluted from a 4% PFA in PBS buffer). Immediately after injection, we counted nociceptive behavior, consisting of flinching or licking the paw, for 5 minutes in capsaicin test or 1 hour in a formalin test. In order to test the effects of blocking P2X7 receptors
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(A740003, Tocris) or gap junctions (carbenoxolone, Sigma) in the dorsal root ganglia, we used a direct intraganglionar (i.gl.) injection, as described elsewhere [14]. A740003 (1 nM, 5 µl in saline + 10%
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DMSO), carbenoxolone (1 nM, 5 µl in saline) or their respective vehicles (control group) were administered in the right L5 dorsal root ganglia 30 minutes before capsaicin or formalin injections. Different animals were used in each experimental group.
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2.4.Statistical analysis
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Behavioral and in vitro fluorescence data are presented as mean ± SEM and compared by t-test
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using GraphPad Prism software. Means were considered significantly different if P<0.05.
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3. Results
The effect of capsaicin on intracellular calcium levels was tested in primary dorsal root ganglia
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cultures using Fluo3-AM as described. In the first 24 hours of primary dorsal root ganglia cultures, most satellite glial are found attached to neuronal cell bodies. When these cultures are incubated with
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Fluo-3 AM for intracellular calcium observation, the fluorophore reaches satellite glial cells first and accumulate in these cells. Therefore, the amount of Fluo-3 AM that reach neurons is reduced, resulting
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in a low basal fluorescence (Figure 1D to 1F). Using confocal microscopy, such difference in fluorescence facilitates visualizing satellite cells surrounding neuronal soma (Figure 1D). Using capsaicin to activate nociceptive neurons, we observed that several neurons responded with intracellular calcium increase after capsaicin administration (Figure 1A). Capsaicin administration induced an immediate intracellular calcium increase in small neurons (Figure 1A, 1E), followed by a
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calcium transient in the surrounding satellite glial cells (Figure 1A, 1C). Pre-treatment of the cells with the antagonist of the P2X7 receptor, A740003 (1 nM) inhibited SGCs response but did not alter neuronal response to capsaicin (Figure 1B, 1C). Furthermore, administration of the P2X7 agonist BzATP (100 µM) induced a calcium transients in cultured SGCs that was reduced in the presence of the antagonist A740003 (1nM) (Figure 1G). Neuronal responses were not observed after
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administration of BzATP (data not shown) to DRG cultures.
To assess the function of the communication between neurons and SGCs observed in vitro, we
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evaluated the participation of glial P2X7 receptors in capsaicin-induced nociception. Capsaicin (10 µg) was injected in the rat’s right hind paw 30 minutes after intraganglionar injection of the P2X7
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antagonist A740003 (1 nM, 5 µL, right L5). Blockage of P2X7 receptors at the dorsal root ganglia
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resulted in a reduced nociceptive response induced by intraplantar capsaicin injection (Figure 2A).
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Capsaicin-induced nociception was also reduced by intraganglionar injections of the gap junctions
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blocker carbenoxolone (1 nM) (Figure 2B). Co-administration of A740003 (1 nM) and carbenoxolone (1 nM) at the DRG did not result in an augmented effect when compared to the effects of each isolated
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drug (Figure 2C).
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The effects of blocking P2X7 receptors and gap junctions in the L5 dorsal root ganglia were also evaluated using the formalin test. Formalin (2.5%, 50 µL, i.pl.) was injected 30 minutes after
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intraganglionar injections of A740003 (1 nM, 5 µL), carbenoxolone (1 nM, 5 µL) or their respective vehicle in rats. Nociceptive behavior (paw flinches) was divided into 2 phases, phase1 comprises the first 15 minutes and is attributed to a direct effect on nociceptors, while phase2 (20 to 60 minutes) is
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related to inflammation and/or central sensitization [15,16]. We observed that blocking P2X7 receptors or gap junctions at the dorsal root ganglia resulted in a reduced phase2 of the formalin test, while not altering the nociceptive response observed in phase1 (Figures 3A and 3B).
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Data presented in the current study showed that communication between SGCs and primary sensory neurons at the dorsal root ganglia participate in a fast processing of acute pain. Cultured neurons were observed, when activated by capsaicin, to release ATP, which activates P2X7 receptor inducing calcium transient in surrounding SGCs. In rats, administration of a P2X7 antagonist or a gap junction blocker directly at the dorsal root ganglia (at L5 level) reduced the acute nocifensive behavior
formalin injection, only the inflammatory phase (phase 2) was reduced.
