Agnès O Martin

Marie-Noëlle Mathieu

Nathalie C Guérineau
email: nathalie.guerineau@ccipe.cnrs.fr

Evidence for long-lasting cholinergic control of gap junctional communication between adrenal chromaffin cells

Keywords: synaptic transmission, gap junctions, nicotinic receptors, rat adrenal slices, splanchnectomized rats, dye coupling, neonates, hexamethonium, brefeldin

We investigated long-lasting interactions that may occur between two forms of intercellular signaling: cholinergic synaptic transmission and gap junction-mediated coupling in the rat adrenal medulla. The junctional coupling between chromaffin cells was studied during reduced or during blocked synaptic transmission in adrenal slices. First, cholinergic synaptic activity was reduced by pharmacological treatment. Bath-application of the nicotinic receptor antagonists (hexamethonium, the oxystilbene derivative F3 or alpha-bungarotoxin) acting at distinct nAChRs significantly increased the incidence of Lucifer yellow (LY) passage (dyecoupling) between chromaffin cells (probability >0.7 in treated-slices versus 0.4 in control).

Dye-coupling was associated with an elevated macroscopic conductance of the junctional current measured by dual patch-clamp. Pharmacological inhibition of protein trafficking from the trans-Golgi network to the plasma membrane by either brefeldin A or nocodazole pretreatment prevented the effects of nAChR antagonists on dye-coupling. Interestingly, this upregulation of gap junction-mediated coupling in response to reduced synaptic activity is of physiological relevance, since it is found in the newborn rat, in which cholinergic synaptic transmission has not yet matured. This mechanism may also be of importance in pathological conditions, as chronic blockade of synaptic transmission after surgical denervation of the adrenal gland also resulted in increased dye-coupling between chromaffin cells. In conclusion, our pharmacological, physiological and pathological data concur to demonstrate that gap junction-mediated intercellular communication between chromaffin cells undergoes persistent adaptation in response to impairment of synaptic activity. These results strongly suggest that gap junctional communication between chromaffin cells is under tonic inhibitory control exerted by cholinergic synaptic inputs.

INTRODUCTION

In integrated biological systems, the precise coordination of cellular signals arising from distinct inputs is critical in maintaining physiological function. In the nervous system, cross talk between chemical synapses and gap junction-mediated cell-to-cell communication is so pervasive [START_REF] Haydon | Glia: listening and talking to the synapse[END_REF] that it is no longer tenable to investigate the mechanisms of neuronal signal integration without considering the intricate interactions between both systems of intercellular contacts. Although synapses represent the main mode of interneuronal communication, it is now well established that gap junctions are widely expressed in neurons [START_REF] Rozental | Temporal expression of neuronal connexins during hippocampal ontogeny[END_REF][START_REF] Schmitz | Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication[END_REF]. The co-existence of synapses and gap junctions in the same cell has raised the question of functional links that may exist between these two forms of cellular communication. Several reports have described short-term regulatory effects of acute application of neurotransmitters or neuromodulators on interneuronal gap junctional communication [START_REF] Piccolino | γ-Aminobutyric acid antagonists decrease junctional communication between L-horizontal cells of the retina[END_REF][START_REF] Piccolino | Decrease of gap junction permeability induced by dopamine and cyclic adenosine 3':5'-monophosphate in horizontal cells of turtle retina[END_REF][START_REF] Pereda | Dopamine enhances both electrotonic coupling and chemical excitatory postsynaptic potentials at mixed synapses[END_REF][START_REF] Mcmahon | Horizontal cell coupling in the giant danio: synaptic modulation by dopamine and synaptic maintenance by calcium[END_REF][START_REF] Hatton | Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H 1 receptors and cyclic nucleotides[END_REF][START_REF] Shinohara | GABAergic modulation of gap junction communication in slice cultures of the rat suprachiasmatic nucleus[END_REF][START_REF] Lagostena | Purinergic control of intercellular communication between Hensen's cells of the guinea-pig cochlea[END_REF].

Physiological or electrically-induced activation of synaptic inputs also acutely modulate intercellular coupling [START_REF] Hatton | Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H 1 receptors and cyclic nucleotides[END_REF][START_REF] Hatton | Function-related plasticity in hypothalamus[END_REF][START_REF] Hatton | Ionotropic histamine receptors and H 2 receptors modulate supraoptic oxytocin neuronal excitability and dye coupling[END_REF], indicating that modulation of coupling by synaptically-released factors likely occurs in situ between excitable cells [START_REF] Hatton | Synaptic modulation of neuronal coupling[END_REF]. The effects on gap junction coupling mainly result from the activation within minutes of intracellular messenger systems (Ca 2+ , cAMP, cGMP), suggesting the modulation of junctional conductances of anchored channels rather than the targeting of a downstream step affecting connexon number or trafficking. We report here that in addition to this very short-term control of gap junctional communication, synaptic inputs can also exert a long-term regulation of the extent of gap junction-mediated intercellular coupling between excitable cells. We addressed this issue in rat adrenal chromaffin cells, because the medulla presents unique features providing an ideal preparation for studying the interactions between gap junctions and synaptic activity. Indeed, although chromaffin cell secretory activity is chiefly regulated transsynaptically via the splanchnic nerve [START_REF] Axelrod | Noradrenaline: fate and control of its synthesis[END_REF], the interaction of gap junction-mediated signal propagation with synaptic transmission represents a complementary mechanism for the exocytosis of catecholamines, as we have shown in slices [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. To demonstrate the effect of cholinergic synaptic activity on gap junction-mediated communication in the adrenal medulla, the junctional coupling between chromaffin cells was examined in slices in which either the synaptic inputs or the nicotinic receptors were manipulated. We found an increase in dye-coupling after pharmacologically impairing or chronically disrupting synaptic transmission to chromaffin cells. This upregulation was abolished when protein-delivery to the plasma membrane was inhibited by agents that disrupt the Golgi apparatus. Of particular physiological interest is the situation observed in neonatal rats. At birth, dye-coupling was upregulated, as could be expected in light of the immaturity of neurochemical synapses. We propose that gap junctional coupling in the adrenal medulla undergoes long-term plasticity as a compensatory response under conditions of reduced cholinergic synaptic transmission. Furthermore, our results indicate that in control conditions gap junctional coupling is under tonic inhibitory control.

