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Research Report

Activity-dependent neuronal control of gap-junctionalcommunication in fibroblasts

Yan Zenga,1, Xiaohua Lvb,1, Shaoqun Zengb,⁎, Jing Shia,⁎aDepartment of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology, HUST, 13 Hangkong Road,Wuhan 430030, ChinabBritton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics,Huazhong University of Science and Technology, Wuhan 430074, China

A R T I C L E I N F O

⁎ Corresponding authors. J. Shi is to be contacTechnology, 13 Hangkong Road, Wuhan 4300Wuhan National Laboratory for Optoelectron

E-mail addresses: sqzeng@mail.hust.edu.Abbreviations: DRG, dorsal root ganglia; G

connexin 43; N/F, Neuron/Fibroblast; LY, L(aminoethylether)tetraacetate; DMSO, dimetDulbecco's modified Eagle's medium; HEPESnoxy) ethane-N,N,N′,N′-tetraacetic acid; HD,1 These authors contributed equally to this

0006-8993/$ – see front matter © 2009 Elsevidoi:10.1016/j.brainres.2009.05.037

A B S T R A C T

Article history:Accepted 13 May 2009Available online 21 May 2009

Intercellular communication through gap junctions plays an important role in fibroblastphysiology. Here we report an activity-dependent neuronal control of gap-junctionalcommunication (GJC) in rat dermal fibroblasts, which is associated with N-methyl-D-aspartate(NMDA) glutamate receptor-mediated elevations in intracellular Ca2+. Both spontaneous andinduced activation of dorsal root ganglion (DRG) neurons, manifested by action potentials andTTX-dependent inward currents, significantly reduced fibroblast GJC in Neuron/Fibroblast (N/F)cocultures. Reduced fibroblast GJC by DRG neurons was prevented by blockade of NMDAreceptors and decrease of intra- and intercellular Ca2+. Immunocytochemistry showed that theNR1 subunit of the NMDA receptor coexisted with CX43 at the plasma membrane offibroblasts. Moreover, glutamate applied to fibroblast cultures triggered NMDA receptor-dependent intracellular Ca2+ elevations and decrease of GJC. These data demonstrate thatNMDA receptor activation contributes to downregulation of GJC in fibroblasts. Sincefibroblasts have been shown to facilitate DRG neurite growth and survival, our findingsprovide a glimpse at the impact that neuronal activation has on fibroblast networks.

© 2009 Elsevier B.V. All rights reserved.

Keywords:DRG neuronDermal fibroblastGap-junctional communicationConnexin 43NMDA receptor

1. Introduction

Fibroblasts form cellular networks extensively interconnectedthrough gap junctions (Hunter and Szigety, 1992; Jester et al.,1995; De Roos et al., 1996; Langevin et al., 2004). Gap-junctionalcommunication (GJC) in fibroblast networks has been found in

ted at Dept. of Neurobiolo30, China. Fax: +86 027 8ics, Huazhong Universitycn (S. Zeng), sj@mails.tjmJC, gap junction communucifer yellow; ATP, adenhyl sulfoxide; [Ca2+]i, intr, N-2-hydroxyethylpiperaHuntington's diseasework.

er B.V. All rights reserved

several tissues including kidney, intestine, and skin (Joyce,1987; Komuro, 1990; Adegboyega et al., 2002). Fibroblasts canalso be coupled to other cells through gap junctions, includingmyocytes (Rook et al., 1992), endothelial (Hunter and Pitts,1981), neuronal (Courbin et al., 1989), and mast cells (Au et al.,2006). Since fibroblasts do not generate action potentials and

gy, Tongji Medical College of Huazhong University of Science and3692608. S. Zeng, Britton Chance Center for Biomedical Photonics,of Science and Technology, Wuhan 430074, China.u.edu.cn (J. Shi).ication; NMDA, N-methyl-D-aspartate; TTX, Tetrodotoxin"; CX43,osine 5′-triphosphate disodium salt; EGTA, ethyleneglycol bisacellular Ca2+ concentration; BSS, balanced salt solution; DMEM,zine-N-2-ethanesulfonic acid; BAPTA-AM, 1, 2-bis (2-aminophe-

.

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are devoid of synaptic contacts, the existence of an exten-sively interconnected cellular network allows communicationamong them. Considerable research has indicated that GJCpatterns in fibroblasts play a key role in regulating the balanceof cytokines, chemokines, and growth factors, further regulat-ingmigration, proliferation, differentiation, andmatrix synth-esis during the process of wound healing or inflammation(Komuro 1990; Ehrlich and Diez, 2003; Ehrlich et al., 2006).

Fibroblast GJC is commonly opened (Furuya et al., 2005;Venance et al., 1997) and depends on extracellular K+ and Ca2+,suggesting that the permeability of gap junctions can bealtered by physiological and pharmacological stimuli. Func-tional studies performed in cultures have demonstrated thatfibroblast GJC is controlled by neurotransmitters, cytokines,growth factors, and other bioactive compounds (Langevin etal., 2004; Daubrawa et al., 2005).

The dermis is tightly connected to the epidermis by abasement membrane, and harbors many nerve endings thatunderlie the sense of touch and heat. Dermal fibroblasts andfibroblast-like cells are in close contact with sensory nerveterminals (Güldner, 1972; Desaki et al., 1984; Joyce 1987). Nerveterminals release signaling substances that may affect fibro-blasts under physiological or pathological conditions. In fact,successful healing of wounds requires sensory innervationand the release of vasoactive neuropeptides that dilate bloodvessels and deliver serum proteins to the wound so as toprevent further injury (Cruise et al., 2004). An importantproblem in neurobiology is the investigation of the mutualinteractions between neurons and fibroblasts. In particular,during the last decade, an active role of fibroblasts in neuronfunction has raised a lot of interest, thanks to several studiesestablishing that fibroblasts may participate in processinginformation and affect neurite outgrowth (Jerregard et al.,2000; Jerregard 2001). The influence neurons have on fibro-blasts, however, has not yet been investigated.