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induced by intraplantar capsaicin injection. When both drugs were administered before intraplantar
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In sensory ganglia, communication between neurons and glia has been implicated in neuronal sensitization and maintenance of chronic pain [3,7, 8,11]. Previous studies report that neurotransmitters, such as glutamate [3] or ATP [1], are released by neurons within the dorsal root
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ganglia and can activate satellite glial cells [11]. In the present study, we detected communication
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between nociceptive neurons and SGCs in primary dorsal root ganglia cultures following capsaicin
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administration. Capsaicin is a selective agonist of the TRPV1, which is a cation channel expressed
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mainly in nociceptors associated with C-fibers [17]. During these experiments, it was observed that
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about 8 seconds after neuronal responses, SGCs exhibited a calcium transient (Figure 1F). Considering SGCs do not express TRPV1 channels, these cells were probably responding to a soluble mediator
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released by activated neurons. The selective P2X7 antagonist, A740003, reduced the activation of SGCs following capsaicin administration. These results are compatible with those described by others
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[1], with the difference that in the previous study, cultured neurons were excited using electric stimulation and not by capsaicin administration. At the dorsal root ganglia, P2X7 receptors are
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selectively expressed in SGCs [18]. Consequently, involvement of ganglionic P2X7 receptors in a process necessarily implicates activation of glial cells. The P2X7 receptor is a distinctive purinergic receptor expressed mainly by cells involved in immune responses [19]. This receptor is a cation channel activated by high ATP concentrations that are usually achieved in injury conditions. Prolonged exposure to ATP induces the formation of a pore structure that is permeable to larger molecules, such
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as organic cations [20]. Activation of P2X7 receptors has been linked to a further ATP release and to the production and release of inflammatory mediators. For instance, P2X7 receptor activation seems to be essential for the release of the cytokine IL-1β [21, 22]. Therefore, these receptors have been studied and reported to play important roles in several pathological conditions. Indeed, genetic disruption of P2X7 receptors suggests an essential role in chronic inflammatory and neuropathic pain [23].
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Considering that capsaicin selectively activates TRPV1 channels expressed by nociceptive neurons [17], we decided to evaluate the functional role of ATP/P2X7 neuronal-glia communication in
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capsaicin-induced nociceptive responses in vivo. Experiments were performed using intraplantar capsaicin administration in rats that had been previously treated with the P2X7 antagonist A740003 administered at the DRG (ipsilateral L5). This administration route was used in previous studies of
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neuron-glia communication [3,12, 24] and allows the administration of drugs at or proximal to the
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right L5 dorsal root ganglia, while not reaching the spinal cord. Most in vivo studies use intrathecal
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administration, which may not distinguish between central or peripheral drug actions. Intraganglionar
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injection of the P2X7 antagonist caused an important reduction of approximately 50% of the
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nocifensive behavior induced by intraplantar capsaicin injection. The behavioral response of flinching or licking the paw is a motor reflex that occurs within 5 minutes of capsaicin administration. The
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neural pathway involved in such reflex is generally described as initiating at the peripheral terminal, where capsaicin directly activates TRPV1 channels at C-fibers, and then transmitted by action
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potentials that will reach central terminals at the dorsal horn of the spinal cord. However, despite the pseudounipolar morphology of primary sensory neurons that allows propagation of action potentials
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directly from the peripheral to the central branch at the T-junction, sensory neurons soma are excitable and the action potentials invade cells soma [4]. The reduction of the nocifensive response induced by the P2X7 antagonist administration at the DRG implies that such fast acute nociceptive response is modulated at peripheral level by SGCs. Therefore, communication between neurons and SGCs, as well
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as activation of the P2X7 receptor, might play a role in basal situations, without injury or other pathological conditions. Satellite glial cells are interconnected by gap junctions that supposedly amplify the signal through cross-excitation or cross-sensitization of neighboring neurons [11]. The expression of connexins is augmented in painful pathological conditions [9,11,25]. In order to test if gap junctions
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are involved in the processing of acute nociception, the gap junction blocker carbenoxolone was administered at the dorsal root ganglia (L5 ipsilateral) before intraplantar capsaicin administration. The
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effect of the gap junction blocker was similar to that observed using the P2X7 antagonist, causing a reduced nocifensive behavior. Besides the blockage of gap junctions, carbenoxolone is also a pannexin hemichannel blocker, and such channels have been proposed to participate in ATP release from SGCs
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[25]. Consequently, administration of carbenoxolone is possibly blocking two processes, gap junction
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communication and ATP release from SGCs. The combination of carbenoxolone and A740003 did
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not result in further inhibition of the capsaicin-induced nociception, considering the actions of each
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drug alone. Therefore, it seems that P2X7 receptors and gap junctions participate in dependent or
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sequential events. In the study that firstly described ATP/P2X7 communication at the DRG [1], the authors observed that electric stimulation of the sciatic nerve, attached to non-dissociated DRGs,
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induced a calcium transient in neurons that was followed by a prolonged intracellular calcium response at SGCs. The [Ca2+]i concentration in SGCs continued to rise after electric stimulus was discontinued
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and remained for 60-80 seconds. This prolonged calcium response was not observed in dissociated cell cultures, possibly because communication via gap junctions is reduced in cultured cells. In vivo, such
