Elsevier

Brain Research

Volume 1006, Issue 2, 1 May 2004, Pages 215-224
Brain Research

Research report
Frequency dependence of synaptic vesicle exocytosis in aortic baroreceptor neurons and the role of group III mGluRs

https://doi.org/10.1016/j.brainres.2003.11.081Get rights and content

Abstract

Synaptic transmission between baroreceptor afferents and the nucleus tractus solitarius (NTS) is essential for reflex regulation of blood pressure. High frequency stimulation of the afferents in vivo leads to a decrease in synaptic strength and is generally attributed to reduction in presynaptic neurotransmitter release. It has been hypothesized that during high frequency stimulation glutamate a major neurotransmitter at the baroreceptor afferent terminals inhibits its own release via presynaptic group III metabotropic glutamate receptors (mGluRs). A key player in modulation of presynaptic release is vesicle exocytosis. The present study utilized cultured aortic baroreceptor neurons and the styryl dye FM2–10 to characterize (1) the dependence of exocytosis at these afferent nerve terminals on the frequency of neuronal activation, (2) the effect of duration of stimulation on the rate of exocytosis and (3) the role of mGluRs in the frequency-dependent modulation of exocytosis. Destaining in the FM2–10 loaded boutons during 3 min of stimulation, a measure of exocytosis, progressively decreased with increasing frequency (0.5, 1.0 and 10 Hz). Blockade of group III mGluRs with 300 μM (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated exocytosis evoked by 10 Hz stimulation but not at 0.5 Hz. The data suggest that aortic baroreceptor terminals exhibit frequency-dependent depression of exocytosis and support a role for group III mGluRs in the frequency-dependent modulation of exocytosis.

Introduction

Sensory nerve endings of the carotid sinus and aortic depressor nerves, located in the arterial walls sense changes in blood pressure and convey this information via the petrosal and nodose ganglia to the nucleus tractus solitarius (NTS). Transmission of information at the “first synapse” between the sensory afferent terminals and neurons in the NTS has been studied extensively in vivo and in vitro [23], [29][1], [4], [9], [20], [24]. These studies show that fidelity of synaptic transmission at this central synapse is influenced by the frequency at which the sensory afferents are activated. High frequency activation (>5 Hz) of the sensory afferents in the solitary tract result in a significant reduction in the amplitude and number of excitatory postsynaptic potentials (EPSPs) evoked in the NTS [1], [20], [24]. As this post-tetanic depression occurred in the absence of concomitant changes in the resting membrane potential of the NTS neurons most investigators put forward arguments in favor of a presynaptic event namely alteration in neurotransmitter release from the presynaptic terminal [4], [24].

Nerve terminals in the central nervous system are inaccessible to approaches used to study synaptic vesicle recycling in giant invertebrate synaptic terminals or in relatively large mammalian neuroendocrine cells [31]. In recent years, development of an optical technique that allows fluorescence imaging of activity-dependent uptake and release of styryl dyes such as FM1–43, FM2–10 by synaptic vesicles has yielded valuable information about dynamics of vesicle recycling at small synaptic terminals [3], [27]. Hay and Hasser [13] have shown that these styryl dyes can be used to study synaptic vesicle exocytosis in primary cultures of baroreceptor afferent neurons of the nodose ganglia. These studies have shown that destaining in FM1–43 loaded synaptic boutons can be evoked by a depolarizing pulse of high K+ or by field stimulation in a calcium-dependent manner. The present study proposes to utilize this technique to determine the dependence of exocytosis at the baroreceptor afferent nerve terminals on the frequency of neuronal activation.

Typically, after exocytosis of the neurotransmitter into the synaptic cleft, empty synaptic vesicles are recycled back into the presynaptic terminal, to be refilled and reused. It is thought that in small nerve terminals with limited capacity for storage, rate of recycling or endocytosis of synaptic vesicles could have an important role in the maintenance of synaptic transmission during high levels of afferent activation. Previous studies from our lab have shown that in aortic baroreceptor neurons, the uptake of FM2–10 by recycling vesicles (a measure of endocytosis) decreased as the frequency of stimulation increased [26]. It can be hypothesized that the rate of exocytosis may not be sustained during prolonged high frequency stimulation. The present study also evaluates the effect of duration of stimulation on the rate of exocytosis during low and high frequency stimulation.

The final aim of this study is to understand the role of metabotropic glutamate receptors (mGluRs) in modulation of exocytosis at different frequencies. Its been hypothesized that glutamate, a major neurotransmitter at baroreceptor afferent terminals, modulates its own release via presynaptic group III mGluRs [12], [19]. In primary cultures of baroreceptor neurons, exocytosis evoked with bath application of 90 mM K+ was inhibited in presence of group III mGluR agonist L-AP4. Group III mGluR antagonist CPPG facilitates endocytosis in baroreceptor neurons but this facilitation was observed at both low and high frequencies [13], [26]. The present study tests the hypothesis that blockade of group III mGluRs reverses the depression of exocytosis observed at high frequency stimulation, by measuring exocytosis evoked at different frequencies in the absence and presence of CPPG.

Section snippets

Methods

The experimental protocols in this work were reviewed and approved by the Animal Care and Use Committee at the University of Missouri. All the experiments were carried out in primary cultures of nodose ganglia obtained from 21-day-old Sprague–Dawley rats.

Frequency dependence of exocytosis and the role of group III mGluRs

As described above, neurons were loaded with the dye FM2–10 by depolarizing the cells with buffer solution containing 90 mM of K+ and 200 μM of FM2–10 for 3 min. Cells were washed for 5–10 min in physiological buffer solution and the fluorescence in the selected boutons (or ROI) was measured during stimulation at different frequencies. Prior to beginning the protocol, control images were acquired once every 3 s for 30 s. The stimulator was set to deliver 1.0 ms pulses at an intensity of 10 V

Results

Results reported here are based on experiments carried out in nodose ganglia neurons labeled with DiI and identified as aortic baroreceptor neurons. Fig. 1A is a bright field image of a nodose ganglia neuron under 60X magnification. Fig. 1B is the same field observed under fluorescent illumination with DiI filter (excitation light filtered at 550 nm and emitted light collected at 565 nm). The DiI labeling is mostly observed in the soma of the neuron. After identifying an aortic baroreceptor

Discussion

The principal findings of the present study are aortic baroreceptor terminals in culture exhibit frequency-dependent depression of exocytosis and the duration of stimulation effects rate of exocytosis even at low frequencies. This study also provides evidence to support a role for group III mGluRs in the frequency-dependent depression of exocytosis.

Most of the information on frequency-dependent depression of synaptic transmission at the afferent terminals in the NTS had been obtained from

Acknowledgements

This work was supported by National Heart, Lung and Blood Institute Grant HL-59676.

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