Thepresent studywasundertaken to explorewhetherdorsalroot ganglion (DRG) neurons can directly regulate intercellularcommunication in dermal fibroblasts. Cocultures were used todetermine the neuronal influence on GJC in dermal fibroblasts.We demonstrate that activity-dependent neurotransmitterrelease downregulates GJC in fibroblasts. Both TTX-sensitivespontaneous or induced neuronal activity reduced fibroblastfunctional coupling. In addition, immunocytochemistry andCa2+ imaging in fibroblasts demonstrated expression of func-tional NMDA receptors. The influence of DRG neurons on GJCwas associated with elevation of cytosolic Ca2+ via NMDAreceptors as application of MK-801, an antagonist of NMDAglutamate receptors, prevented the reduction in GJC. Ourfindings suggest that communication amongdermal fibroblastsis significantly affected by neuronal activity.

2. Results

2.1. DRG neurons reduce gap-junctional permeabilityin fibroblasts

The effects of DRG neurons on dye coupling betweenconfluent fibroblasts were investigated by comparing GJC

in pure fibroblast cultures with that in mixed N/F cultures.Fibroblast cultures could reach 95% purity whereas cocul-tures contained ∼33% DRG neurons and 67% fibroblasts,identified by their morphological features under light andfluorescence microscopy. GJC was first investigated in purefibroblast cultures with a patch pipette loaded with LuciferYellow (LY). The dye was delivered into fibroblasts viaiontophoresis. In cultures of fibroblasts devoid of DRGneurons, the average incidence of dye coupling per experi-ment was 27% for 1–10 coupled cells and 51% for >10coupled cells, whereas in the remaining 24% of the cells, nodye coupling was observed (1a, 1b, and 1e). In N/F cocultures,the average incidence of dye coupling was 21% for 1–10coupled cells, 30% for >10 coupled cells, and 49% of cells didnot show dye coupling (Figs. 1c, d, and e), which suggested asignificant reduction of GJC in N/F cocultures. Theseobservations were supported by testing dye coupling usingscrape-loading in N/F cocultures. In these conditions, junc-tional permeability in fibroblasts was reduced only whenneurons were kept for one week or more in cocultures,suggesting that a long-term presence of DRG neurons wasnecessary. This time-dependent action of DRG neurons onfibroblast GJC was confirmed by using the N/F model at acritical time, 5–6 d after neuronal adherence (1f, n=15). LYdiffusion in N/F cocultures was markedly reduced by apotent inhibitor of GJC heptanol (500 μM, 10 min). Thisreduction in dye diffusion reached 77% (data not shown,n=4) as compared with controls. This indicates that theneuron-induced decrease in dye coupling is mediated bygap-junctional channels. In addition to fibroblast–fibroblastjunctions, we also occasionally observed gap junctioncoupling between DRG neurons and fibroblasts, as reportedby others (Courbin et al., 1989).

Because a high percentage of DRG neurons in culture arespontaneously active (Bergey et al., 1984; Beaudu et al., 2000),we asked whether blocking the electrical activity of DRGneurons in N/F cocultures by chronic treatment with a Na+

channel blocker, tetrodotoxin (1 μM, TTX) would prevent theaction of DRG neurons on GJC in fibroblasts. When recorded intheir culture medium, about one-half of the DRG neuronsdisplayed medium amplitude spontaneous action potentialsand inward currents (Figs. 2a and b, n=19). We define theseDRG neurons with spontaneous inward currents as ‘active’neurons. Treatment for 24 h–4 d with 1 μM TTX abolishedboth spontaneous inward currents in DRG neurons and inparallel significantly enhanced the percentage of the inci-dence of gap junction permeability in fibroblasts (Fig. 2c,n=13). This result indicates that the DRG neuronal effects onfibroblast GJC is activity-dependent.

2.2. Stimulated DRG neurons reduced gap-junctionalpermeability in fibroblasts

Since the neuron-induced downregulation of fibroblast wasactivity-dependent, attempts were made to determinewhether induced excitation of neurons produced similareffects on fibroblast GJC in coculture models. First, underwhole-cell current clamp conditions, at a membrane potentialof −70 mV, current trains (1s, 10 Hz) were applied to DRGneurons to induce activation. After 6–7 d in cocultures, over

Fig. 1 – Dye coupling between fibroblasts is downregulated by DRG neurons. Dye coupling was determined by diffusion of apipette solution containing 0.2% LY. a–d, Light micrographs taken with DIC of (a) fibroblast culture and (c) coculture andfluorescence micrographs of the same fields (b and d, respectively) taken 5 min after withdrawal of the patch pipette. Thenumber of dye-coupled cells is decreased in the presence of DRG neurons, indicating a decrease in the permeability of gapjunctions. e, summary diagram of dye coupling experiments obtained from 144 fibroblasts filled with LY and classified in threecategories, noncoupled (0), weakly coupled (1–10; stained cells), and strongly coupled (>10 stained cells). TheΧ2 comparison testfor the three categories reveals a significant difference in the distribution of fibroblast coupling measured in the absence andpresence of neurons (P<0.01). f, the effect of DRG neurons on fibroblast GJC is time-dependent. The level of GJC (fibroblastcoupling) was evaluated in N/F cocultures using the scrape-loading dye transfer technique, and was expressed as arbitraryunits referring to the fluorescence area. A significant decrease in fibroblast GJCwas first detected 6 d after neuronal plating. Theratios between the fluorescence areas of tests (N/F cocultures) and internal controls (fibroblast cultures) are: 1.51, 1.42, and 1.03for 1–5, 6, and 9 d, respectively. Statistical analysis was carried out using a t test. Bar, 20 μm.