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prolonged stimulation of SGCs might result from propagation of calcium waves between adjacent cells through gap junctions and result in a further ATP release from these glial cells. Taken together, data suggest that satellite glial cells are activated by ATP released by excited neurons and the signal can be transmitted through neighboring satellite cells by means of calcium or other substances that cross through gap junctions. Although such process had been previously
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detected, this is the first report of such communication occurring in acute nociception. In basal conditions gap junctions connecting SGCs are mainly found among cells that envelope the same neuron, while after nerve damage or inflammation such connection is also observed between cells surrounding neighboring neurons [9,10]. Therefore, at basal conditions, such as in acute pain, SGCs are more likely providing a positive feedback to the same neuron that was excited. The involvement of
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this type of neuron-SGC communication seems to be dependent on the nociceptive stimulus, as blockage of P2X7 receptors or gap junctions did not affect the first phase of the formalin test. The
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intraganglionar administration of the P2X7 antagonist or the gap junction blocker was tested before formalin (2.5%) injection in the rat paw. Administration of both drugs resulted in a reduced phase2 of the formalin test without affecting phase1. The first phase of the formalin test is considered to be
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induced by direct excitation of nociceptive neurons while the second phase is an inflammatory phase
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that depends on peripheral and/or central sensitization [15,16]. Using a high concentration of formalin
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(2.5%), as used in the present study, there is a predominant activation of Aδ fibers at phase 1, while at the second phase all C-fibers are activated and predominate over Aδ fibers [26]. Differential activation
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of nociceptive fibers might be an explanation for the absence of P2X7 and gap junction involvement in the first phase of the formalin test. In agreement with this hypothesis, one study described that mainly
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small nociceptive neurons, characteristic of amyelinic C-fibers, express the vesicular nucleotide
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transporter (VNUT) that is essential for neuronal ATP release at the DRG [27]. Therefore, one possible explanation for the differential participation of P2X7 receptors and gap junctions expressed at SGCs is the involvement of C-fibers in pain responses. Reinforcing this hypothesis, we observed that
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the P2X7 antagonist administered at the dorsal root ganglia was also able to reduce nocifensive behavior induced by intraplantar menthol injection (data not shown). Menthol selectively activates TRPM8 receptors, which are cold receptors expressed preferentially by nociceptors associated to Cfibers [28].
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Most studies regarding SGCs involvement in pain report that these cells are activated, which is usually observed as an augmented GFAP expression, and release inflammatory mediators or cytokines, such as TNF-α and IL1-β, which in turn sensitize nociceptive neurons. Results presented in this study indicate that SGCs also influence nociceptive signal as it passes through the dorsal root ganglia, amplifying the signal between neighboring neurons or acting as a positive feedback between activated
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neurons and surrounding SGCs in order to increase neuron excitation. This sort of fast processing is not likely dependent on the release of inflammatory cytokines, which usually depend on slow and
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prolonged effects, affecting neuronal sensitization. On the other hand, SGCs are able to release neurotransmitters, such as ATP [1] and glutamate [24,29], that might have a fast influence on neuronal excitation. It is possible that ATP released by SGCs modulates P2X3 receptors expressed in
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nociceptive neurons, since it was described that this purinergic receptor is functionally linked to P2X7
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activation in SGCs [30]. Understanding neuronal-glia interactions at the dorsal root ganglia might
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present interesting possibilities for analgesic drug development. Since the dorsal root ganglia are
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located outside the blood-brain barrier, some drugs could have analgesic effects with reduced central
5. Conclusions
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side effects.
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The present study indicates that P2X7 receptors at satellite glial cells are activated by ATP released by nociceptive neurons during acute nociception. In addition, the communication among
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adjacent satellite glial cells via gap junctions or by pannexin hemichannels plays a role in modulating nociceptive response. This process seems to be dependent on activation of nociceptive C-fibers.
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Therefore, data presented in this study suggest that the communication between neurons and satellite glial cells is involved not only in pathological conditions or in inflammatory pain but also in acute pain.
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Acknowledgments This work received financial support from Coordenação de Aperfeiçoamento de Pessoal de
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Nível Superior (CAPES) and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG).
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Figure Captions
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Figure 1. Calcium transients induced by capsaicin in neurons and satellite cells of primary dorsal root ganglia cultures – involvement of P2X7 receptors in neuronal-glia communication. (A) Effect of capsaicin (1 μM) on intracellular calcium in neurons (blue curves) and glial cells (red curves) observed using Fluo-3AM fluorescence emission in confocal microscopy. (B) A740003 (10 nM) administered 5 minutes before capsaicin administration (1 µM) reduced calcium transients in satellite glial cells (red curves) but not in neurons (blue curves). (C) Maximal fluorescence variation (ΔF/F0) induced by capsaicin (1 μM) on neurons and SGCs in cultures pre-treated (5 min) with A740003 (10 nM) or vehicle. Data shown as means ± SEM of cells from 3 different cultures, n indicated above each bar. *P<0.05 (t-test). (D) Basal Fluo-3fluorescence emission by a neuron (arrow) and surrounding satellite glial cells (arrowhead) in primary cultured dorsal root ganglia (24 hours). (E) Fluorescent increase indicating calcium transient in the neuron after 8 seconds of capsaicin (1 µM) administration. (F) Calcium transient observed as an increase in SGCs fluorescence after 16 seconds of capsaicin administration. (G) Maximal fluorescence variation (ΔF/F0) induced by the selective P2X7 agonist BzATP (100 μM) on SGCs in cultures pre-treated (5 min) with A740003 (10 nM) or vehicle. Data shown as means ± SEM of 30 cells from 3 different cultures. *P<0.0001 (t-test).