MATERIALS AND METHODS

Adrenal slice preparation. Acute adrenal slices were prepared as previously reported [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. Briefly, the adrenal glands were removed from new born and adult (12-to16week-old) female Wistar rats killed by decapitation after cervical dislocation. After keeping the glands in ice-cold saline for 2 min, a gland was glued onto an agarose cube and transferred to the stage of a vibratome (Microslicer ® , DTK-1000, D.S.K, Dosaka EM CO. LTD, Kyoto, Japan). Slices of 250 µm thickness were then cut with a razor blade and transferred to a storage chamber maintained at 32°C containing Ringer's saline (in mM): 125 NaCl, 2,5 KCl, 2 CaCl 2 , 1 MgCl 2 , 1,25 NaH 2 PO 4 , 26 NaHCO 3 , 12 glucose and buffered to pH 7.4. The saline was continuously bubbled with carbogen (95% O 2 / 5% C0 2 ). In some experiments acute slices were prepared from splanchnectomized adult female rats (n = 5). The left adrenal gland was denervated by transecting the left splanchnic nerve (surgery performed by Iffa Credo, L'Arbresle, France). Splanchnectomized rats were used 2 to 4 weeks after surgery. The efficacy of the adrenal denervation was confirmed by the absence of postsynaptic currents in chromaffin cells during electrophysiological recordings.

For optical and/or electrophysiological recordings, slices were then transferred to a recording chamber attached to the stage of an upright microscope fitted with differential interference contrast optics (Axioskop FS, Zeiss, Le Pecq, France) and continuously superfused with Ringer's saline at 30°C.

Real-time imaging of cytosolic calcium. Ca 2+ changes were monitored using a confocal laser scanning microscope equipped with an Ar/Kr laser (Odyssey XL with InterVision 1.5.1 software, Noran Instruments Inc., Middleton, WI) [START_REF] Guérineau | Synchronized spontaneous Ca 2+ transients in acute anterior pituitary slices[END_REF]. Cells were viewed with a 63x 0.9 NA achroplan water immersion objective (Zeiss). For [Ca 2+ ]i measurements the largest detection slit (100 µm) of the confocal microscope was used, giving bright images with a 3.1 µm axial resolution. Slices were loaded with the Ca 2+ -sensitive fluorescent probe Oregon Green 488 BAPTA-1 by intermittent exposure to 15 µM Oregon Green 488 BAPTA-1 acetoxymethyl ester (Molecular Probes, Eugene, OR) for 20-30 min at 32°C, delivered onto a cell field with a blunt pipette [START_REF] Bonnefont | Rhythmic bursts of calcium transients in acute anterior pituitary slices[END_REF]. Oregon Green 488 BAPTA-1 was excited through a 488 nm-band-pass-filter and the emitted fluorescence was collected through a 515 nm-barrier filter. To follow the time course of Oregon Green 488 BAPTA-1 emission changes, the bright-over-time tool of the software package was applied to live images (120 images/s with averaging 4 frames). [Ca 2+ ]i changes were expressed as the F/Fmin ratio where Fmin was the minimum fluorescence intensity measured during the recording. Acquired data and images were then processed for analysis with Igor Pro 3.16 (Wavemetrics Inc., Lake Oswego, OR) and NIH Image 1.6.0 software.

Electrophysiology.

All experiments were performed in the whole-cell configuration of the patch-clamp technique [START_REF] Hamill | Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches[END_REF]. Patch pipettes were pulled to a resistance of 5-8 MΩ from borosilicate glass (1.5 mm outer diameter, 1.17 mm inner diameter) and filled with the following internal solution (in mM): 140 potassium-gluconate, 2 MgCl 2 , 1.1 EGTA, 5 HEPES, that was titrated to pH 7.2 with KOH. The membrane potential of chromaffin cells was recorded under current-clamp conditions using an EPC-9 patch-clamp amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) and filtered at 3 kHz. Spontaneous excitatory postsynaptic currents (EPSCs) were recorded in chromaffin cells voltage-clamped at -80 mV in normal external [K + ], and were filtered at 1 kHz. Signals were acquired and analyzed using Pulse + PulseFit software (version 8.5, HEKA Electronik) on a G4 Macintosh computer. For combined experiments in which membrane potential was simultaneously recorded with Ca 2+ events, Oregon Green 488 BAPTA-1 in free acid form (10 µM) was directly added to the internal pipette solution [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. Membrane potentials of chromaffin cell pairs were recorded under current-clamp conditions using an EPC-9 dual patch-clamp amplifier (HEKA Electronik) and filtered at 3 kHz. Intercellular junctional currents were monitored under dual voltage-clamp conditions [START_REF] Neyton | Single-channel currents of an intercellular junction[END_REF] using a modified intrapipette solution (in mM: 140 Cs + -gluconate, 2 MgCl 2 , 1.1 EGTA, 5 HEPES). To determine the macroscopic junctional conductance (Gj), the I/V curve in which Ij amplitude was plotted as a function of the transjunctional voltage (Vj) was fitted by a computed linear regression y = ax + b (where y corresponds to Ij and x to Vj). Gj was then given by the slope (a) of the linear regression.

