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Am J Physiol Heart Circ Physiol 281: H387-H395, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 1, H387-H395, July 2001

Frequency dependence of endocytosis in aortic baroreceptor neurons and role of group III mGluRs

Jaya Pamidimukkala1 and Meredith Hay1,2

1 Dalton Cardiovascular Research Center and 2 Department of Biomedical Sciences, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Synaptic transmission between baroreceptor afferents and the nucleus tractus solitarius (NTS) is known to exhibit frequency-dependent depression. Reductions in neurotransmitter release and alterations in mechanisms regulating synaptic transmission are hypothesized to be involved in the activity-dependent depression observed in baroreceptor afferent neurons. The present study utilized cultured aortic baroreceptor neurons and the fluorescent dyes FM1-43 and FM2-10 to characterize the process of endocytosis or vesicle retrieval and its dependence on 1) frequency of neuronal activation, 2) metabotropic glutamate receptor (mGluR) activation, and 3) calcium concentrations inside and outside the cell. Endocytosis per spike, measured in fluorescence units after a 10-s stimulus applied at frequencies of 0.5 (53 ± 4), 1.0 (23 ± 1), and 10.0 Hz (2.7 ± 0.2), was significantly depressed at higher frequencies. Blockade of group III mGluRs with (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) facilitated endocytosis at all frequencies, suggesting that this receptor subtype may be involved in the inhibition of endocytosis. Manipulating the extracellular and intracellular calcium concentrations subsequent to exocytosis had no effect on endocytosis. These results suggest that frequency-dependent depression of endocytosis observed in vitro could contribute to the frequency-dependent depression of baroreceptor afferent neurotransmission and that group III mGluRs inhibit endocytosis.

FM1-43; synaptic transmission; nodose ganglia; metabotropic glutamate receptors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BARORECEPTOR AFFERENTS with sensory endings in the aortic arch and carotid sinus relay information concerning the level of blood pressure via the nodose and the petrosal ganglia to the medullary neurons within the nucleus tractus solitarius (NTS). Appropriate integration of this baroreceptor information at the NTS is the first step in the normal regulation of cardiovascular reflex function (44). In both intact animals and brain slices taken from the NTS, intracellular recordings from neurons receiving direct synaptic inputs from sensory afferents have shown depression of evoked excitatory postsynaptic potentials (EPSPs) after a high-frequency stimulation of the afferents (2, 29, 44). Alteration of synaptic transmission between the baroreceptor afferents and the NTS neurons has been suggested to contribute to inappropriate reflex control of sympathetic outflow (23).

Synaptic depression or fatigue after repeated neuronal stimulation is a form of synaptic plasticity observed not only at the baroreceptor synapses but in a wide variety of synapses, including neuromuscular junctions and mammalian central synapses (5, 12, 26). Reduction in the neurotransmitter release from the presynaptic nerve terminals could potentially be responsible for this synaptic depression (14, 41, 48).

The frequency-dependent depression observed at the first synapse in the NTS has also been attributed to presynaptic mechanisms (28, 31). It has been hypothesized that glutamate, the major neurotransmitter at the baroreceptor afferent terminals, under conditions of high afferent activity, may act presynaptically to inhibit neurotransmitter release and affect synaptic fidelity. Likely candidates for mediating these presynaptic effects are the metabotropic glutamate receptors (mGluRs). These G protein-coupled receptors are involved in modulating excitatory and inhibitory synaptic transmission in the hippocampus, cerebral corticostriatal pathways, and visual cortex in rats (4, 9, 15, 32, 34). Both in vivo and in vitro studies suggest a role for mGluRs in mediating the synaptic depression at the baroreceptor afferent terminals at the level of the NTS (16, 28). Nodose ganglion cells in adult rats express mainly group III mGluRs, mGluR7 and -8, with lower expression of mGluR4 (24). Activation of these receptors with the group III specific agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) inhibits exocytosis from synaptic terminals in a primary culture of nodose ganglion cells (21).

Several factors could contribute to the reduction in exocytosis observed in the presence of L-AP4, including reduction in the 1) recycling capacity or endocytosis, 2) number of vesicles in the releasable and reserve pools, and 3) rate of mobilization of the vesicles from the reserve pool to the releasable pool (36, 43). Sustaining neurotransmission during periods of intense activity requires the nerve terminals to maintain pools of synaptic vesicles in reserve that can be recruited to active zones (37). Because most of the central neurons have only a few hundred vesicles, retrieval of vesicles or endocytosis after exocytosis plays a major role in preserving synaptic transmission (19, 43).