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Figure 2. In vivo blockage of P2X7 receptors or gap junctions at the dorsal root ganglia affects nociceptive behavior induced by intraplantar injection of capsaicin. (A) Blockage of P2X7 receptors at DRG by A74003. (B) Blockage of gap junctions at DRG by carbenoxolone. (C) Co-administration of the P2X7 antagonist, A740003, and the gap junction blocker, carbenoxolone, at DRG. The P2X7 receptor antagonist A740003 (1 nM, 5 µL in saline + 10% DMSO), the gap junctions blocker carbenoxolone (1 nM, 5 µL in saline) or their respective vehicles (control) were injected at the right L5 dorsal root ganglia 30 minutes before intraplantar injections of capsaicin (10 µg in 50 µL of saline). The number of paw flinches exhibited by each animal was recorded during 5 minutes. Results presented as means ± SEM of the number of animals indicated inside each bar. *P<0.05 (t-test).
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Figure 3. In vivo blockage of P2X7 receptors or gap junctions at the dorsal root ganglia affect nociceptive behavior induced by intraplantar injection of formalin. (A) Blockage of P2X7 receptors at DRG by A740003 administration at DRG. (B) Blockage of gap junctions by carbenoxolone administration at DRG. The P2X7 receptor antagonist A740003 (1 nM, 5 µL in saline + 10% DMSO), the gap junctions blocker carbenoxolone (1 nM, 5 µL in saline) or their respective vehicles (control) were injected at the right L5 dorsal root ganglia 30 minutes before formalin (2.5% in 50 µL of saline). Nociceptive response (flinching or licking the paw) was counted in 5 minutes blocks that were averaged in phase1 (first 15 minutes) and phase2 (20 to 60 minutes). Results presented as means ± SEM of the number of animals indicated inside bars. *P<0.001, (t-test).
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S0304-3940(18)30258-1 https://doi.org/10.1016/j.neulet.2018.04.003 NSL 33526
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
24-11-2017 29-3-2018 2-4-2018
Please cite this article as: J´ulia Borges Paes Lemes, Tais de Campos Lima, D´ebora Oliveira Santos, Amanda Ferreira Neves, Fernando Silva de Oliveira, Carlos Almicar Parada, Celina Monteiro da Cruz Lotufo, Participation of satellite glial cells of the dorsal root ganglia in acute nociception, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.04.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Participation of satellite glial cells of the dorsal root ganglia in acute nociception. Júlia Borges Paes Lemesa,*, Tais de Campos Limaa, Débora Oliveira Santosa, Amanda Ferreira Nevesb, Fernando Silva de Oliveiraa, Carlos Almicar Paradab, Celina Monteiro da Cruz Lotufoa,*
aDepartment b
of Physiological Science, Universidade Federal de Uberlândia, Minas Gerais, Brazil Department of Structural Biology, Universidade Estadual de Campinas, São Paulo, Brazil.
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Corresponding author. Federal University of Uberlândia, Institute of Biomedical Sciences. Pará 1720, Umuarama, Brazil Tel.: +55 34 98855-7679; e-mail address: [email protected] (Lotufo, CM).
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*
Highlights
Nociceptors communicate with satellite glial cells in dorsal root ganglia through ATP release.
Activation of glial P2X7 receptors by ATP participates in acute nociception.
Communication through gap junctions in satellite glial cells is involved in acute pain
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Abstract
At dorsal root ganglia, neurons and satellite glial cells (SGC) can communicate through ATP release and P2X7 receptor activation. SGCs are also interconnected by gap junctions and have been previously
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implicated in modulating inflammatory and chronic pain.We now present evidence that SGCs are also involved in processing acute nociception in rat dorsal root ganglia. Using primary dorsal root ganglia
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cultures we observed that calcium transients induced in neurons by capsaicin administration were followed by satellite glial cells activation. Only satellite glial cells response was reduced by
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administration of the P2X7 receptor antagonist A740003. In vivo, acute nociception induced by intraplantar injection of capsaicin in rats was inhibited by A740003 or by the gap junction blocker carbenoxolone administered at the dorsal root ganglia (L5 level). Both drugs also reduced the second phase of the formalin test. These results suggest that communication between neurons and satellite
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glial cells is not only involved in inflammatory or pathological pain, but also in the transmission of the nociceptive signal, possibly in situations involving C-fiber activation.
Keywords Nociception, satellite glial cells, purinergic P2X7 receptor, Adenosine Triphosphate, Gap Junctions.