Lucifer yellow diffusion. The fluorescent dye Lucifer yellow (Lucifer yellow-CH, LY, 1 mM) was introduced into chromaffin cells using patch pipettes. Dye transfer between gap junction-coupled cells was visualized with confocal microscopy using the 488 nm-centered wavelength of the laser beam. The extent of LY diffusion between chromaffin cells was estimated by counting the number of labeled cells. The probability of LY diffusion was expressed as a ratio corresponding to the number of injected cells that show dye transfer to adjacent cells over the total number of injected cells. Transmitted images were acquired using the longer wavelength of the laser beam (647 nm), which penetrated deeply into adrenal slices.

Immunofluorescence and image processing. After dye-coupling experiments, adrenal slices were fixed in 4% paraformaldehyde for 1h at room temperature prior to immunofluorescence. Background noise was reduced by quenching free aldehyde groups with 50 mM NH 4 Cl in phosphate buffered saline (PBS, pH 7.4) for 2 x 15 min. Permeabilization was achieved with PBS containing 0.5% Triton X-100 and 20% horse serum for 1h at room temperature. Adrenal slices were then incubated with the primary antibodies to TGN-38 (Transduction Laboratories product, Interchim, Montluçon, France, 1/100) or β-tubulin (Sigma, 1/2000) for 48h at 4°C in PBS + 0.1% Triton + 20% horse serum. The slices were washed three times for 10 min in PBS and incubated with the secondary antibody (Cy3conjugated mouse IgG, 1/1000, Jackson product, Interchim) for 2h at room temperature in PBS + 0.1% Triton + 20% horse serum. Slices were washed three time for 10 min with PBS, and mounted on glass coverslips in Mowiol. 1,4-diazobicyclo-[2.2.2]-octane (DABCO, 2.5% Sigma) was added to the mounting medium to reduce fading. Immunolabeling without primary antibody was used as control. Slices were imaged with the Odyssey XL (Noran Instruments Inc) laser confocal microscope. The 568 nm line of the laser beam was selected and the emitted fluorescence was collected through the 590 nm barrier filter (low pass filter).

Cells were viewed with an 100x 1.3 NA plan-neofluar oil immersion objective (Zeiss, France). For image acquisition, the 25 µm detection slit of the confocal microscope was used, giving bright images with a 0.7 µm z-axis resolution. Optical slices of the chromaffin cells were recorded using the normal scan mode (30 images/s with averaging 256 frames) with an image size of 308 x 239 pixels and an 400 ns dwell rate/pixel. To improve resolution of confocal images, extraction of unblurred images was performed by a 2D blind deconvolution (deconvolution module of the InterVision software package).

Solutions and chemicals. The nicotinic receptor blockers hexamethonium (Sigma), F3 and alpha-bungarotoxin (α-Bgt, Sigma) were bath-applied (5 minutes before testing their effects on spontaneous EPSCs and 30 minutes before dye spread experiments). The oxystilbene derivative F3 compound was synthetized in Dr. Francesco Clementi's laboratory (CNR, Cellular and Molecular Pharmacology Center, University of Milan, Italy) and was firstly reported to be a selective ligand for neuronal nicotinic α-bungarotoxin-sensitive receptors [START_REF] Gotti | 4-oxystilbene compounds are selective ligands for neuronal αbungarotoxin receptors[END_REF]. Lucifer yellow (LY), the gap junction blocker carbenoxolone and the non-selective P2 purinergic receptor antagonists were purchased from Sigma (St Louis, MO). Brefeldin A (BFA, 2 µg/ml,) purchased from Fluka (L'isle d'Abeau Chesnes, France) and nocodazole (25 µM) from Tebu-France (Le Perray en Yveline, France) were used to inhibit the Golgi apparatus [START_REF] Dinter | Golgi-disturbing agents[END_REF]. To fully disrupt the Golgi complex and microtubules, adrenal slices were treated either with BFA or nocodazole for at least 30 min at 37°C before and during incubation in hexamethonium-containing saline.

Statistics. Numerical data are expressed as the mean ± SEM. The Student's t-test was used to compare means when appropriate. Differences between groups were assessed by using the non-parametric Mann-Whitney U test. Percentages were compared using a contingency table and the chi-square test. Differences with p<0.01 were considered significant.

RESULTS

As recently reported, the presence of gap junctions between chromaffin cells can be accurately determined by examining the diffusion of the low molecular weight fluorescent dye Lucifer yellow [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. This approach was used here to study the gap junction-mediated intercellular coupling between chromaffin cells under various experimental conditions, in which the level of synaptic transmission at the splanchnic nerve terminals was manipulated.