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90% of DRG neurons exhibited large action potentials inresponse to membrane depolarizations (Fig. 3b, n=15). Afterneuronal activation, we performed dye transfer assays infibroblasts using LY. The average incidence of dye coupling

was 24% for 1–10 coupled cells, 17% for >10 coupled cells, and59% of cells did not show dye coupling (Fig. 3a, n=17), whichsuggests a significant reduction in fibroblast GJC followingactivation of DRG neurons. These results indicate that

Fig. 2 – Patch-clamp recordings of spontaneous actionpotentials and Na+ inward currents recorded from DRGneurons in N/F cocultures 7 d (a and b) after neuronal plating.a, occurrence of spontaneous action potentials illustrated bya continuous voltage trace recorded at a membrane potentialof −60mV and the blocking effect of TTX on action potentials.b, occurrence of spontaneous inward currents illustrated by acontinuous current trace recorded at a holding potential of−60mV and the blocking effect of TTX on the current. Currenttraces shown in a and b were from the same cells and wereobtained with pipettes filled with a CsCl solution. c, the roleof DRG neurons on dye coupling was reversed by addition of1 μM TTX into N/F cocultures.

Fig. 3 – MK-801 prevented the action of excited andspontaneously active DRG neurons on the fibroblast GJC. Athree-way comparison test for the three categories reveals asignificant difference in the distribution of fibroblast couplingmeasured in the with and without MK-801 groups (**P<0.01),and with and without stimulation in N/F cocultures.

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intracellular stimulation of DRG neurons acutely affectedfibroblast GJC.

Interestingly, we also found that blockade of N-methyl-D-aspartate (NMDA) receptors in N/F cocultures prevented theeffects of DRG neurons on fibroblasts by increasing theincidence of GJC. The NMDA receptor non-competitive antago-nist dizocilpine maleate (MK-801, 10 μM), applied to the bathbefore the DRG neuron stimulation, prevented the reduction offibroblast GJC. The average incidence of dye coupling was 28%for 1–10 coupled cells, 47% for >10 coupled cells, and 25%of cellsdidnot showdyecoupling (Fig. 3a,n=13).MK-801alsopreventedthe effects of non-stimulated DRG neurons when applied for along-term to the N/F cocultures (Fig. 3a, n=13). We found thatMK-801 reduced gap junction communication in fibroblastcultures alone (data not showed). These data indicate that thereduction of GJC strength between the dermal fibroblasts wasdue to the long-termor transient activation ofNMDAglutamatereceptors located in fibroblasts, and the likely glutamate releasefrom excited or spontaneously active DRG neurons.

2.3. Role of DRG neurons on fibroblast GJC isCa2+ dependent

To elucidate whether the effects of DRG neurons on fibroblastGJC are dependent on Ca2+ influx through NMDA receptorchannels, two paradigms were utilized to examine inhibitionof GJC: incubating the cocultures with 1, 2-Bis (2-aminophe-noxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) (100 μM)and using a Ca2+-free buffer for at least 5 min (2 mM EGTA). InCa2+-free bath, depolarization of DRG neurons still inducedsmall-amplitude action potentials, but the average incidenceof dye coupling per injection was 33% for 1–10 coupled cellsand 43% for >10 coupled cells (Fig. 4, n=8), whereas in 24% ofthe cells no dye coupling was observed. This result wassignificantly different from that with Ca2+ in the bath, andwas consistent with that from pure fibroblast cultures (Fig. 4,n=7). In cocultures with BAPTA loading into fibroblasts, theaverage incidence of dye coupling per experiment was 31%for 1–10 coupled cells and 46% for >10 coupled cells, whereasin 23% of the cells no dye coupling was observed (Fig. 4, n=6).Interestingly, we also found that both EGTA and BAPTAreduced the dye coupling in fibroblast culture alone (Fig. 4,n=6), but there are no significant difference compared tofibroblast alone control. Together, these results allow us toconclude that both intracellular and extracellular Ca2+ isnecessary fibroblast GJC.

Fig. 4 – the role of DRG neurons on fibroblast GJC is Ca2+

dependent. Removing extracellular Ca2+ with EGTA orintracellular Ca2+with BAPTA could prevent the action of DRGneurons on fibroblast GJC.

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2.4. Co-localization of CX43 and NMDA-R1 (NR1)glutamate receptor protein and its functional role in fibroblasts

Since the downregulation of fibroblast GJC is related to NMDAreceptor activation in fibroblasts, the expression and functionof the NMDA receptors in fibroblasts was characterized by

Fig. 5 – Expression and function of NR1 subunits of the NMDA recin fibroblast cultures. Green, fibroblasts were probed with NR1 ayellow, overlapping images of the same region displaying NR1 anco-localization of the proteins. Bar, 20 μM. b, Ca2+ imaging withwere taken by confocal microscope from fibroblasts exposed to 1did not cause [Ca2+]i increase after applying MK-801 or EGTA. Ba

using double labeling immunofluorescence and confocal Ca2+

imaging. Confocal immunofluorescence microscopy revealedwidespread co-localization of CX43 and NR1 subunit of theNMDA receptor (Fig. 5a, n=7). NR1 subunits were found inclusters of transmembrane particles in the plasmamembrane,and distinctive densities were in close proximity to CX43labeling. These results provide further evidence that NMDAreceptor activation has the potential to regulate the activity offibroblast gap junctions.

Next, we examined the functional role of NMDA recep-tors in fibroblasts using Ca2+ imaging with confocal micro-scopy (Olympus, FV-1000). After treatment with glutamate(100 μM), sustained and significant elevations of intracel-lular Ca2+ ([Ca2+]i) levels were observed and these elevationswere suppressed by MK-801(10 μM) (Fig. 5b, n=12). This dataindicate that NMDA receptors expressed in fibroblastswere functional.

