Synaptotagmin-7 facilitates acetylcholine release in splanchnic nerve-chromaffin cell synapses during nerve activity

Abstract

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca²⁺ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca²⁺-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in synapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

Keywords: Adrenal medulla; Preganglionic synapses; Synaptotagmin; Synaptotagmin-7.

Fig. 1.. Comparison of evoked EPSCs in WT and Syt7 KO synapses.

EPSCs were evoked by stimulating preganglionic input to the adrenal medulla with a bipolar stimulating electrode. Models created with BioRender (https://www.biorender.com). B. EPSCs recorded in a chromaffin cell evoked by stimulating the preganglionic nerve terminals (black) were blocked by the cholinergic antagonist hexamethonium (red), and recovered during washout (blue), *p = 0.032, Kruskal-Wallis, Dunn’s multiple comparisons (n = 5 slices; 3 independent preps). Representative EPSCs at WT synapses: Control (black), during block by Hexamethonium (Hex, red), and after Washout (blue) (inset). C. EPSCs recorded in chromaffin cells by stimulating the WT (black) and Syt7 KO (red) preganglionic nerve terminals. D. Averaged peak amplitudes of evoked EPSCs from Syt7 KO (red) are decreased compared to WT (black). ***p < 0.001, Mann Whitney test (n = 14 wt and n = 9 KO slices; > 6 independent preps). E. Average EPSC charge transfer from Syt7 KO (red) mice are decreased compared to WT (black) mice. ***p < 0.001, Mann Whitney test (n = 14 wt and n = 9 KO slices; > 6 independent preps) F. Average decay time constants of evoked EPSCs in chromaffin cells after stimulation of WT (black) and Syt7 KO (red) axons. p = ns, Mann Whitney test (n = 14 wt and n = 9 KO slices; >6 independent preps). G. Charge transfer normalized to peak EPSC amplitude from evoked EPSCs are not different in WT (black) mice compared to Syt7 KO (red) mice. p = ns, Mann Whitney test (n = 14 wt and n = 9 KO slices; >6 independent preps). H. CV⁻² of the EPSC amplitude was significantly greater in chromaffin cells from WT (black) compared to Syt7 KO (red) mice. **p < 0.05, Mann Whitney test (n = 14 wt and n = 9 KO slices; > 6 independent preps, those records were obtained at 2 mM external calcium).

Fig. 2.. Synaptic facilitation is evident in WT but not Syt7 KO medullae.

A. Representative traces (top) and averaged paired-pulse ratio (PPRs) ± SEM from evoked EPSCs at different interstimulus intervals (ISIs). PPRs at WT (black) and Syt7 KO (red) synapses are significantly different at ISIs of 60, 100 and 200 ms intervals but not at 500 ms. **p = 0.002, ***p < 0.001, Two-way ANOVA (n = 13 wt and n = 13 KO slices; >6 independent preps). B. Experiments were repeated at low extracellular Ca²⁺ (0.5 mM) and PPRs calculated as in A. *p = 0.01, Two-way ANOVA (n = 13 wt and n = 15 KO slices; >6 independent preps). Experiments at different calcium concentrations were performed on independent samples.

Fig. 3.. Tonic current is reduced at Syt7 KO synapses.

A, B. Synaptic responses to 20 Hz stimulation recorded from WT (black) and Syt7 KO (red) preparations. Expanded traces show the tonic current. C. Summary graph of individual EPSC amplitudes normalized to the first EPSC amplitude during a train (WT in black and Syt7 KO in red). D. Summary graph of the average tonic current, tonic current amplitudes was significantly greater in chromaffin cells from WT (black) compared to Syt7 KO (red) mice***p < 0.001, t = 3.840, df = 26 Student’s t-test (n = 12 wt and n = 16 KO slices; > 6 independent preps). (n = 7 wt and n = 11 KO slices; >4 independent preps). E. Average of PPRs calculated by dividing the second EPSC in the train by the first is. Facilitation is reduced in in chromaffin cells from Syt7 KO (red) compared with WT(black) mice ***p = 0.005, t = 6.515, df = 26 Student’s t-test (n = 12 wt and n = 16 KO slices; >6 independent preps.

Fig. 4.. Syt7 is expressed in splanchnic processes and chromaffin cells in the adrenal medulla.

A. A WT and Syt7 KO adrenal sections were stained with antibodies against Syt7 and ChAT, then imaged as shown. B. Boxed region in A is expanded. C. Fluorescence emission from the Syt7 channel was averaged across 8 wt and KO fields. Significantly less Syt7-positive fluorescence was detected in KO sections compared to WT sections. *** p < 0.001 Student’s t test, t = 4.765, df = 14 (n = 8 wt and n = 8 KO sections; 2 independent preps) D. The fluorescence intensity of Syt7 in discrete ROIs within a ChAT-positive object (i.e., axon) was measured. These ROIs were positioned in an image of a different Syt7-labeled field. Syt7 fluorescence was measured in these ROIs and used to define the null hypothesis (labeled “Control”). This process was repeated across 8 pairs of images on separate experimental days from sections obtained from two WT mice. E. The experimental distribution of intensities was compared to the null distribution of intensities. ****p < 0.0001 Student’s t test, t = 6.281, df = 14 (n = 8 ROI and n = 8 Ctrl images; 2 independent preps).