Increase in gap junctional communication between chromaffin cells after acute impairment of synaptic excitatory cholinergic transmission

When chromaffin cells were voltage-clamped at -80 mV, spontaneous inward synaptic currents were observed, which are likely to reflect acetylcholine release from the presynaptic terminals contacting chromaffin cells [START_REF] Barbara | Quantal release at a neuronal nicotinic synapse from rat adrenal gland[END_REF]. In control saline (2.5 mM K + ), 12% of chromaffin cells exhibited spontaneous excitatory postsynaptic currents (EPSCs), when recorded over 10 minutes (n = 75 cells, figure 1Aa, b andc, left traces). The mean EPSC amplitude and frequency were 89.6 ± 5.6 pA (n = 252 EPSCs, 9 cells) and 0.08 ± 0.04 Hz (n = 9 cells; range from 0.001 to 0.34 Hz), respectively. It has previously been reported that inward synaptic currents are mediated by the activation of neuronal-like postsynaptic nicotinic receptors (nAChRs) [START_REF] Barbara | Quantal release at a neuronal nicotinic synapse from rat adrenal gland[END_REF]. Because the α3 subunit-containing nAChRs represent the major subtype transducing the ACh-evoked postsynaptic electrical response in chromaffin cells [START_REF] Nooney | Inhibition by cyclothiazide of neuronal nicotinic responses in bovine chromaffin cells[END_REF], we tested the effect of hexamethonium, which specifically targets this receptor subtype. Bath-application of hexamethonium (200 µM) significantly reduced the amplitude and frequency of spontaneous EPSCs (figure 1Aa, right trace and histograms). The percentage of inhibition of both EPSC frequency and amplitude was 41.4 ± 10.6% and 56.6 ± 10.5%, respectively (n = 8 cells recorded before and in the presence of hexamethonium). These effects were partly reversible within 10-20 minutes (data not shown). The frequency of spontaneous EPSCs was also significantly decreased in the presence of the oxystilbene derivative F3 (30 nM, bathapplication, figure 1Ab, right trace), a compound recently reported to antagonize α3 subunitcontaining nAChRs in the rat adrenal gland (Di [START_REF] Angelantonio | Antagonism of nicotinic receptors of rat chromaffin cells by N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2propylammonium iodide: a patch clamp and ligand binding study[END_REF]. No significant effect on the amplitude of the remaining EPSCs was observed (n = 10). The effect of F3 was reversible within several minutes (data not shown). Because both hexamethonium and F3 did not fully inhibit spontaneous EPSCs, and rat adrenomedullary chromaffin cells also express the α7 subunit-containing AChRs [START_REF] Rust | Expression of neuronal nicotinic acetylcholine receptor subunit genes in the rat autonomic nervous system[END_REF][START_REF] Criado | Differential expression of α-bungarotoxin-sensitive neuronal nicotinic receptors in adrenergic chromaffin cells: a role for transcription factor Egr-1[END_REF][START_REF] Mousavi | Expression of nicotinic acetylcholine receptors in human and rat adrenal medulla[END_REF], the snake neurotoxin α-bungarotoxin, an irreversible antagonist of α7 nAChRs [START_REF] Sine | Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding[END_REF], was tested. As illustrated in figure 1Ac, α-Bgt (1.5 µM) significantly decreased EPSC frequency (p<0.01, n = 4). These results indicate that hexamethonium, F3 and α-Bgt efficiently reduce excitatory cholinergic synaptic transmission between presynaptic nerve terminals and chromaffin cells.

To investigate possible signaling interactions between synaptic transmission and gap junctionmediated intercellular coupling, we next studied the effects of applying hexamethonium or F3 or α-Bgt for 10-30 minutes on LY diffusion between chromaffin cells. As shown in figure 1B, the probability of observing LY diffusion between nearby chromaffin cells was significantly increased when slices were incubated with nAChR blockers (0.74, 0.67 and 0.70 for hexamethonium, F3 and α-Bgt, respectively versus 0.44 in control slices, p<0.01). Incubation in a medium containing a combination of hexamethonium and α-Bgt did not significantly increase the probability of dye-coupling between chromaffin cells when compared to the effect of either blocker alone (figure 1B). In the presence of nAChR antagonists, LY-labeled cell clusters (≥ 3 cells) were more frequently observed (21.1% in hexamethonium-treated slices versus 2.4% in control slices). An example of prominent dye-coupling in an F3-bathed slice is illustrated in figure 1C.

Could this upregulation of dye-coupling be mimicked by other synaptically released excitatory factors? Acetylcholine and ATP are stored together in some synaptic vesicles, including those in sympathetic splanchnic nerve terminals [START_REF] Zimmermann | Signalling via ATP in the nervous sytem[END_REF]. We therefore investigated the effect of blocking postsynaptic purinergic receptors in chromaffin cells on the spreading of LY. Thirty minute incubation in the presence of 100 µM suramin, a non-selective blocker of P 2 purinergic receptors modified neither the incidence of dyecoupling (probability 0.44, n = 16, data not shown) when compared to control slices (0.44) nor the extent of coupling (data not shown). To further investigate the role of potential nonnicotinic purinergic EPSCs, slices were exposed to suramin (100 µM), hexamethonium (200 µM) and α-Bgt (1.5 µM) for at least 30 min before LY injections. Dye coupling to adjacent chromaffin cells was observed in 73.7% of the injected cells (n = 19). This was similar to the percentage found with saline containing nAChR antagonists (p>0.01). This result indicates that the increase in gap junctional coupling is mediated by a specific pathway involving postsynaptic nAChRs and is of interest when considering that acetylcholine is the main synaptically-released transmitter that stimulates catecholamine exocytosis in chromaffin cells.

To confirm that the increase in dye coupling reflects increased functional communication between chromaffin cells, depolarization-evoked action potentials were triggered in a single chromaffin cell and subsequent [Ca 2+ ]i signals were simultaneously monitored in neighboring cells in hexamethonium-treated slices (figure 2A). As reported earlier [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF], this paradigm led to propagated electrically-triggered [Ca 2+ ]i rises between gap junctioncoupled cells. As a result, synchronous multiple [Ca 2+ ]i rises were frequently observed in neighboring cells (68.7% of cell fields, n = 22 of 32, figure 2B) in the presence of hexamethonium. In untreated slices, the percentage dropped to 48.1% (n = 13 of 27), as previously shown [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. In addition, the number of neighboring cells in which the [Ca 2+ ]i rose in response to a single cell depolarization was higher in hexamethoniumcontaining saline than in control conditions. These data are in agreement with those reported in figure 1.