2.5. Neuron-induced downregulation of GJC was linkeddirectly to the effect of glutamate released from DRG neurons

To determine whether the TTX-sensitive neuron-induceddownregulation of GJC in fibroblasts was linked directly tothe effect of glutamate released from DRG neurons, fibroblastcultureswere exposed for either a brief (10min) or a long (72 h)period to glutamate. We found that the incubation of

eptor in fibroblast cultures. a, co-localization of NR1 and CX43ntibody; Red, fibroblasts were probed with CX43 antibody;d CX43 specific labeling weremerged to visually demonstrateFluo-3 in fibroblasts. The representative time course curves00 μM glutamate with or without 10 μM MK-801. Glutamater, 20 μm.

Table 1 – Effects of glutamate and conditioned culture media on fibroblast GJC.

Coupledcells

Number of injections

Control Glutamate(10 min)

Glutamate(72 h)

Neuronalmedia

N/F coculturemedia

Glutamate+MK-801

N/F coculture media+MK-801

0 25±1.4 47±3.4 ⁎ 51±4.5 ⁎ 48±3.2 ⁎ 46±3.3 ⁎ 27±1.4 28±2.10–10 29±1.7 28±1.9 ⁎ 26±1.5 ⁎ 25±1.6 ⁎ 26±1.7 ⁎ 35±1.8 35±2.7>10 54±4.3 25±1.3 ⁎ 23±1.2 ⁎ 23±1.2 ⁎ 24±1.2 ⁎ 48±3.4 48±3.4

Data were expressed as mean±SEM.⁎ P<0.05 versus single fibroblast cultures (control).

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fibroblast cultures with glutamate (400 μM, 10 min) wasfollowed by a large decrease in the diffusion of LY (Table 1.n=9). Long-term exposure to glutamate (100 μM, 72 h) alsoreduced significantly GJC in fibroblast cultures (Table 1, n=9).The effects of glutamate could be prevented in both cases byco-treatment with 10 μM MK-801. Interestingly, a significantchange in LY diffusion was observed when conditionedmediafrom neuronal cultures (Table 1, n=9) or N/F cocultures (Table1, n=9) were added for 48 h onto two-week-old fibroblastcultures. This strongly suggests that glutamate is normallyreleased from DRG neurons to the external medium andthrough NMDA receptors is responsible for the neuron-induced downregulation of GJC in fibroblasts. These resultsindicate that glutamate via stimulation of NMDA glutamatereceptors inhibits fibroblast GJC. Treatment with glutamateincreased cytoplasmic Ca2+ in fibroblasts, further arguing forthe presence of functional NMDA receptors on fibroblasts.

3. Discussion

These present study provides conclusive evidence that DRGneurons downregulate GJC in dermal fibroblasts via therelease of a diffusible factor, glutamate acting on NMDAreceptors of fibroblasts to increase intracellular Ca2+. We showthat the DRGneuron-induced downregulation of fibroblast GJCoccurs when neurons are in a non-stimulated or in anactivated state, and blockade of the NMDA subtype ofglutamate receptors prevented the neuron-induced down-regulation of fibroblast GJC. Furthermore, NMDA receptor-dependent changes in fibroblast gap junction channels wereattributable to an elevation of [Ca2+]i. Together, these observa-tions indicate that GJC in fibroblasts represent an importanttarget in the neuron-fibroblast partnership. DRG neurons arekey elements in sensory signaling under physiological andpathological conditions. The activation of these neurons hasbeen previously shown to regulate electrical coupling inSchwann cells further to promote cell proliferation andmyelination of axons by Schwann cells (Guenard et al., 1994;Huang et al., 2005). Our findings argue for an activity-dependent effect of DRG neurons on gap junction permeationof surrounding non-neuronal cells.

GJC is widespread in dermal fibroblasts and important forhomeostasis, growth control, inflammation and development(Langevin et al., 2004). Several mechanisms known to regulatethe permeability of gap junction channels in fibroblasts havebeen considered to elucidate the process involved in the

neuron-induced decrease of GJC. Our study demonstrates anactivity-dependent neuronal regulation of fibroblast GJC. DRGneurons in vivo or in vitro exhibit spontaneous electricalactivity (Bergey et al., 1981; Mathers and Barker, 1984; Russelland Burchiel, 1988; Kajander et al., 1992; Beaudu et al., 2000).Using patch-clamp recordings of DRG neurons, we showedthat DRG neurons exhibit spontaneous action potentials. Thiselectrical activity was suppressed by TTX treatment. This is inagreement with other studies indicating that TTX blocksneuronal activity (Enomoto et al., 2006). The neurons exhibit-ing spontaneous inward Na+ currents were considered as‘active’ DRG neurons. By inhibiting neuronal activity, long-term applications of TTX (>24 h) significantly suppressed theneuronal stimulatory effect on fibroblast GJC (Fig. 2). There-fore, our observations demonstrated that DRGneurons controlGJC in fibroblasts and this was likely related to spontaneousspiking properties. Nathalie Rouach et al. demonstrated thatneurons upregulate GJC and the expression of CX43 inastrocytes, which depended to some degree on spontaneousfiring of neurons (Rouach et al., 2000). In addition, we foundthat excitation of DRG neurons by electrical stimulationreduced GJC of fibroblasts in N/F cultures more significantly(Fig. 3), further demonstrating that the effects of DRG neuronson fibroblast GJC are activity-dependent.

