Real-time monitoring of chemical transmission in slices of the murine adrenal gland

Abstract

The real-time electrochemical detection of catecholamine secretion from murine adrenal slices using fast-scan cyclic voltammetry (FSCV) and amperometry at carbon fiber microelectrodes is described. Bright-field and immunofluorescent microscopy supported that chromaffin cells in the adrenal medulla are organized into clusters and positively stain for tyrosine hydroxylase confirming that they are catecholaminergic. Spontaneous exocytotic catecholamine events were observed inside chromaffin cell clusters with both FSCV and amperometry and were modulated by the nicotinic acetylcholine receptor antagonist hexamethonium and low extracellular calcium. Reintroduction of extracellular calcium and pressure ejection of acetylcholine caused the frequency of spikes to increase back to predrug levels. Electrical stimulation caused the synchronous secretion from multiple cells within the gland, which were modulated by nicotinic acetylcholine receptors but not muscarinic receptors or gap junctions. Furthermore, electrically stimulated release was abolished with perfusion of low extracellular calcium or tetrodotoxin, indicating that the release requires electrical excitability. An extended waveform was used to study the spontaneous and stimulated release events to determine their chemical content by FSCV. Consistent with total content analysis and immunohistochemical studies, about two thirds of the cells studied spontaneously secreted epinephrine, whereas one third secreted norepinephrine. Whereas adrenergic sites contained mostly epinephrine during electrical stimulation, noradrenergic sites contained a mixture of the catecholamines showing the heterogeneity of the adrenal medulla.

Figure 1

Adrenomedullary microenvironments. A, Representative bright-field image of adrenomedullary microenvironments. AM, Adrenal medulla; CV, central vein; CX, adrenal cortex. Asterisks mark four chromaffin cell clusters. Scale bar, 100 μm. Bright-field images B and F show two adrenomedullary microenvironments identified as chromaffin cell clusters. Fluorescent images C–E, A negative control experiment in which the primary antibody to TH was omitted from the immunohistochemical protocol. Images G–I show the localization of the TH immunofluorescence to cytoplasm of chromaffin cells contained within a cluster. In both experiments, the nuclei were stained with Hoechst 33342 (C and G). In H, TH antibody conjugated to Alexa Fluor 647 labels chromaffin cells contained within a cluster. E and I show C/D and G/H overlays respectively. Scale bar, 25 μm.

Figure 2

Bright-field micrograph of an electrochemical recording setup. Carbon-fiber microelectrode (arrow points to the microelectrode tip) is positioned within a chromaffin cell cluster; the stimulating electrode consisting of two tungsten electrodes (tip of each is marked with an asterisk) is placed on top a slice straddling the electrochemical sensor. Scale bar, 100 μm.

Figure 3

Representative electrically evoked catecholamine release event in a chromaffin cell cluster. The color plot shows current changes observed before (−5 to 0 sec), during (0–1 sec), and after (1–50 sec) the delivery of an electrical stimulus. Catecholamines are oxidized at +600 mV vs. Ag/AgCl (green) and the resultant o-quinone is reduced at −200 mV (dark blue). The trace above the color plot shows the changes in catecholamine concentrations that occur as a result of the electrical stimulation (extracted at +600 mV, solid horizontal line). The delivery of electrical stimulation at time 0 sec is indicated by a red bar; the second red bar at time 1 sec marks the end of stimulation. Cyclic voltammograms (inset) that confirm signal’s catecholaminergic chemical identity were extracted from the color plot at the time of the maximum concentration change (striped vertical lines). The black arrow points to the maximum concentration change for electrically stimulated release, and it corresponds to the cyclic voltammogram plotted in black; the gray arrow points to the maximum concentration change for spontaneous catecholamine release event and it corresponds to the gray cyclic voltammogram.

Figure 4

Modulation of electrically stimulated catecholamine release. A, Transmural electrical stimulation of tissue slices during perfusion of low Ca²⁺ buffer (0.1 mm) results in the absence of catecholamine release. Top panel, Representative catecholamine concentration vs. time traces and their corresponding color plots before (left), during (middle), and after (right) low Ca²⁺ buffer perfusion. The inset cyclic voltammograms confirm catecholaminergic nature of the detected signal. Lower panel, Summary of the modulation of catecholamine release by low Ca²⁺ buffer and TTX. B, Slice perfusion with HEX abolishes electrically stimulated release. Similar to A, top panel of B shows representative catecholamine concentration vs. time traces and their corresponding color plots before (left), during (middle), and after (right) 100 μm HEX perfusion. Lower panel summarizes the modulation of release by the nicotinic (HEX) and muscarinic (ATR and 4-DAMP) AChR antagonists as well as the gap junction blocker COX. ***, P < 0.001.

Figure 5

Biological origin of spontaneous catecholamine secretion. The representative amperometric recording of spontaneous catecholamine release in A was obtained by holding the microelectrode at a DC potential of +700 mV; in B, the number of spontaneous release events was reduced by about 70% after a 20-min perfusion of the slice preparation with HEX, a cholinergic nicotinic receptor blocker. A 20-min perfusion with a low Ca²⁺ (0.1 mm) buffer reduced the number of spontaneous events by about 90% (C and D) and was recoverable by pressure ejection of 1 mm acetylcholine (Ach; E and F).

Figure 6

Electrochemical analysis of spontaneous catecholamine release. Catecholamine concentration vs. time traces and their corresponding color plots show spontaneous adrenergic (top panel) and noradrenergic (bottom panel) secretory activity observed within two separate cell clusters. Top (green, extracted at +1300 mV) and bottom (blue, extracted at +600 mV) concentration traces reveal chemical identity of the catecholamine released: spontaneous secretion of epinephrine is established by the presence of voltammetric spikes at both +1300 mV (striped white line in color plot) and +600 mV (solid white line in color plot), whereas spontaneous norepinephrine secretion is confirmed by the presence of spikes at +600 but not +1300 mV. Catecholamine concentration vs. time traces for +600 and +1300 mV are offset for clarity.

Figure 7

Electrochemical analysis of electrically evoked catecholamine release. Catecholamine concentration vs. time traces and their corresponding color plots reveal the chemical identity of electrically evoked release in locations that have been identified based on spontaneous secretory activity as either adrenergic (top) or noradrenergic (bottom). The site that was previously found to spontaneously release only norepinephrine displays the presence of a second oxidation event as evident from the color plot on the right as well as the concentration vs. time trace at +1300 mV.