Evidence for Long-Lasting Cholinergic Control of Gap Junctional Communication between Adrenal Chromaffin Cells

Propagation of action potential-induced [Ca²⁺]_i rises between chromaffin cells in hexamethonium-containing saline. A, Electrical activity-driven multicellular [Ca²⁺]_iincreases were visualized by real-time scanning laser confocal imaging (120 images per second, averaging 4 frames) in five chromaffin cells loaded with Oregon Green 488 BAPTA-1 as the Ca²⁺-sensitive fluorescent probe. The adrenal slice was continuously perfused with 200 μm hexamethonium (for at least 30 min before recording). The plots of relative Oregon Green 488 BAPTA-1 emission changes show a [Ca²⁺]_i rise in either the stimulated cell (1*, burst of action potentials triggered by an injection of a 500 msec depolarizing current) or three nearby cells (cells 2–4). Note that cell 5 remained silent. Dotted lines indicate the baseline.B, Histogram illustrating the percentage of cell fields in which the [Ca²⁺]_i rise was propagated to adjacent cells in control and hexamethonium-treated slices. *p < 0.01 compared with control.

Electrical coupling between chromaffin cell pairs in hexamethonium-containing extracellular medium. Membrane potential was monitored in chromaffin cell pairs using the dual patch-clamp technique. A, Illustration of a cell pair in which the triggering of action potentials in cell 1 resulted in small membrane depolarizations in cell 2 (left traces) and vice versa (right traces). The two cells were current-clamped at −65 mV. B, Example of a cell pair in which action potentials were transmitted to the nonstimulated cell. The two cells were current-clamped at −68 mV.C, Histograms illustrating the wide distribution range of the coupling ratio calculated in 22 chromaffin cell pairs (7 noncoupled pairs and 15 coupled pairs) from current-clamp measurements of voltage amplitude in response to a hyperpolarizing current injection in cell 1 (stepped cell) and cell 2 (target cell) (from 0 for noncoupled pairs to 1 for highly coupled pairs).

Macroscopic junctional currentsI _j between chromaffin cell pairs in hexamethonium-containing saline. Junctional currents (I _j) were measured in Cs⁺-loaded (140 mmCs⁺-gluconate) cell pairs voltage-clamped at −60 mV (transjunctional potential from −120 to +60 mV; 150 msec duration).A, Example of a low I _jamplitude indicating weak coupling (macroscopic junctional conductance,G _j = 50 pS, as calculated from the slope of the I–V curve). B, Robust electrical coupling evidenced by high I _jamplitude (G _j = 6.6 nS).C, I–V relationships from pooled data of six and five cell pairs exhibiting weak and robust coupling, respectively. The linear regression used to fit the data (dotted line) was y = −0.042x − 0.41 (r ² = 0.94) for the weak coupling and y = −12.4x− 6.37 (r ² = 0.99) for the robust coupling, given a G _j of 42 pS and 12.4 nS, respectively. D, Pooled data ofG _j calculated in five cell pairs bathed in control saline and 11 pairs in hexamethonium-containing saline (logarithmic scale on y-axis). The determination ofG _j in control medium includes data fromMartin et al. (2001). E, Percentage of appearance of weak and robust coupling and control (ctrl) and hexamethonium (hex)-treated slices.

Effect of inhibiting connexon delivery to the plasma membrane on the increase in gap junctional coupling in hexamethonium-treated slices. Connexon trafficking from the Golgi apparatus to the plasma membrane was abolished by treating slices with either BFA (2 μg/ml) or the cytoskeletal disrupting agent nocodazole (25 μm) for at least 30 min before adding 200 μm hexamethonium. A, a, Immunofluorescent labeling of the TGN using an antibody directed against TGN38. As expected, the labeling appeared as a fluorescent crescent near the nucleus (inset: scale bar, 5 μm) in untreated slices, whereas the Golgi network was dramatically disorganized in BFA-treated slices.A, b, Microtubules were immunolabeled with an antibody directed against β-tubulin. Nocodazole treatment strongly disrupted the cytoskeleton, as seen by the absence of β-tubulin detection.B, Pooled data showing that treatment with either BFA or nocodazole abolished the increase in LY spreading in hexamethonium-containing medium. *p < 0.01 compared with control.

Greater gap junction-mediated intercellular communication between chromaffin cells in neonates. A, Chart recording of spontaneous excitatory synaptic activity in chromaffin cells in neonates. B, Increased probability of LY diffusion in neonates compared with adults (*p < 0.01). C, Increase in the extent of coupling in neonates compared with adults. C, a, Examples of in situ LY diffusion between chromaffin cells in adults (2 cells) and in neonates (up to 5 cells in the same optical plane). 1* represents the patched cell. C, b, pooled data.

Propagation of electrical activity-linked [Ca²⁺]_i rise between chromaffin cells: a functional role for gap junctions in neonates. A, An acute adrenal slice from a neonatal rat was loaded with a Ca²⁺-sensitive probe to simultaneously image [Ca²⁺]_i changes in multiple neighboring chromaffin cells. The plots of relative Oregon Green 488 BAPTA-1 emission changes show a [Ca²⁺]_i rise in either the stimulated cell (1*, action potential triggered by an injection of a 500 msec depolarizing current) or in several nearby cells (cells 2–6). Dotted lines indicate the baseline. B, Histogram illustrating the percentage of cell fields in which the [Ca²⁺]_i rise was propagated to adjacent cells in neonates and in adults.