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1. Introduction Sensory ganglia are the first station in the somatosensory pathway in which pain can be modulated. Dorsal root ganglia (DRG) are located near the posterior horn of the spinal cord and contain the cell bodies of primary afferent neurons, which transmit the pain signal from the periphery into the central nervous system. Sensory neurons do not form proper synapses at the DRG, but the
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neuronal soma is able to release neurotransmitters, such as glutamate, ATP and Substance P, and it also expresses a variety of receptors for neurotransmitters and inflammatory mediators [1,2,3,4]. At
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DRG, the neuronal soma is enwrapped by satellite glial cells (SGCs) [5,6]. These glial cells play an active role in modulating neuronal activity in painful pathologies. In inflammatory and neuropathic
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conditions, SGCs exhibit morphological changes that are described as SGC activation [7,8]. These
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cells enlarge, proliferate and increase glial fibrillary acidic protein (GFAP) expression. Also, in the
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same pain conditions there is an increase in the expression of connexins that form gap junctions
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between adjacent SGCs [9, 10,11]. Furthermore, activated SGCs in pathological conditions might modulate neuronal sensitivity via production and release of inflammatory mediators, such as IL-1β,
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TNF-α, and prostaglandins [12,1]. It was previously described that sensory neurons are able to communicate with SGCs by releasing neurotransmitters. Electric stimulation of DRG cultures was
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shown to promote neuronal ATP release, which activates P2X7 receptors that are selectively expressed in SGCs [1]. The involvement of SGCs in pain and hyperalgesia has been evaluated using P2X7
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antagonists or gap junction blockers that promotes analgesia and reduces SGCs activation in animal pain models [7,12,13]. In the present study, we describe evidence that P2X7 receptors and gap
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junctions, expressed by SGCs at the DRG, participate in processing acute nociceptive responses in the absence of neuronal sensitization or pathological pain condition.
2. Materials and Methods 2.1. Animals
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Male Wistar rats (200-250g) were housed with food and water ad libitum in a controlled environment of 12/12h light/dark cycle. All experiments were approved by the Committee for Ethical Use of Animals from Universidade Federal de Uberlândia (CEUA/UFU).
2.2. Primary cell culture of Dorsal Root Ganglion
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Rats were euthanized under isoflurane anesthesia and lumbar and thoracic DRGs were harvested and transferred to Hank’s buffered saline solution containing Hepes (10 mM). Ganglia were
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incubated in 0.28 U/mL collagenase (type II; Sigma) for 75 min and in 0.25% trypsin (Sigma) for 12 min. After three washes with DMEM containing 10% of fetal bovine serum, the ganglionic cells were dissociated using a fire-polished glass Pasteur pipette and plated in glass bottom dishes coated with
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Matrigel (BD). Cultures were maintained in DMEM plus 10% of fetal bovine serum and penicillin (50
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U/mL)/streptomycin (50 mg/mL) for 48h at 37 °C with 5% CO2 atmosphere. For intracellular calcium
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detection, cultures were incubated with 5 μM Fluo-3AM (Invitrogen) for 40 minutes and then washed
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with Hanks/HEPES solution. Serial images were acquired using a confocal microscope (LSM 510
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meta, Zeiss). Capsaicin was initially diluted and aliquoted at 10 mM of ethanol and was further diluted in saline for administration at the cell cultures. Capsaicin (1 μM) was administered after vehicle
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(Hanks) or the P2X7 receptor antagonist A740003 (1 nM). Administrations were performed during serial image acquisition using an automatic pipette by adding 10 µl of 10x the final concentration of
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the drug to 90 µl of the Hanks solution bathing the cultured cells. Neuronal and SGCs responses were analyzed by selecting individual cells as ROIs (Region of Interest) and calculating mean gray value
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variations using the ImageJ software (NIH). Each experiment was repeated in 3 different plates, obtained from at least two animals, and individual cells responses were considered for statistics.
2.3.Behavioral tests
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Acute nociception was accessed using the capsaicin and formalin tests. Animals received either intraplantar (i.pl., right hind paw) injections of 50 µl of sterile saline solution (NaCl 0.9%) containing capsaicin (10 µg, Sigma) or formalin (2.5% diluted from a 4% PFA in PBS buffer). Immediately after injection, we counted nociceptive behavior, consisting of flinching or licking the paw, for 5 minutes in capsaicin test or 1 hour in a formalin test. In order to test the effects of blocking P2X7 receptors
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(A740003, Tocris) or gap junctions (carbenoxolone, Sigma) in the dorsal root ganglia, we used a direct intraganglionar (i.gl.) injection, as described elsewhere [14]. A740003 (1 nM, 5 µl in saline + 10%
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DMSO), carbenoxolone (1 nM, 5 µl in saline) or their respective vehicles (control group) were administered in the right L5 dorsal root ganglia 30 minutes before capsaicin or formalin injections. Different animals were used in each experimental group.
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2.4.Statistical analysis
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Behavioral and in vitro fluorescence data are presented as mean ± SEM and compared by t-test
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using GraphPad Prism software. Means were considered significantly different if P<0.05.
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3. Results
The effect of capsaicin on intracellular calcium levels was tested in primary dorsal root ganglia
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cultures using Fluo3-AM as described. In the first 24 hours of primary dorsal root ganglia cultures, most satellite glial are found attached to neuronal cell bodies. When these cultures are incubated with
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Fluo-3 AM for intracellular calcium observation, the fluorophore reaches satellite glial cells first and accumulate in these cells. Therefore, the amount of Fluo-3 AM that reach neurons is reduced, resulting
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in a low basal fluorescence (Figure 1D to 1F). Using confocal microscopy, such difference in fluorescence facilitates visualizing satellite cells surrounding neuronal soma (Figure 1D). Using capsaicin to activate nociceptive neurons, we observed that several neurons responded with intracellular calcium increase after capsaicin administration (Figure 1A). Capsaicin administration induced an immediate intracellular calcium increase in small neurons (Figure 1A, 1E), followed by a
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calcium transient in the surrounding satellite glial cells (Figure 1A, 1C). Pre-treatment of the cells with the antagonist of the P2X7 receptor, A740003 (1 nM) inhibited SGCs response but did not alter neuronal response to capsaicin (Figure 1B, 1C). Furthermore, administration of the P2X7 agonist BzATP (100 µM) induced a calcium transients in cultured SGCs that was reduced in the presence of the antagonist A740003 (1nM) (Figure 1G). Neuronal responses were not observed after
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administration of BzATP (data not shown) to DRG cultures.