The aims of the present study were to 1) characterize endocytosis at the baroreceptor afferents and its dependence on the frequency of neuronal activation, 2) evaluate the role of mGluRs in the modulation of endocytosis, and 3) evaluate the role of extracellular Ca2+ and intracellular Ca2+ in endocytosis at the aortic baroreceptor nerve terminals.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General Methods

The methods used involved imaging of fluorescent styryl FM dyes, which specifically label recycling synaptic vesicles. This method allows us to study synaptic vesicle recycling at previously inaccessible nerve terminal compartments and the ability to distinguish between exocytosis and endocytosis (6, 7, 10). FM dyes are amphipathic molecules that can insert reversibly into the surface of lipid membranes. They emit minimal fluorescence in aqueous medium but fluoresce intensely upon membrane binding. When incubated with neuronal preparations, the dyes insert into the plasma membrane and are incorporated into the synaptic vesicle membrane during stimulus-evoked vesicle endocytosis. The dye remaining on the membrane that has not been internalized can be quickly washed away, leaving labeled synaptic vesicles inside the nerve terminal. Monitoring the uptake of the dye reflects endocytosis. The loss of fluorescence from these labeled regions on subsequent stimulation is a measure of vesicle exocytosis.

The kinetics of endocytosis, measured using simultaneous whole cell capacitance and styryl dyes, revealed a brief delay between exocytosis and endocytosis (3). Furthermore, at most fast synapses, exocytosis precedes endocytosis. Exocytosis ceases as soon as the stimulation is stopped, but endocytosis has been shown to continue for up to 2 min after stimulation (38). This sustained endocytosis was utilized in the present study to evaluate group III mGluR and Ca2+-mediated modulation of endocytosis in aortic baroreceptor neurons. In the present study, the drugs were added after cessation of electrically evoked action potential stimuli, thereby ensuring the effects observed were exclusively due to modulation of endocytosis.

Neuronal Culture and Preparation

All the experiments were carried out in primary cultures of nodose ganglia neurons from 3-wk-old Sprague-Dawley rats. Methods for labeling and dissociation of nodose ganglia neurons were similar to those previously described (22, 30). Briefly, the aortic baroreceptor neurons were anterogradely labeled by discrete application of the fluorescent anatomic dye Fast DiI (Molecular Probes) to the aortic arch of 15- to 18-day-old Sprague-Dawley rats. The rats were anesthetized with Metafane and artificially ventilated. After a thoracotomy, a small piece of Parafilm was placed underneath the aortic arch. With the use of a glass micropipette, small amounts of dye were placed on the aortic arch, close to the origin of the aortic depressor nerves (ADNs). To prevent the dye from spilling out to adjacent tissue, the area was covered with Wacker-Silicone gel, which was allowed to dry before the surgical closure. One week was allowed for the dye to be anterogradely transported via the ADNs to the cell bodies in the nodose ganglia. The right and the left nodose ganglia were isolated from all the rats and placed together in cold minimum essential medium (MEM) supplemented with 5% fetal bovine serum (Hyclone) and 0.1% serum extender (Collaborative). The tissue was then transferred to Earle's balanced salt solution (EBSS) containing 10 mg/ml of collagenase and incubated in a water bath for 30 min at 37°C. Papain (100 µl; Worthington) was added to the collagenase solution, and the tissue was incubated for an additional 25 min. After the collagenase solution was aspirated, the tissue was triturated in 2-3 ml of MEM containing 4% BSA and serum extender with serially smaller pipettes until most of the tissue was dissociated. After the remaining trituration media was added, the cell suspension was underlayed with 4% BSA in MEM and centrifuged. The pellet was resuspended in supplemented MEM containing nerve growth factor and finally plated on collagen-coated coverslips. The neuronal cultures were maintained for 7-14 days.

Optical Measurement of Endocytosis

Nodose ganglia neurons labeled with DiI fluoresced under an excitation wavelength of 550 nm and were identified as aortic baroreceptor neurons. These aortic baroreceptor neurons were then loaded with styryl FM dyes using a modification of the methods previously described (21). Briefly, the coverslips were transferred to a low-volume (0.7 ml) recording chamber fitted with two conducting wires connected to a stimulator (SD79, Grass Instruments). The buffer solution consisted of (in mM) 138 NaCl, 5.0 KCl, 1.8 CaCl2, 0.1 MgSO4, 0.9 NaH2PO4, 10 HEPES, and 28 glucose; pH 7.4. The stimulator was set to generate pulses of 1.0 ms duration at a given frequency. Immediately before the protocols were initiated, the regular bath solution was switched to a bath solution containing 100 µM FM2-10 or 2 µM FM1-43. The neurons were stimulated at a given frequency for a given duration of time. Stimulation was stopped, and the dye was allowed to remain in the bath for another 180 s. The cells were then washed for 5-10 min. After the wash, a total of five images (250 ms of exposure each) were acquired.