All these results show that reduced excitatory cholinergic synaptic activity resulting in a decrease in postsynaptic nAChR activation rapidly (in less than 1 hour) upregulates gap junctional coupling between chromaffin cells.

Electrical coupling and gap junction currents between chromaffin cells in hexamethonium-treated slices: increased incidence of highly electrically coupled cells

What is the mechanism underlying the short-term increase in gap junction-mediated coupling between chromaffin cells in the presence of nAChR antagonists? To address this issue, membrane potentials were recorded from chromaffin cell pairs using the dual patch-clamp technique. In 68.2% of cell pairs (n = 15 of 22), the voltage changes in response to hyperpolarizing/depolarizing current injected into the stimulated cell were reflected as variably attenuated responses in the unstepped cell (figure 3A andB, left traces) and viceversa (right traces). In 80% of coupled pairs, an action potential in the injected cell induced only a small depolarization in the neighboring cell (figure 3A) indicating weak coupling, whereas in the remaining pairs, robust coupling led to the transmission of suprathreshold responses resulting in action potentials (figure 3B). Consequently, the coupling ratios exhibited a wide distribution range from 0.01 to 0.96 (mean 0.24 ± 0.09, n = 15 coupled pairs, figure 3C) that appeared to be bimodal. This reflects the presence of two chromaffin cell populations, a weakly coupled cell population and a highly coupled cell population. It is interesting to note that the value of the coupling ratio for a given cell pair remained constant independent of the amplitude or polarity of the current step. These findings were confirmed in Cs + -loaded chromaffin cell pairs voltage clamped at -60 mV. Junctional currents were recorded in 64.7% (11 out of 17) of the cell pairs (figure 4), a value similar to that found in control slices (61%, Martin et al., 2001, p>0.01). Delivering voltage steps with command pulses of both polarities induced macroscopic junctional currents (Ij) in the unstepped cell that appeared to remain constant for the duration of the step, but again exhibiting variable degrees of attenuation. In 56.5% of recorded pairs (n = 6), Ij amplitude did not exceed several picoamperes (figure 4A), whereas in 45.5% of cell pairs (n = 5) Ij could reach 1-2 nA (figure 4B). When plotted as a function of the transjunctional potential, the I/V curve of Ij displayed a linear relationship within the membrane potential range of -200 to +150 mV. The curve used to fit the data was derived from a linear regression, given a mean macroscopic conductance of ~42 pS (n = 6) for the weak coupling and ~12 nS (n = 5) for the robust coupling (figure 4C).

In comparison to our previous study [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF], the main effect of hexamethonium was to significantly increase the incidence of highly electrically coupled cells. Indeed, a robust Ij was less frequently observed in control conditions (20% versus 45% in control and hexamethonium-treated slices, respectively, p<0.01, figures 4D and E).

Effects of inhibiting connexon trafficking to the plasma membrane by disrupting the trans-Golgi network and microtubules

We further examined possible mechanisms that could underlie the increase of the macroscopic Ij conductance. Based on the fact that the effects of nAChR antagonists occurred within 30 min, we first tested the hypothesis of a protein trafficking-mediated effect by manipulating intracellular connexon trafficking. As recently shown [START_REF] Lauf | Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells[END_REF], newly synthesized connexons are transported from the Golgi apparatus to the plasma membrane via vesicular transport along microtubules. Because the trans-Golgi network (TGN) is involved in the sorting and targeting of proteins to the plasma membrane, we first attempted to inhibit membrane insertion of new connexons by disrupting the TGN. This was achieved by treating the adrenal slices for at least 30 min (at 37°C) before and during nAChR antagonist treatment with brefeldin A (BFA, 2 µg/ml), an inhibitor of vesicular export from the TGN. BFA has been reported to disassemble the Golgi apparatus into tubules by inhibiting the guanine nucleotide exchange on ADP-ribosylating factors [START_REF] Klausner | Brefeldin A: insights into the control of membrane traffic and organelle structure[END_REF][START_REF] Helms | Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF[END_REF].

Golgi disruption was verified by immunolabeling of TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network [START_REF] Luzio | Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38)[END_REF]. As illustrated in figure 5Aa, in BFA-treated slices TGN38 appeared homogeneously distributed within the cytosol, while it showed the expected perinuclear localization in untreated slices. Under these experimental conditions, the increase in the probability of dye-coupling in the presence of hexamethonium was abolished and was not significantly different from that found in control slices (p>0.01, 0.41 versus 0.44 for BFA-treated and control slices, respectively, figure 5B,n = 39). This result was further confirmed in slices treated with the potent microtubule depolymerizing agent nocodazole (25 µM, at least 30 min at 37°C before incubation in hexamethonium). As expected, nocodazole treatment strongly disrupted the microtubule network visualized by the immunofluorescent detection of β-tubulin protein (figure 5Ab).

Similarly to what we found in BFA-treated slices, the probability of observing LY coupling in the presence of hexamethonium dropped to control conditions (p>0.01, 0.44 for nocodazoletreated and control slices, respectively, figure 5B, n = 29). Note that BFA or nocodazole treatment did not modify per se either the resting membrane potential or the ability of chromaffin cells to exhibit spontaneous or electrically-evoked action potentials (data not shown).