To assess the function of the communication between neurons and SGCs observed in vitro, we
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evaluated the participation of glial P2X7 receptors in capsaicin-induced nociception. Capsaicin (10 µg) was injected in the rat’s right hind paw 30 minutes after intraganglionar injection of the P2X7
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antagonist A740003 (1 nM, 5 µL, right L5). Blockage of P2X7 receptors at the dorsal root ganglia
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resulted in a reduced nociceptive response induced by intraplantar capsaicin injection (Figure 2A).
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Capsaicin-induced nociception was also reduced by intraganglionar injections of the gap junctions
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blocker carbenoxolone (1 nM) (Figure 2B). Co-administration of A740003 (1 nM) and carbenoxolone (1 nM) at the DRG did not result in an augmented effect when compared to the effects of each isolated
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drug (Figure 2C).
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The effects of blocking P2X7 receptors and gap junctions in the L5 dorsal root ganglia were also evaluated using the formalin test. Formalin (2.5%, 50 µL, i.pl.) was injected 30 minutes after
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intraganglionar injections of A740003 (1 nM, 5 µL), carbenoxolone (1 nM, 5 µL) or their respective vehicle in rats. Nociceptive behavior (paw flinches) was divided into 2 phases, phase1 comprises the first 15 minutes and is attributed to a direct effect on nociceptors, while phase2 (20 to 60 minutes) is
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related to inflammation and/or central sensitization [15,16]. We observed that blocking P2X7 receptors or gap junctions at the dorsal root ganglia resulted in a reduced phase2 of the formalin test, while not altering the nociceptive response observed in phase1 (Figures 3A and 3B).
4. Discussion 5
Data presented in the current study showed that communication between SGCs and primary sensory neurons at the dorsal root ganglia participate in a fast processing of acute pain. Cultured neurons were observed, when activated by capsaicin, to release ATP, which activates P2X7 receptor inducing calcium transient in surrounding SGCs. In rats, administration of a P2X7 antagonist or a gap junction blocker directly at the dorsal root ganglia (at L5 level) reduced the acute nocifensive behavior
formalin injection, only the inflammatory phase (phase 2) was reduced.
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induced by intraplantar capsaicin injection. When both drugs were administered before intraplantar
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In sensory ganglia, communication between neurons and glia has been implicated in neuronal sensitization and maintenance of chronic pain [3,7, 8,11]. Previous studies report that neurotransmitters, such as glutamate [3] or ATP [1], are released by neurons within the dorsal root
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ganglia and can activate satellite glial cells [11]. In the present study, we detected communication
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between nociceptive neurons and SGCs in primary dorsal root ganglia cultures following capsaicin
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administration. Capsaicin is a selective agonist of the TRPV1, which is a cation channel expressed
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mainly in nociceptors associated with C-fibers [17]. During these experiments, it was observed that
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about 8 seconds after neuronal responses, SGCs exhibited a calcium transient (Figure 1F). Considering SGCs do not express TRPV1 channels, these cells were probably responding to a soluble mediator
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released by activated neurons. The selective P2X7 antagonist, A740003, reduced the activation of SGCs following capsaicin administration. These results are compatible with those described by others
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[1], with the difference that in the previous study, cultured neurons were excited using electric stimulation and not by capsaicin administration. At the dorsal root ganglia, P2X7 receptors are
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selectively expressed in SGCs [18]. Consequently, involvement of ganglionic P2X7 receptors in a process necessarily implicates activation of glial cells. The P2X7 receptor is a distinctive purinergic receptor expressed mainly by cells involved in immune responses [19]. This receptor is a cation channel activated by high ATP concentrations that are usually achieved in injury conditions. Prolonged exposure to ATP induces the formation of a pore structure that is permeable to larger molecules, such
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as organic cations [20]. Activation of P2X7 receptors has been linked to a further ATP release and to the production and release of inflammatory mediators. For instance, P2X7 receptor activation seems to be essential for the release of the cytokine IL-1β [21, 22]. Therefore, these receptors have been studied and reported to play important roles in several pathological conditions. Indeed, genetic disruption of P2X7 receptors suggests an essential role in chronic inflammatory and neuropathic pain [23].