Synaptic boutons were viewed with a Nikon Diaphot 300 epifluorescence microscope (×100 oil immersion lens, 1.3 numerical aperture) and illuminated with a 100-W mercury lamp. Fluorescence imaging was achieved with 475- to 490-nm excitation and 525- to 550-nm emission filters. Digital images were acquired with a Photometric Sensys camera at preset times and stored on a computer. Each individual bouton cluster was composed of a zone selected to include the largest number of individual fluorescent spots while including the minimal background area. This region was digitally marked, and the average pixel intensity within this region was representative of the fluorescence intensity in an individual bouton cluster. The fluorescence in each loaded cluster across five images were averaged and used as a measure of endocytosis. Image averaging is a technique used to reduce random readout noise relative to the signal, and averaging five images improves the signal-to-noise ratio by ~2.2-fold. In our experiments, images were averaged after acquisition during analysis. For successful averaging, we ensured that each pixel got information from the same structural element or bouton cluster in the field of view during averaging. Each bouton cluster is a collection of several hundreds of fluorescent spots representative of several synaptic vesicles loaded with fluorescent dye. In these experiments, the Photometric Sensys camera configuration utilized a 1,313 × 1,035-pixel array with 6.8 × 6.8-µm pixels. Thus the resolution of the pixels was not good enough to image an individual bouton but did adequately image the bouton clusters. In Axon Imaging Workbench, each pixel has a value representing intensity. The greater the fluorescence intensity, the greater the pixel value. The average of all the pixel values in a bouton cluster is a measure of fluorescence intensity of an individual bouton cluster.

To account for fluctuations in lamp intensity, images were also acquired from dark regions not on the synaptic terminal and were subtracted from each image. On average, 8-12 regions were selected for each field of study. The FM dyes also passively and faintly stained the neuronal cell membrane without stimulation. FM2-10 was used in most of the experimental protocols because it required a shorter wash period compared with FM1-43 to remove nonspecific staining. Both FM1-43 and FM2-10 produced similar loading in the boutons. To account for nonspecific staining after each loading protocol, the loaded boutons were unloaded by replacing the bath solution with 90 mM KCl, and the residual fluorescence was subtracted from the initial fluorescence to obtain the final fluorescence. Seventy percent destaining occurs in the first 60 s, with the remaining ten to fifteen percent occurring over the next 2 min. In some experiments, nonspecific staining was determined by adding the FM dyes to the bath for 180 s without stimulating the neurons. Irrespective of the method used, nonspecific staining was always <15% of the total staining. Data were analyzed with Microsoft Excel and are expressed as means ± SE. Statistical differences were calculated using unpaired or paired t-test wherever appropriate using the StatsView program.

Experimental Protocols

Frequency dependence of endocytosis and role of group III mGluRs. In these experiments, neurons were stimulated at a given frequency (0.5, 1.0, or 10 Hz) for 10 s in presence of FM2-10 in the bath. Stimulation was stopped, and the dye was left in the bath for an additional 180 s. Cells were then washed for 5-10 min, and the fluorescence in the selected boutons was measured. The loaded boutons were then unloaded with 90 mM KCl for 3 min, and the residual staining was considered nonspecific staining. The role of group III mGluRs was evaluated by adding 300 µM of (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG) to the bath along with FM2-10 after the stimulation was stopped. The cells were then washed and unloaded with KCl as before. In all the experiments, the residual staining was subtracted from the initial fluorescence. To facilitate comparison between different stimulation protocols, the data were normalized to the number of spikes given by the stimulator during a protocol.

Modulation of endocytosis by group III mGluR agonist L-AP4. To assess the effects of the specific group III mGluR agonist L-AP4 on endocytosis, the neurons were first stimulated at 10 Hz for 10 s in presence of FM2-10 in the bath. Stimulation was stopped, and the bath solution was immediately replaced with bath solution containing FM2-10 and 200 µM L-AP4 for 180 s. Cells were then washed for 5-10 min, and fluorescence in the selected boutons was measured. The loaded boutons were then unloaded with 90 mM KCl for 3min. The residual staining was considered nonspecific staining and subtracted from the initial fluorescence. In these experiments, endocytosis was measured in arbitrary fluorescence units, and statistical comparison between paired groups was carried out using a t-test.