Taken together, these results indicate that connexon trafficking from the trans-Golgi to the plasma membrane is a likely mechanism mediating the effects of hexamethonium on gap junctional coupling between chromaffin cells.

Gap junctional coupling is modulated by the functional state of synaptic transmission: the physiological significance in newborn rats

At birth the innervation of chromaffin cells by splanchnic nerve fibers is not fully mature [START_REF] Millar | Catecholamine-storing cells in the adrenal medulla of the preand postnatal rat[END_REF], although the majority, if not all of the pre-and postsynaptic components are already present prenatally [START_REF] Daikoku | Development of the adrenal medullary cells in rats with reference to synaptogenesis[END_REF][START_REF] Tomlinson | The innervation of the adrenal gland. IV. Innervation of the rat adrenal medulla from birth to old age. A description and quantitative morphometric biochemical study of the innervation of the chromaffin cells and adrenal medullary neurons in Wistar rats[END_REF]. Spontaneous synaptic activity was recorded in adrenal slices from newborn rats. In neonates, about 22% of recorded cells spontaneously exhibited EPSCs (figure 6A, n = 23).

The mean EPSC amplitude was 35.7 ± 3.2 pA (n = 59, 5 cells) and the frequency was 0.02 ± 0.01 Hz (n = 5). With respect to LY coupling, the probability of observing dye spreading between chromaffin cells was significantly higher in neonates than in adults (~0.6 versus ~0.4, respectively, p<0.01, figure 6B). LY coupling was indeed mediated by gap junctions, as shown by the dramatic reduction in LY diffusion in the presence of carbenoxolone (200 µM, 10-60 min, figure 6B), a decoupling agent [START_REF] Ishimatsu | Synchronous activity in locus coeruleus results from dendritic interactions in pericoerulear regions[END_REF]. The spatial extent of LY staining was also higher in neonates as compared to adult rats. After injecting dye into a single cell up to 4-5 adjacent cells were labeled, whereas coupling was confined to 1-2 cells in adults (figure 6C).

What might the physiological significance for gap junction-mediated coupling in neonates be? We previously reported that gap junctions are involved in cell-to-cell propagation of electrical events and subsequent [Ca 2+ ]i increases in the adrenal medulla of adult rats [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. To address the role of gap junctions in the intercellular transfer of biological signals in neonates, multicellular [Ca 2+ ]i changes were monitored with real-time confocal microscopy using Oregon Green 4800 BAPTA-1-loaded chromaffin cells. As illustrated in figure 7, the triggering of an action potential resulted in a [Ca 2+ ]i rise in the stimulated cell and in several adjacent cells (up to 5 cells in the same optical plane). Such a simultaneous [Ca 2+ ]i increase in neighboring chromaffin cells in response to depolarizationevoked action potentials was observed in 62.5% of recorded cell fields (n = 10 of 16), a percentage significantly higher in neonates than in adult (p<0.01, [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. In addition, the number of cells in which [Ca 2+ ]i simultaneously increased was higher in the neonate than in the adult. This result is in agreement with the data illustrated in figure 6C showing enhanced LY diffusion in neonates.

The propagation of action potential-induced [Ca 2+ ]i elevations likely reflects a physiological phenomenon as synchronized [Ca 2+ ]i transients spontaneously occurred between adjacent chromaffin cells in the absence of stimulation (data not shown). Based on the kinetics of [Ca 2+ ]i transients, i.e. a fast peaking rising phase followed by a return to baseline within a few seconds, we proposed that these are likely to reflect Ca 2+ entry through voltage-gated Ca 2+ channels opening during action potentials.

Long-lasting persistence of the upregulation of gap junction-mediated intercellular coupling between chromaffin cells: a study in splanchnectomized rats

To study the effects of chronic disruption of synaptic activity, we monitored dye-coupling in denervated adrenal glands after unilateral lesion of the splanchnic nerve, which represents the preganglionic input to the chromaffin cells. As shown in figure 8A, 2-4 weeks after surgery, synaptic transmission was fully disrupted in the denervated medulla, since no more spontaneous synaptic currents occurred (figure 8A, medium trace, n = 15 cells). Furthermore, increasing the external K + concentration to 20 mM to induce a Ca 2+ rise in the nerve terminals had no effect on synaptic activity, thus confirming the effectiveness of the denervation (data not shown). In contrast, spontaneous EPSCs persisted in the contralateral adrenal gland (figure 8A, lower trace) and the percentage of cells exhibiting synaptic events did not differ from that found in unoperated animals (17.6% versus 12%, respectively, p>0.01). However, the mean EPSC amplitude was 138.5 ± 9.6 pA (n = 80, 3 cells) and was significantly higher when compared to that found in unoperated rats (p<0.01, figure 8A, upper trace, n = 9 cells), although EPSC frequency was not altered (0.04 ± 0.02 Hz, n = 3 for contralateral gland versus 0.08 ± 0.04 Hz, n = 9 for unoperated animals, p>0.01). The probability of dye-coupling in splanchnectomized rats was significantly higher in denervated glands (~0.7) as compared to either the contralateral gland (~0.4) or glands from unoperated rats (~0.4, figure 8B, p<0.01).

In the denervated adrenal gland the extent of LY diffusion from the patched cell to a neighboring cell was also significantly increased when compared to the contralateral innervated gland or the glands in unoperated rats (figure 8C, p<0.01).