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Considering that capsaicin selectively activates TRPV1 channels expressed by nociceptive neurons [17], we decided to evaluate the functional role of ATP/P2X7 neuronal-glia communication in
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capsaicin-induced nociceptive responses in vivo. Experiments were performed using intraplantar capsaicin administration in rats that had been previously treated with the P2X7 antagonist A740003 administered at the DRG (ipsilateral L5). This administration route was used in previous studies of
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neuron-glia communication [3,12, 24] and allows the administration of drugs at or proximal to the
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right L5 dorsal root ganglia, while not reaching the spinal cord. Most in vivo studies use intrathecal
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administration, which may not distinguish between central or peripheral drug actions. Intraganglionar
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injection of the P2X7 antagonist caused an important reduction of approximately 50% of the
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nocifensive behavior induced by intraplantar capsaicin injection. The behavioral response of flinching or licking the paw is a motor reflex that occurs within 5 minutes of capsaicin administration. The
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neural pathway involved in such reflex is generally described as initiating at the peripheral terminal, where capsaicin directly activates TRPV1 channels at C-fibers, and then transmitted by action
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potentials that will reach central terminals at the dorsal horn of the spinal cord. However, despite the pseudounipolar morphology of primary sensory neurons that allows propagation of action potentials
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directly from the peripheral to the central branch at the T-junction, sensory neurons soma are excitable and the action potentials invade cells soma [4]. The reduction of the nocifensive response induced by the P2X7 antagonist administration at the DRG implies that such fast acute nociceptive response is modulated at peripheral level by SGCs. Therefore, communication between neurons and SGCs, as well
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as activation of the P2X7 receptor, might play a role in basal situations, without injury or other pathological conditions. Satellite glial cells are interconnected by gap junctions that supposedly amplify the signal through cross-excitation or cross-sensitization of neighboring neurons [11]. The expression of connexins is augmented in painful pathological conditions [9,11,25]. In order to test if gap junctions
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are involved in the processing of acute nociception, the gap junction blocker carbenoxolone was administered at the dorsal root ganglia (L5 ipsilateral) before intraplantar capsaicin administration. The
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effect of the gap junction blocker was similar to that observed using the P2X7 antagonist, causing a reduced nocifensive behavior. Besides the blockage of gap junctions, carbenoxolone is also a pannexin hemichannel blocker, and such channels have been proposed to participate in ATP release from SGCs
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[25]. Consequently, administration of carbenoxolone is possibly blocking two processes, gap junction
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communication and ATP release from SGCs. The combination of carbenoxolone and A740003 did
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not result in further inhibition of the capsaicin-induced nociception, considering the actions of each
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drug alone. Therefore, it seems that P2X7 receptors and gap junctions participate in dependent or
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sequential events. In the study that firstly described ATP/P2X7 communication at the DRG [1], the authors observed that electric stimulation of the sciatic nerve, attached to non-dissociated DRGs,
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induced a calcium transient in neurons that was followed by a prolonged intracellular calcium response at SGCs. The [Ca2+]i concentration in SGCs continued to rise after electric stimulus was discontinued
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and remained for 60-80 seconds. This prolonged calcium response was not observed in dissociated cell cultures, possibly because communication via gap junctions is reduced in cultured cells. In vivo, such
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prolonged stimulation of SGCs might result from propagation of calcium waves between adjacent cells through gap junctions and result in a further ATP release from these glial cells. Taken together, data suggest that satellite glial cells are activated by ATP released by excited neurons and the signal can be transmitted through neighboring satellite cells by means of calcium or other substances that cross through gap junctions. Although such process had been previously
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detected, this is the first report of such communication occurring in acute nociception. In basal conditions gap junctions connecting SGCs are mainly found among cells that envelope the same neuron, while after nerve damage or inflammation such connection is also observed between cells surrounding neighboring neurons [9,10]. Therefore, at basal conditions, such as in acute pain, SGCs are more likely providing a positive feedback to the same neuron that was excited. The involvement of
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this type of neuron-SGC communication seems to be dependent on the nociceptive stimulus, as blockage of P2X7 receptors or gap junctions did not affect the first phase of the formalin test. The
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intraganglionar administration of the P2X7 antagonist or the gap junction blocker was tested before formalin (2.5%) injection in the rat paw. Administration of both drugs resulted in a reduced phase2 of the formalin test without affecting phase1. The first phase of the formalin test is considered to be
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induced by direct excitation of nociceptive neurons while the second phase is an inflammatory phase
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that depends on peripheral and/or central sensitization [15,16]. Using a high concentration of formalin
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(2.5%), as used in the present study, there is a predominant activation of Aδ fibers at phase 1, while at the second phase all C-fibers are activated and predominate over Aδ fibers [26]. Differential activation
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of nociceptive fibers might be an explanation for the absence of P2X7 and gap junction involvement in the first phase of the formalin test. In agreement with this hypothesis, one study described that mainly
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small nociceptive neurons, characteristic of amyelinic C-fibers, express the vesicular nucleotide
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transporter (VNUT) that is essential for neuronal ATP release at the DRG [27]. Therefore, one possible explanation for the differential participation of P2X7 receptors and gap junctions expressed at SGCs is the involvement of C-fibers in pain responses. Reinforcing this hypothesis, we observed that
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the P2X7 antagonist administered at the dorsal root ganglia was also able to reduce nocifensive behavior induced by intraplantar menthol injection (data not shown). Menthol selectively activates TRPM8 receptors, which are cold receptors expressed preferentially by nociceptors associated to Cfibers [28].