Calcium dependence of endocytosis. In these experiments, the neurons were stimulated at 10 Hz for 10 s. The stimulation was stopped, and 2.0 µM FM1-43 was added to the bath for 180 s. Cells were then washed for 10-15 min, and five images of the loaded boutons were acquired. The effect of extracellular Ca2+ on endocytosis was determined by adding FM1-43 to the bath in presence of low Ca2+ (no added calcium plus 2 µM EGTA) or CdCl2 (100 µM). The cells were then washed and imaged as described before. The role of intracellular Ca2+ in endocytosis was determined by adding FM1-43 to the bath in presence of caffeine (10 mM) or thapsigargin (500 nM). Caffeine increases intracellular calcium by releasing the calcium stores, and incubation with thapsigargin depletes calcium stores over time. The cells were then washed for 10 min and imaged. At the end of the experiments, the boutons were maximally loaded by depolarizing the cells with 2.0 µM FM1-43 in 90 mM KCl solution for 180 s. Loading with KCl was saturated at 3 min, and no further increases were observed after longer incubation periods. All the data are a percentage of this maximal intensity.

Cytosolic calcium imaging. To confirm that the doses of caffeine (10 mM) and thapsigargin (500 nM) were sufficient to produce the required changes in intracellular calcium pools, aortic baroreceptor neuron intracellular Ca2+ concentration ([Ca2+]i) levels were monitored using fura 2 fluorescence as described earlier (20). Briefly, nodose ganglion neurons were loaded with fura 2 by incubating the cells for 30 min in buffer solution containing 2 µM fura 2-AM and 20 µg/ml pluronic F127. Cells were illuminated with light emitted by 75-W Xenon lamp alternately filtered with narrow band-pass filters at 340 nm (Ca2+ sensitive) and then at 380 nm (Ca2+ insensitive). Emitted light from the fura 2 in the cells was filtered at 510 nm and captured with a Nikon charge-coupled device camera. Images were digitized and analyzed with the help of Axon Imaging Workbench. The changes in [Ca2+]i were determined by acquiring image pairs at 340/380 nm at intervals of 20 s for 5 min after stimulating the loaded cells with KCl (90 mM), caffeine (10 mM), and thapsigargin (500 nM). The effects of KCl and caffeine were also tested after emptying the intracellular calcium pools with thapsigargin. Conversion of the fluorescence intensity into calcium ion concentration was achieved by calibrating with buffer solution containing 0 and 100 µM Ca2+.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results reported here are based on experiments carried out in nodose ganglia neurons labeled with DiI and identified as aortic baroreceptor neurons (30). Figure 1A is a bright-field photomicrograph of a nodose ganglia neuron under a ×100 objective. Figure 1B is the same field observed under fluorescent illumination with a rhodamine filter. The DiI labeling is mostly observed in the soma of the neuron. After an aortic baroreceptor neuron was identified, the nerve process arising from that neuron was brought into the view field, and endocytosis was studied in the nerve terminals. Figure 1C shows the staining and destaining of the nerve terminals with FM2-10 in aortic baroreceptor neurons. Figure 1C, top, shows the nerve process before loading with FM2-10. Figure 1C, middle, shows the punctate staining of boutons in the process after they were loaded with FM2-10 and washed for 5 min. Figure 1C, bottom, shows the unloading or exocytosis of FM2-10 after depolarization with 90 mM KCl.


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  		Fig. 1.   A: bright-field image of a nodose ganglia neuron observed under a ×100 oil immersion objective. B: same field observed under fluorescent illumination with a rhodamine filter. The DiI labeling is mostly observed in the soma of the neuron. C: staining and destaining of the nerve terminals with FM2-10 in aortic baroreceptor neurons. Top, nerve process before loading with FM2-10. Middle, punctate staining of boutons in the process after they were loaded with FM2-10 and washed for 10 min. Bottom, unloading or exocytosis of FM2-10 after depolarization with 90 mM KCl.