Another finding in our study was that the reduction offibroblast GJC depends on the activation of NMDA receptorsand the consequent increase in [Ca2+]i in fibroblasts, as DRGneuron effects could be prevented by the NMDA receptorantagonist MK-801 and intracellular injections of the Ca2+

chelator BAPTA-AM into fibroblasts (Figs. 4 and 5). Thepossible explanation of our findings is that GJC inhibitionmay be the result of NMDA receptor-mediated [Ca2+]i eleva-tions. We provide evidence by confocal microscopy for thepresence of the NR1 subunit of the NMDA receptor subtype,proposed to be a key regulatory element of [Ca2+]i, and thatthis subunit is colocalized with CX43 at the plasmamembraneof cultured fibroblasts. In addition, the profile obtained withCa2+-imaging experiments also suggests NMDA receptorsmediated sustained and large [Ca2+]i elevations in fibroblasts.A change in Ca2+ homeostasis seems to be involved in theneuron-induced decrease in fibroblast GJC. [Ca2+]i elevation isgenerally considered to inhibit cell coupling in fibroblastsexpressing CX43 (Dakin and Li, 2006) as well as in other cells(Baux et al., 1978). Furthermore, directly increasing the levelsof cytosolic Ca2+ in hippocampal neurons leads to occlusion ofdye coupling (Rao et al., 1987). These effects may be mediateddirectly via Ca2+-activated phosphorylation of the connexinsubunits (Rorig and Sutor, 1996). Indeed, when we depleted

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intercellular and intracellular Ca2+ to prevent [Ca2+]i eleva-tions, DRG neurons did not affect fibroblast GJC anymore.Studies have shown that when hypothalamic cultures arechronically treated with the NMDA receptor antagonist MK-801, developmental uncoupling of gap junctions and down-regulation of connexin 36 are abolished (George et al., 2002).

Since the reduction of GJC strength between dermalfibroblasts could be induced by both transient and long-term activation of NMDA receptors, glutamate release fromexcited DRG neurons appeared likely. Glutamate is present inDRG neurons of rats (Yang et al., 1998) and can be releasedfrom excited and spontaneously ‘active’ DRG neurons inresponse to stimuli that induce increases in intracellular Ca2+

levels. In cultured and isolated DRG neurons, it has beenfound that glutamate is released in response to adenosinetriphosphate (ATP), potassium (K+), bradykinin (BK)- orcapsaicin (CAPS) (Gu and MacDermott, 1997; Rydh et al.,2001), or accumulated in the extracellular space withoutstimulation (Barakat and Droz, 1990; Rydh et al., 2001; Jeftinijaet al., 1991). Moreover, cultured fibroblasts isolated fromembryonic muscle, skin and peripheral nerve tissues werealso found to accumulate [3H] L-glutamate (Balcar et al., 1994).Previous studies have shown that neurotransmitters acutelymodulate gap-junctional coupling in both neurons and glialcells (Rorig and Sutor, 1996; Roerig and Feller, 2000). In the ratsomatosensory cortex, dye coupling is significantly reducedby noradrenaline and 5-HT (Roerig and Feller, 2000). Earlypostnatal blockade of the NMDA glutamate receptors usingMK-801 arrested the developmental decrease in electrotonicand dye coupling during the first postnatal week (George etal., 2002). In addition, activation of Ca2+-permeable ionotropicglutamate receptors has been shown to result in a decrease ingap-junctional conductance in cerebellar Bergman glia (Mul-ler et al., 1996), an effect that could be blocked in Ca2+-freemedium. Thus, it is possible that sustained activation ofNMDA receptors may lead in the short-term to occlusion andin the longer term to degradation or downregulation of gap-junctional proteins located in fibroblasts.

In fact, glutamate has been found to regulate the functionof fibroblasts, although the exact mechanism remains to beelucidated. For example, human cultured skin fibroblastsundergo rapid cellular degeneration and cell death whenexposed to moderate levels of glutamate (10 mM–30 mM).Moreover, Huntington's disease (HD) skin fibroblast culturesaremore sensitive to the toxic effects of glutamate (Gray et al.,1980) and the toxic mechanisms may relate to degenerativeprocesses occurring in the HD brain (May and Gray, 1985; Stahlet al., 1984). Thus, we can speculate that glutamate is involvedin the regulation of fibroblast function in physiological andpathological conditions. On the other hand, our study suggeststhat DRG neurons affect fibroblast-fibroblast communicationby multiple signaling mechanisms. Fibroblasts express manykinds of receptors, such as angiotensin II, ATP, ADP, bradyki-nin, serotonin, substance P (Furuya et al., 1994). It is possiblethat other transmitters such as substance P andATP, known tobe released by fibroblasts, could also be involved in regulatinggap-junctional coupling and further studies investigatingblockade of their receptors are warranted.

In conclusion, we investigated the effects of DRG neuronson fibroblast GJC. Our results indicate that glutamate

released by DRG neurons can act directly on Ca2+ levels offibroblasts, resulting in modulation of GJC. Glutamate acti-vates this DRG neuron-to-fibroblast signaling pathway uponstimulation of NMDA receptors. Both spontaneous anddepolarization-evoked activity of neurons results in thereduction of fibroblast GJC.

4. Experimental procedures

4.1. Fibroblast cultures

Normal dermal fibroblasts were prepared from SpragueDawley rats within 24 h postnatally. The dorsal skin wasdissected in PBS buffered salt solution, and incubated in asolution of collagenase in DMEM medium for 16 h at 4 °C. Thedermis was peeled off the epidermis with forceps and gentlystirred in 0.05% trypsin for 15min at 37 °C. Cells were seeded ata density of 2×10 4 cells/ml onto poly-L-lysine-coated(25 μg/ml) glass coverslips. The medium contained minimalessential medium and was supplemented with antibiotics(0.17% penicillin V, 0.1% gentamycin sulfate, and 0.01 μg/mlamphotericin) and 10% fetal bovine serum. The cells werecultured at 37 °C in a humidified atmosphere of 95% air and 5%CO2. The medium was changed every 3 days.

4.2. DRG neuron cultures

Primary cultures of DRG neurons were prepared from SpragueDawley rats within 24 h postnatally. The DRG was dissected inPBS buffered salt solution and incubated in 0.25% trypsin inDMEM medium at 37 °C for 15 min, washed three times inDMEM medium at room temperature, and triturated 30–40times using a fire-polished Pasteur pipette. Neurons wereseeded at a density of 2×104 cells/ml onto poly-L-lysine-coated(25 μg/ml) glass coverslips. After 24 h, this medium wascompletely replaced with a maintenance medium containing96% Neurobasal, 2% B27, and 1% L-glutamine (GIBCO BRL); thismedium was subsequently given half-changes every 3 days.With the use of this protocol, 75–80% of the cultured cells wereDRG neurons.