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Most studies regarding SGCs involvement in pain report that these cells are activated, which is usually observed as an augmented GFAP expression, and release inflammatory mediators or cytokines, such as TNF-α and IL1-β, which in turn sensitize nociceptive neurons. Results presented in this study indicate that SGCs also influence nociceptive signal as it passes through the dorsal root ganglia, amplifying the signal between neighboring neurons or acting as a positive feedback between activated
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neurons and surrounding SGCs in order to increase neuron excitation. This sort of fast processing is not likely dependent on the release of inflammatory cytokines, which usually depend on slow and
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prolonged effects, affecting neuronal sensitization. On the other hand, SGCs are able to release neurotransmitters, such as ATP [1] and glutamate [24,29], that might have a fast influence on neuronal excitation. It is possible that ATP released by SGCs modulates P2X3 receptors expressed in
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nociceptive neurons, since it was described that this purinergic receptor is functionally linked to P2X7
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activation in SGCs [30]. Understanding neuronal-glia interactions at the dorsal root ganglia might
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present interesting possibilities for analgesic drug development. Since the dorsal root ganglia are
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located outside the blood-brain barrier, some drugs could have analgesic effects with reduced central
5. Conclusions
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side effects.
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The present study indicates that P2X7 receptors at satellite glial cells are activated by ATP released by nociceptive neurons during acute nociception. In addition, the communication among
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adjacent satellite glial cells via gap junctions or by pannexin hemichannels plays a role in modulating nociceptive response. This process seems to be dependent on activation of nociceptive C-fibers.
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Therefore, data presented in this study suggest that the communication between neurons and satellite glial cells is involved not only in pathological conditions or in inflammatory pain but also in acute pain.
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Acknowledgments This work received financial support from Coordenação de Aperfeiçoamento de Pessoal de
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Nível Superior (CAPES) and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG).
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Figure Captions
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Figure 1. Calcium transients induced by capsaicin in neurons and satellite cells of primary dorsal root ganglia cultures – involvement of P2X7 receptors in neuronal-glia communication. (A) Effect of capsaicin (1 μM) on intracellular calcium in neurons (blue curves) and glial cells (red curves) observed using Fluo-3AM fluorescence emission in confocal microscopy. (B) A740003 (10 nM) administered 5 minutes before capsaicin administration (1 µM) reduced calcium transients in satellite glial cells (red curves) but not in neurons (blue curves). (C) Maximal fluorescence variation (ΔF/F0) induced by capsaicin (1 μM) on neurons and SGCs in cultures pre-treated (5 min) with A740003 (10 nM) or vehicle. Data shown as means ± SEM of cells from 3 different cultures, n indicated above each bar. *P<0.05 (t-test). (D) Basal Fluo-3fluorescence emission by a neuron (arrow) and surrounding satellite glial cells (arrowhead) in primary cultured dorsal root ganglia (24 hours). (E) Fluorescent increase indicating calcium transient in the neuron after 8 seconds of capsaicin (1 µM) administration. (F) Calcium transient observed as an increase in SGCs fluorescence after 16 seconds of capsaicin administration. (G) Maximal fluorescence variation (ΔF/F0) induced by the selective P2X7 agonist BzATP (100 μM) on SGCs in cultures pre-treated (5 min) with A740003 (10 nM) or vehicle. Data shown as means ± SEM of 30 cells from 3 different cultures. *P<0.0001 (t-test).
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Figure 2. In vivo blockage of P2X7 receptors or gap junctions at the dorsal root ganglia affects nociceptive behavior induced by intraplantar injection of capsaicin. (A) Blockage of P2X7 receptors at DRG by A74003. (B) Blockage of gap junctions at DRG by carbenoxolone. (C) Co-administration of the P2X7 antagonist, A740003, and the gap junction blocker, carbenoxolone, at DRG. The P2X7 receptor antagonist A740003 (1 nM, 5 µL in saline + 10% DMSO), the gap junctions blocker carbenoxolone (1 nM, 5 µL in saline) or their respective vehicles (control) were injected at the right L5 dorsal root ganglia 30 minutes before intraplantar injections of capsaicin (10 µg in 50 µL of saline). The number of paw flinches exhibited by each animal was recorded during 5 minutes. Results presented as means ± SEM of the number of animals indicated inside each bar. *P<0.05 (t-test).
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Figure 3. In vivo blockage of P2X7 receptors or gap junctions at the dorsal root ganglia affect nociceptive behavior induced by intraplantar injection of formalin. (A) Blockage of P2X7 receptors at DRG by A740003 administration at DRG. (B) Blockage of gap junctions by carbenoxolone administration at DRG. The P2X7 receptor antagonist A740003 (1 nM, 5 µL in saline + 10% DMSO), the gap junctions blocker carbenoxolone (1 nM, 5 µL in saline) or their respective vehicles (control) were injected at the right L5 dorsal root ganglia 30 minutes before formalin (2.5% in 50 µL of saline). Nociceptive response (flinching or licking the paw) was counted in 5 minutes blocks that were averaged in phase1 (first 15 minutes) and phase2 (20 to 60 minutes). Results presented as means ± SEM of the number of animals indicated inside bars. *P<0.001, (t-test).
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