Taken together, the data obtained from splanchnectomized animals strengthen our hypothesis that synaptic activity tonically controls the degree of gap junction-mediated intercellular communication between chromaffin cells. This modulation, which is likely to involve multiple mechanisms (see discussion) takes place within an hour of disrupting synaptic transmission and appears to persist for several weeks.

DISCUSSION

We show here that the gap junction-mediated coupling between rat adrenal chromaffin cells is dynamically regulated by the functional state of excitatory nicotinic synaptic transmission.

Experimental conditions in which postsynaptic nAChRs are weakly stimulated or blocked (acute pharmacological blockade of nAChRs, surgically-induced adrenal denervation or immature nicotinic synaptic transmission in neonates) lead to a significant increase in dyecoupling between chromaffin cells. This suggests that synaptic inputs, when cholinergic transmission is normal or mature, exert a tonic inhibitory effect on gap junction-mediated communication. Taken together, the present study provides strong support for our previously proposed hypothesis according to which gap junction-mediated signaling and synaptic transmission act in a coordinated manner to ensure normal cell function [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF].

The trafficking machinery as a possible mediator of upregulated gap junctional coupling following blockade of nicotinic synaptic transmission

Dye-coupling experiments performed in the presence of bath-applied nAChR antagonists show that the gap junction-dependent networks between chromaffin cells rapidly respond (within an hour) to modifications in nicotinic synaptic transmission efficiency. As revealed by the recording of the junctional current Ij, the enhanced dye-coupling observed in nAChR antagonist-containing saline is associated with an increase in macroscopic junctional conductance (Gj). We propose two hypotheses, which are not mutually exclusive, to account for this finding: i) an increase in the mean open probability of unitary gap junction channels and/or ii) a change in the number or composition of channels. Although we cannot definitively rule out an effect on the open probability, our data with brefeldin A or nocodazole strongly implicate connexon trafficking or scaffolding protein or connexon traffickingassociated regulatory proteins [START_REF] Giepmans | The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein[END_REF][START_REF] Toyofuku | Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes[END_REF][START_REF] Toyofuku | c-Src regulates the interaction between connexin-43 and ZO-1 in cardiac myocytes[END_REF][START_REF] Barker | Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions[END_REF] in the increased expression of gap junctions. The present study does not allow us to identify the intracellular components targeted by nAChR antagonists. Nevertheless, the following mechanisms can be considered. Connexon trafficking involves both their delivery from the TGN to the plasma membrane and their internalization/degradation via proteasomal and lysosomal pathways [START_REF] Lampe | Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly[END_REF]. The rapid insertion (within less than 1 hour) of existing 'nascent' connexons into the chromaffin cell membrane seems plausible, given that the delivery of newly synthetized connexons from the Golgi complex to the plasma membrane can occur within 2 hours [START_REF] Lauf | Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells[END_REF]. This delivery step, which as shown for Cx43 [START_REF] Giepmans | Gap junction protein connexin-43 interacts directly with microtubules[END_REF] depends on a direct interaction with the microtubules, may be a target for modulation by nicotinic receptor signaling. Our results with nocodazole would be consistent with such an effect on microtubule function. Because gap junctions are constantly being renewed and rapidly turnover [START_REF] Fallon | Five-hour half-time of mouse liver gap-junction protein[END_REF][START_REF] Laird | The life cycle of a connexin: gap junction formation, removal, and degradation[END_REF], gap junction assembly at the plasma membrane may be subject to regulation at the level of connexin turnover [START_REF] Musil | Regulation of connexin degradation as a mechanism to increase gap junction assembly and function[END_REF]. Reduction in connexin cycling could therefore increase gap junctional coupling by reducing endocytic traffic of connexins. Alternatively, connexon assembly at the plasma membrane may be affected by nicotinic receptor signaling. The assembly process is dynamically regulated by connexin phosphorylation [START_REF] Cooper | Casein kinase 1 regulates connexin-43 gap junction assembly[END_REF] as well as by microtubules (Johnson et al., 2002) and by adhesion molecules such as α-catenin [START_REF] Govindarajan | Impaired trafficking of connexins in androgen-independent human prostate cancer cell lines and its mitigation by α-catenin[END_REF]. A further mechanism to account for the increase in gap junctional conductance may be a change in the subunit composition of the connexons. Such a remodeling would imply that new connexins with a high unitary conductance would preferentially replace existing connexins. A good candidate for a high conductance connexin would be Cx43, which is expressed in rat adrenal chromaffin cells [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF].

Because slices were bathed with nAChR antagonists for at least 30 min before dyecoupling experiments or Ij measurement, very short-term effects of the blockers on the biophysical properties of gap junction channels were not investigated. Recordings of unitary electrical activity from gap junction channels would be required to definitively address this issue. Nevertheless, we cannot exclude that intracellular second messengers (Ca 2+ and downstream Ca 2+ -dependent kinases or phosphatases) may also affect the gating properties of gap junctions [START_REF] Lampe | Regulation of gap junctions by phosphorylation of connexins[END_REF].

Long-term persistence of changes in gap junction-mediated communication

Our experiments on splanchnectomized rats indicate that up-regulation of gap junctional coupling can persist for as long as synaptic activity is impaired (at least 3 weeks in this study).