Activity-dependent loading of the FM dyes was observed not only at sites where the neurites form contacts with other cell bodies or other neurites but also in discrete puncta on the axonal processes. With the use of immunocytochemistry, we (13) have previously shown that these aortic baroreceptor neurons express multiple synaptic proteins such as SNAP-25, synapsin I, and synaptotagmin in the neuronal processes as well as the cell bodies. These brightly stained puncta on the axonal processes were identified as functional synapses because exocytosis of these FM-loaded vesicles was dependent on calcium influx through voltage-gated Ca2+ channels (21). In the present study, endocytosis was studied only in those regions that released the dye on subsequent stimulation with KCl. Because we did not observe any differences between the puncta on processes and those on terminals in the present study, the data quantified include endocytosis observed at both the sites.

Frequency Dependence of Endocytosis and Role of Group III mGluRs

To determine whether the frequency-dependent depression of endocytosis is observed in aortic baroreceptor neurons, we studied the effects of increased stimulation frequency on absolute endocytosis. Figure 2 illustrates averaged endocytosis measured in baroreceptor terminals after stimulation of neurons at increasing frequencies with pulses of 1 ms duration for 10 s in the presence or absence of CPPG. In the absence of CPPG, endocytosis normalized to the number of spikes or pulses delivered during each frequency protocol was significantly smaller at higher frequencies (Fig. 2; P < 0.05). Endocytosis per spike, expressed in arbitrary fluorescence units at 0.5, 1.0, and 10.0 Hz, was 53 ± 4 [number of bouton clusters (n) = 128 from 10 experiments], 23 ± 1 (n = 122 from 10 experiments), and 2.74 ± 0.2 (n = 98 from 9 experiments), respectively. To determine whether activation of mGluRs is involved in the frequency-dependent depression of endocytosis, endocytosis was evaluated in the presence of a group III mGluR antagonist. Blockade of group III mGluRs with 300 µM CPPG increased endocytosis by 121 ± 16% at 0.5 Hz (from 53 ± 4 to 102 ± 9), 57 ± 18% at 1.0 Hz (P < 0.05 vs. 0.5 Hz, from 23 ± 1 to 30 ± 3), and 185 ± 109% at 10 Hz (from 2.74 ± 0.2 to 4.45 ± 0.5), respectively. These data suggest that group III mGluRs are involved in control of endocytosis at all the frequencies tested.


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  		Fig. 2.   Endocytosis after stimulation-induced depolarization of the nodose ganglia neurons is dependent on the frequency of stimulation. The neurons were stimulated with pulses of 1.0 ms duration at frequencies of 0.5 Hz (n = 128), 1.0 Hz (n = 122), 10.0 Hz (n = 98) for 10 s in the presence or absence of (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG). Endocytosis normalized to the number of pulses given during the stimulation period was significantly reduced at higher frequencies (*P < 0.05). In the presence of CPPG, endocytosis was facilitated at all the frequencies. dagger P < 0.05, significantly higher compared with the endocytosis at corresponding frequencies in the absence of CPPG.

Modulation of Endocytosis by L-AP4

To determine whether direct activation of group III mGluRs mimics the inhibition of endocytosis seen with increased frequencies, we evaluated the effects of L-AP4 on endocytosis. Activation of group III mGluRs with 200 µM L-AP4 after stimulation of the neurons with pulses of 1.0 ms duration at 10 Hz for 10 s resulted in heterogenous effects on endocytosis in the aortic baroreceptor neurons. In 4 of 12 experiments, L-AP4 inhibited endocytosis (Fig. 3, top; n = 42). Endocytosis, expressed in arbitrary fluorescence units, was 645 ± 48 under control conditions and 331 ± 31 in presence of L-AP4. In 7 of 12 experiments, L-AP4 facilitated endocytosis (Fig. 3, middle; n = 113). Control and L-AP4 values were 436 ± 22 and 582 ± 26, respectively. When the data were pooled together (Fig. 3, bottom; n = 168), endocytosis in presence of L-AP4 (509 ± 22) was not statistically different from control values (492 ± 22).


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  		Fig. 3.   Activation of group III mGluRs with the selective agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) had heterogenous effects on endocytosis. A: endocytosis, measured in arbitrary fluorescence units, was significantly inhibited in presence of 200 µM L-AP4 (4 of 12 experiments, n = 42, *P < 0.05). B: in 7 of 12 experiments, L-AP4 (200 µM) significantly facilitated endocytosis (n = 113, *P < 0.05). C: pooled data suggest that L-AP4 (200 µM) overall did not have any effect on endocytosis in the aortic baroreceptor neurons (12 experiments, n = 168).