4.3. DRG neuron/dermal fibroblasts (N/F) cocultures

N/F cocultures were obtained by adding dissociated DRG cellsto 1-wk-old primary cultures of confluent fibroblasts. Theinternal control was provided by incubating sister dishes ofconfluent fibroblasts originating from the same culture. Poly-L-lysine-coated (1.5 μg/ml) 35-mm diameter culture dishes(2×106cells/dish; NUNC) were used for scrape-loading.12-mm coverslips (3×105 cells/coverslip) were employed forimmunocytochemistry, electrophysiogy and Ca2+ imagingexperiments. Unless otherwise stated, the number of disso-ciated cells added to the primary cultures of 1-wk-oldfibroblasts represented half the number of originally platedcells. This second plating mainly resulted in neurons, as thenumber of fibroblastswas similar in fibroblasts cultures andN/F cocultures. After 7 d in coculture under these conditions, 67%of the cells were fibroblasts and 33% were DRG neuronsaccording to phase contrast microscopy identification of

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typical morphology of fibroblasts and DRG neurons. In addi-tion, identification of morphology of fibroblasts and DRGneurons was facilitated by safranine staining. For this purposecells were exposed to safranine (1%) during 5 min and thenrinsed with water and alcohol. Field images were analyzedwith computer image analysis software (NIH ImageJ) using aCCD camera connecting the microscope to the computer. Theperimeter of the cell body, the number of primary neurites, thetotal length of neurites, and the number of branching pointswere measured. Six neurons per coverslip were analyzed.

4.4. Electrical stimulation

Patch-clamp recordings were performed in the whole-cellconfiguration by using a PC2/C patch-clamp amplifier and apCLAMP software (Huangzhong University of Science andTechnology, China). Patch pipette resistance was 2–4 MΩ.Membrane voltage was held at −60 mV and the spontaneousand induced membrane electrical activities in DRG neuronswere recorded. In order to activate DRGneurons, current trains(1 s, 10 Hz) were applied. Standard external solution contained(in mM): NaCl 150, KCl 5.0, CaCl2 2.0, MgCl2 1.0, HEPES 10, andglucose 10 (pH 7.4 adjusted with NaOH). For a Ca2+-free bath,we removed CaCl2 from the external solution and added 2mMEGTA. The standard internal solution contained (in mM) CsCl153, MgCl21, HEPES 10 and ATP 4 (pH 7.2). All experiments wereperformed at room temperature (22–25 °C).

4.5. Confocal Ca2+ imaging

To examine the ability of glutamate to evoke Ca2+ spikes infibroblasts, cells were loaded with Fluo-3AM (5 μM, MolecularProbes) via incubation with acetoxymethyl ester Pluronic-127for 30 min and were then subjected to confocal line scanningimaging (LSCM system FV1000, Olympus) and a randomaccesstwo-photon microscopy (Zeng et al., 2006; Lv et al., 2006), andimaged using a 40× water immersion lens (numerical aperture0.9). A single neuron of 15–40 μm in diameter was selected andexcited at 488 nm, whereas fluorescence was detected at530±15 nm. Only one neuron per coverslip was used forexperiments. The fluorescence signals were normalized withrespect to the resting fluorescence intensity (F0) and expressedas F/F0. Extracellular solution contained (in mM) NaCl 150, KCl5.0, MgCl21.1, glucose10, HEPES 10, and CaCl2 2.0 (pH 7.4). For aCa2+-free bath, we removed CaCl2 from the external solutionand added 2 mM EGTA. Image processing and data analysiswere performed by using FV-1000 (Olympus).

4.6. Determination of gap-junctional permeability

To conduct dye transfer assay, fibroblasts were iontophor-etically injected with the low molecular weight tracer Luciferyellow (LY, 2%) using depolarizing current pulses (0.5–3 nA,200 ms duration at 3.3 Hz for 3 min). The pipette was thenwithdrawn and the microscopic field around the injected cellwas photographed 5 min later under epifluorescence illumi-nation using a CCD camera with appropriate filters. Gap-junctional permeability was quantified by determining thenumber of adjacent fluorescent fibroblasts. In cases wheremore than one fibroblast was impaled per preparation, care

was taken to separate the second electrode track by at least500 μm in the rostro-caudal axis in order to avoid accidentallabeling of other cells. Transient impalement, where the cellwas held hyperpolarized for less than 5 min, did not result infibroblast labeling. GJC was also studied using the scrape-loading technique, as previously described (Venance et al.,1997). For each trial, data were quantified by counting the cellsloadedwith fluorescence in 5 consecutive fields from digitizednegatives using image analysis software (NIH ImageJ).

4.7. Immunocytochemistry

Cells were fixed with 4% paraformaldehyde at 4 °C for 20 minand permeabilized with Tween-20 (0.05%, pH 7.5). Immunos-taining for NR1 and CX43 was performed by incubating thecultures with antibodies diluted in the Tween-20 buffer.Cultures were simultaneously incubated with primary anti-bodies against NR1 (polyclonal rabbit–goat), diluted 1:200;Sigma-Aldrich) and against CX43 (monoclonal mouse, diluted1:200). Secondary antibodies were applied in different combi-nations and included rhodamine-conjugated goat anti-mouseIgG1 (diluted 1:250) and fluorescein-conjugated goat anti-rabbit IgG (diluted 1:200). Rinsing was performed betweenevery incubation step. After staining, coverslips weremounted on glass slides in moviol and examined withconfocal microscopy (FV-1000 Japan).