This result clearly shows that gap junctional coupling present in adults can undergo both short-term changes as previously reported and long-term changes. Such prolonged effects on gap junctional coupling are consistent with a previous study showing that the reestablishment of gap junctional coupling after injury can persist for many weeks [START_REF] Chang | Nerve injury induces gap junctional coupling among axotomized adult motor neurons[END_REF]. In splanchnectomized animals sustained changes in sympathetic transmission may affect the level of mRNA encoding connexins (or other proteins interacting with connexins), as reported for tyrosine hydroxylase activity after chemical sympathectomy [START_REF] Mueller | Compensatory increase in adrenal tyrosine hydroxylase activity after chemical sympathectomy[END_REF]. Such a compensatory increase in connexin-encoded gene activity could lead to long-term upregulation in the number of gap junction plaques between chromaffin cells. Additional approaches including immunostaining and in situ hybridization in the denervated adrenal gland will be required to evaluate this hypothesis. Alternatively, splanchnectomy may result in the modulation of connexin assembly into functional channels and/or the modification of existing gap junction plaques [START_REF] Chang | Nerve injury induces gap junctional coupling among axotomized adult motor neurons[END_REF].

Roles of gap junctions between chromaffin cells in neonates: gap junctions as relays before the establishment of functional synaptic inputs

It is known that at birth synaptic transmission between the splanchnic nerve and chromaffin cells is not fully competent [START_REF] Millar | Catecholamine-storing cells in the adrenal medulla of the preand postnatal rat[END_REF][START_REF] Slotkin | Development of the sympathoadrenal axis[END_REF], although pre-and postsynaptic elements are already present prenatally [START_REF] Daikoku | Development of the adrenal medullary cells in rats with reference to synaptogenesis[END_REF]. From a functional point of view, we show here that the main difference observed in neonatal tissue as compared to adults was a reduction in EPSC amplitude. Under the same experimental conditions, gap junctional coupling was up-regulated. Furthermore, we found that action potential-induced [Ca 2+ ]i increases imaged in multiple cells resulted in synchronized signal propagation between nearby chromaffin cells, demonstrating that gap junctions are functional. Because transsynaptic transmission is not fully functional at birth, other processes are likely to mediate catecholamine release in neonatal rats. Intercellular communication mechanisms between chromaffin cells within the medulla would be good candidates. Although paracrine and/or endocrine interactions cannot be definitively ruled out, our findings that altered synaptic activity leads to an increase in gap junction coupling strongly suggest that this mode of communication contributes to catecholamine release in neonates. Gap junctions would thus act as a substitute for chemical synaptic transmission, thereby compensating for reduced synaptic activity. This hypothesis is consistent with the fact that gap junction-coupled neuronal assemblies often precede synaptically-connected networks [START_REF] Kandler | Coordination of neuronal activity by gap junctions in the developing neocortex[END_REF] and that the expression of gap junctions gradually decreases during postnatal development, coinciding with the establishment of functional synapses [START_REF] Personius | Loss of correlated motor neuron activity during synaptic competition at developing neuromuscular synapses[END_REF]Balice-Gordon, 2001, Mentis et al., 2002).

Functional significance of an interaction between synaptic transmission and gap junction coupling in the adrenal medulla

This study strengthens our view that a prominent interplay between cholinergic synaptic activity and gap junctional coupling is necessary for normal function of the rat adrenal medulla. Coordination of cellular responses is crucial in maintaining syncytial function in various tissues. The innervation density, the frequency of stimulation, and the extent of intercellular coupling are among the parameters that must be integrated for normal activity, as recently modeled [START_REF] Ramanan | Neuronal innervation, intercellular signal transduction and intercellular coupling: a model for syncytial tissue responses in the steady state[END_REF]. Finally, we propose that the modulation of gap junction coupling is important under pathological conditions where synaptic transmission is reduced. By compensating for the loss in chemical neurotransmission, gap junction signaling could sustain catecholamine release by maintaining communication within the chromaffin cell network. electrical coupling evidenced by high Ij amplitude (Gj = 6.6 nS). C, I/V relationships from pooled data of 6 and 5 cell pairs exhibiting weak and robust coupling, respectively. The linear regression used to fit the data (dotted line) was y = -0.042x -0.41 (correlation coefficient r 2 = 0.94) for the weak coupling and y = -12.4x -6.37 (r 2 = 0.99) for the robust coupling, given a Gj of 42 pS and 12.4 nS, respectively. D, Pooled data of Gj calculated in 5 cell pairs bathed in control saline and 11 pairs in hexamethonium-containing saline (logarithmic scale on y axis).

FIGURE LEGENDS

The determination of Gj in control medium includes data from [START_REF] Martin | Gap junctions mediate electrical signaling and ensuing cytosolic Ca 2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release[END_REF]. E, Percentage of appearance of weak and robust coupling and control and hexamethoniumtreated slices.

Figure 1 :

 1 Figure 1: Acute impairment of synaptic activity-induced upregulation of LY diffusion

Figure 2 :

 2 Figure 2: Propagation of action potential-induced [Ca 2+ ]i rises between chromaffin cells

Figure 3 :

 3 Figure 3: Electrical coupling between chromaffin cell pairs in hexamethonium-

Figure 4 :

 4 Figure 4: Macroscopic junctional currents Ij between chromaffin cell pairs in

Figure 5 :

 5 Figure 5: Effect of inhibiting connexon delivery to the plasma membrane on the increase

Figure 6 :

 6 Figure 6: Greater gap junction-mediated intercellular communication between

Figure 7 :

 7 Figure 7: Propagation of electrical activity-linked [Ca 2+ ]i rise between chromaffin cells:

Figure 8 :

 8 Figure 8: Effect of splanchnectomy on gap junctional coupling in chromaffin cells

F3

  Figure 1

  Figure 6

technical assistance. We are also indebted to Dr. C. Gotti for kindly providing the F3 compound. This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, Région Languedoc-Roussillon, Association pour la Recherche sur le Cancer and Fondation pour la Recherche Médicale.