4.8. Drugs

Chemicals used; Glutamate, BAPTA-AM, EGTA, MK-801, tryp-sin, heptanol, and TTX were purchased from Sigma (St. Louis,MO). For compounds dissolved in dimethyl sulfoxide (DMSO),the control cultures included 0.1% DMSO in the medium. LYand Fluo-3AMwere purchased fromMolecular Probes (Eugene,OR). Cell culture materials were bought from GIBCO BRL,Invitrogen (Gaithersburg, MD). NR1, (polyclonal goat) and CX43(monoclonal mouse) were purchased from Sigma-Aldrich andChemicon. Secondary antibodies were purchased from South-ern Biotechnology Associates. Other reagents were purchasedfrom Shanghai Life Technology Company.

4.9. Statistics

Data were expressed as mean±SEM. n refers to the number ofindependent experiments. All statistical analyses were per-formed on raw data. One-way ANOVA was used determinestatistical significance acrossmultiple groupmeans. Student'st test was used for two-group comparisons. And the Χ2

comparison was used for to test the three categories.Statistical significance was established at P<0.05 and P<0.01as indicated.

Acknowledgments

We are grateful to Dr. Carlos Cepeda for invaluable assistancein manuscript preparation. We also thank Dr. Shunlian Tianfor help in cell cultures. This work was supported by theNational Natural Science Foundation of China.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.brainres.2009.05.037.

R E F E R E N C E S

Adegboyega, P.A., Mifflin, R.C., DiMari, J.F., Saada, J.I., Powell, D.W.,2002. Immunohistochemical study ofmyofibroblasts in normalcolonic mucosa, hyperplastic polyps, and adenomatouscolorectal polyps. Arch. Pathol. Lab. Med. 126, 829–836.

Au, S.R., Au, K., Saggers, G.C., Karne, N., Ehrlich, H.P., 2006. Ratmast cells communicate with fibroblasts via gap junctionintercellular communications. J. Cell. Biochem. 100, 1170–1177.

Balcar, J., Shen, J., Bao, S., King, N.J., 1994. Na+-dependent highaffinity uptake of L-glutamate in primary cultures of humanfibroblasts isolated from three different types of tissue. FEBSLett. 339, 50–54.

Barakat, W.I., Droz, B., 1990. Glutamine synthetase is expressed byprimary sensory neurons from chick embryos in vitro but notin vivo: influence of skeletal muscle extract. Eur. J. Neurosci. 2,836–844.

Baux, G., Simonneau, M., Tauc, L., Segundo, J.P., 1978. Uncouplingof electrotonic synapses by calcium. Proc. Natl. Acad. Sci.U. S. A. 75, 4577–4581.

Beaudu, L.C., Colomar, A., Israel, J.M., Coles, J.A., Amedee, T., 2000.Spontaneous neuronal activity in organotypic cultures ofmouse dorsal root ganglion leads to upregulation of calciumchannel expression on remote Schwann cells. Glia 29, 281–287.

Bergey, G.K., Fitzgerald, S.C., Schrier, B.K., Nelson, P.G., 1981.Neuronal maturation in mammalian cell culture is dependenton spontaneous electrical activity. Brain Res. 207, 49–58.

Courbin, P., Koenig, J., Ressouches, A., Beam, K.G., Powell, J.A.,1989. Rescue of excitation–contraction coupling in dysgenicmuscle by addition of fibroblasts in vitro. Neuron 2, 1341–1350.

Cruise, B.A., Xu, P., Hall, A.K., 2004. Wounds increase activin inskin and a vasoactive neuropeptide in sensory ganglia. Dev.Biol. 271, 1–10.

Dakin, K., Li, W.H., 2006. Local Ca2+ rise near store operated Ca2+channels inhibits cell coupling during capacitative Ca2+ influx.Cell Commun. Adhes. 13, 29–39.

Daubrawa, F., Sies, H., Stahl, W., 2005. Astaxanthin diminishes gapjunctional intercellular communication in primary humanfibroblasts. J. Nutr. 135, 2507–2511.

De Roos, A.D.G., van Zoelen, E.J.J., Theuvenet, A.P.R., 1996.Determination of gap junctional intercellular communicationby capacitance measurements. Pflügers Arch. 431, 556–563.

Desaki, J., Fujiwara, T., Komuro, T., 1984. A cellular reticulum offibroblast-like cells in the rat intestine: scanning andtransmission electron microscopy. Arch. Histol. Jpn. 47,179–186.

Ehrlich, H.P., Diez, T., 2003. Role for gap junctional intercellularcommunications in wound repair. Wound Repair Regen. 11,481–489.

Ehrlich, H.P., Sun, B., Saggers, G.C., Kromath, F., 2006. Gap junctioncommunications influence upon fibroblast synthesis of Type Icollagen and fibronectin. J. Cell. Biochem. 98, 735–743.

Enomoto, A., Han, J.M., Hsiao, C.F., Wu, N., Chandler, S.H., 2006.Participation of sodium currents in burst generation andcontrol of membrane excitability in mesencephalic trigeminalneurons. J. Neurosci. 26, 3412–3422.

Furuya, K., Furuya, S., Yamagishi, S., 1994. Intracellular calciumresponses and shape conversions induced by endothelin incultured subepithelial fibroblasts of rat duodenal villi. PflugersArch. 428, 97–104.

Furuya, S., Furuya, K., Sokabe, M., Hiroe, T., Ozaki, T., 2005.Characteristics of cultured subepithelial fibroblasts in the ratsmall intestine. II. Localization and functional analysis ofendothelin receptors and cell-shape-independent gap junctionpermeability. Cell Tissue Res. 319, 103–119.

George, Z.M., Eugenia, D., Linda, B., Roberto, N., 2002. Increasedincidence of gap junctional coupling between spinalmotoneurones following transient blockade of NMDAreceptors in neonatal rats. J. Physiol. 544, 757–764.

Gray, P.N., May, P.C., Mundy, L., Elkins, J., 1980. L-Glutamatetoxicity in Huntington's disease fibroblasts. Biochem. Biophys.Res. Commun. 95, 707–714.

Guenard, V., Gwynn, L.A., Wood, P.M., 1994. Astrocytes inhibitSchwann cell proliferation and myelination of dorsal rootganglion neurons in vitro. J. Neurosci. 14, 2980–2992.

Gu, J.G., MacDermott, A.B., 1997. Activation of ATP P2X receptorselicits glutamate release from sensory neuron synapses.Nature 389, 749–753.

Guldner, F.H., 1972. Characteristics of neuro-glial synapses-likecontacts in eminentia mediana in the rat. Verh. Anat. Ges. 67,279–283.

Huang, T.Y., Cherkas, P.S., Rosenthal, D.W., Hanani, M., 2005. Dyecoupling among satellite glial cells in mammalian dorsal rootganglia. Brain Res. 1036, 42–49.

Hunter, G.K., Pitts, J.D., 1981. Non-selective junctionalcommunication between some differentmammalian cell typesin primary culture. J. Cell Sci. 49, 163–175.

Hunter, G.K., Szigety, S.K., 1992. Effects of proteoglycan onhydroxyapatite formation under non-steady-state andpseudo-steady-state conditions. Matrix 12, 362–368.

Jeftinija, S., Jeftinija, K., Liu, F., Skilling, S.R., Smullin, D.H.,Larson, A.A., 1991. Excitatory amino acids are released fromrat primary afferent neurons in vitro. Neurosci. Lett. 125,191–194.

Jerregard, H., 2001. Sensory neurons influence the expression ofcell adhesion factors by cutaneous cells in vitro and in vivo.J. Neurocytol. 30, 327–336.

Jerregard, H., Akerud, P., Arenas, E., Hildebrand, C., 2000.Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from ratsensory neurons in vitro. J. Neurocytol. 29, 653–663.

Jester, J.V., Petroll, W.M., Barry, P.A., Cavanagh, H.D., 1995.Temporal, 3-dimensional, cellular anatomy of corneal woundtissue. J. Anat. 186, 301–311.

Joyce, B., (1987) Bridging the gap. Miss RN. 49, 29–30.Kajander, K.C., Wakisaka, S., Bennett, G.J., 1992. Spontaneous

discharge originates in the dorsal root ganglion at the onset ofa painful peripheral neuropathy in the rat. Neurosci. Lett. 138,225–228.

Komuro, T., 1990. Re-evaluation of fibroblasts and fibroblast-likecells. Anat. Embryol. 182, 103–112.

Langevin, H.M., Cornbrooks, C.J., Taatjes, D.J., 2004. Fibroblastsform a body-wide cellular network. Histochem. Cell Biol. 122,7–15.

Lv, X., Zhan, C., Zeng, S., Chen, W.R., Luo, Q., 2006. Construction ofmultiphoton laser scanning microscope based on dual-axisacousto-optic deflector. Rev. Sci. Instrum. 77 (046101).

Mathers, D.A., Barker, J.L., 1984. Spontaneous voltage and currentfluctuations in tissue cultured mouse dorsal root ganglioncells. Brain Res. 293, 35–47.

May, P.C., Gray, P.N., 1985. The mechanism of glutamate-induceddegeneration of cultured Huntington's disease and controlfibroblasts. J. Neurol. Sci. 70, 101–112.

Muller, T., Moller, T., Neuhaus, J., Kettenmann, H., 1996. Electricalcoupling among Bergmann glial cells and its modulation byglutamate receptor activation. Glia 17, 274–284.

Rao, G., Barnes, C.A., McNaughton, B.L., 1987. Occlusion ofhippocampal electrical junctions by intracellular calciuminjection. Brain Res. 408, 267–270.

22 B R A I N R E S E A R C H 1 2 8 0 ( 2 0 0 9 ) 1 3 – 2 2

Roerig, B., Feller, M.B., 2000. Neurotransmitters and gap junctionsin developing neural circuits. Brain Res. Brain Res. Rev. 32,86–114.

Rook, M.B., van Ginneken, A.C.G., de Jonge, B., El Aoumari, A., Gros,D., Jongsma, H.J., 1992. Differences in gap junction channelsbetween cardiac myocytes, fibroblasts, and heterologous pairs.Am. J. Physiol. 263, 959–977.

Rorig, B., Sutor, B., 1996. Regulation of gap junction coupling in thedeveloping neocortex. Mol. Neurobiol. 12, 225–249.

Rouach, N., Glowinski, J., Giaume, C., 2000. Activity-dependentneuronal control of gap-junctional communication inastrocytes. J. Cell Biol. 149, 1513–1526.

Russell, L.C., Burchiel, K.J., 1988. Spontaneous activity inafferent and efferent fibers after chronic axotomy:response to potassium channel blockade. Somatosens. Mot.6, 163–177.

Rydh, R.M., Kerekes, N., Svensson, M., Hokfelt, T., 2001.Glutamate release from adult primary sensory neurons in

culture is modulated by growth factors. Regul. Pept. 102,69–79.

Stahl, W.L., Ward, C.B., Casper, J.B., Bird, T.D., 1984. Effects ofL-glutamate on viabilities of cultured diploid skin fibroblastsand lymphocytes. Increased toxicity not observed inHuntington's disease. J. Neurol. Sci. 66, 183–191.

Venance, L., Stella, N., Glowinski, J., Giaume, C., 1997. Mechanisminvolved in initiation and propagation of receptor-inducedintercellular calcium signaling in cultured rat astrocytes.J. Neurosci. 17, 1981–1992.

Yang, K., Wang, G.D., Li, Y.Q., Shi, J.W., Zhao, Z.Q., 1998.Co-existence of glutamate and substance P inelectrophysiologically identified dorsal root ganglion neuronsof rats. Sheng Li Xue Bao 50, 453–459.

Zeng, S., Lv, X., Zhan, C., Chen, W.R., Xiong, W., Jacques, S.L., Luo,Q., 2006. Simultaneous compensation for spatial and temporaldispersion of acousto-optical deflectors for two-dimensionalscanning with asingle prism. Opt. Lett. 31, 1091–1093.