Calcium Dependence of Endocytosis

One of the variables that is altered during high-frequency stimulation that may effect endocytosis is the free calcium ion concentration ([Ca2+]i) in the cytoplasm of the synaptic terminal. [Ca2+]i has been shown to increase during repetitive nerve stimulation in a wide variety of synaptic terminal preparations (25, 35, 47). At the frog neuromuscular junction, endocytosis is influenced by extracellular calcium levels but not [Ca2+]i (51). In retinal bipolar cells, recycling of vesicles back to the reserve pools and the releasable pools has been shown to be dependent on [Ca2+]i (18). In hippocampal neurons, endocytosis is either not dependent on extracellular or intracellular Ca2+ or inhibited by high internal Ca2+ (27, 39, 49). To determine if calcium has a role in endocytosis in aortic baroreceptor neurons, endocytosis was measured after manipulating the extracellular calcium concentrations and [Ca2+]i.

Endocytosis and Extracellular Calcium

Figure 4A illustrates endocytosis in the presence and the absence of calcium in the extracellular bath solution during endocytosis. In the absence of extracellular calcium (n = 25), endocytosis after stimulation of the neurons at 10 Hz for 10 s was reduced to 59 ± 3% from 70 ± 7% observed in the presence of normal levels of calcium in the bath solution. However, this reduction in endocytosis was not statistically significant. Figure 4B shows the effects of the calcium channel blocker CdCl2 on nodose ganglia neuron somatic voltage-gated Ca2+ currents after step depolarizations to 0 mV from a holding potential of -80 mV. The inward Ca2+ current is nearly abolished by application of 100 nM CdCl2, a voltage-gated Ca2+ channel blocker. However, even 10-fold higher concentrations of CdCl2 (100 µM, n = 34) did not affect endocytosis after a 10-Hz stimulation protocol (Fig. 4C; 52 ± 6% vs. a control value of 46 ± 5%). The data suggest that synaptic vesicle endocytosis can occur even after extracellular calcium is subsequently removed or calcium influx is blocked after depolarization.


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  		Fig. 4.   Removal of extracellular Ca2+ or inhibition of Ca2+ influx with Cd2+ subsequent to exocytosis had no effect on endocytosis. A: cells were stimulated at 10 Hz for 10 s before FM1-43 was added to the bath solution with or without Ca2+ (n = 25). B: effects of CdCl2 on nodose ganglia neuron somatic voltage-gated Ca2+ currents after step depolarizations to 0 mV from a holding potential (Vh) of -80 mV. The inward Ca2+ current was totally abolished by application of 100 nM of the voltage-gated Ca2+ channel blocker CdCl2. C: cells were stimulated at 10 Hz for 10 s before FM1-43 was added to the bath solution with or without 100 µM CdCl2 (n = 34).

Intracellular Calcium and Endocytosis

Figure 5 illustrates the role of intracellular calcium in endocytosis at the aortic baroreceptor neurons. Transient increases in intracellular calcium produced by bath application of 10 mM caffeine after depolarization of the neurons had no effect on endocytosis (Fig. 5A; 50 ± 6% compared with a control value of 46 ± 4%, n = 30).


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  		Fig. 5.   Modulation of intracellular Ca2+ concentrations ([Ca2+]i) with caffeine (10 mM, n = 30; A) or thapsigargin (TPG; 500 nM, n = 26; B) subsequent to the stimulation train had no effect on endocytosis. C: pseudocolor images of [Ca2+]i distribution in fura 2-loaded nodose neurons before and after application of KCl (top) and caffeine (bottom). D: effects of KCl (top) and caffeine (bottom) on [Ca2+]i in the presence of 500 nM TPG. The average changes in intracellular calcium produced by KCl and caffeine (n = 4) in the absence and presence of TPG are shown in E and F, respectively.

To confirm the effects of caffeine on intracellular Ca2+, in a separate group of experiments, changes in [Ca2+]i were measured in fura 2-loaded nodose ganglia neurons. Depolarization of neurons with a bath application of KCl produced peak changes in [Ca2+]i within 45 s (Fig. 5C, top). Application of 10 mM caffeine in Ca2+-free solution to the bath for 10 s produced transient increases in [Ca2+]i (Fig. 5C, bottom). Average changes in [Ca2+]i in response to KCl and caffeine are shown in Fig. 5E (n = 4).

Depletion of the intracellular pools by preincubation with 500 nM thapsigargin also did not effect endocytosis (Fig. 5B; 60 ± 5% vs. a control value of 55 ± 4%, n = 26) in aortic baroreceptor neurons. These data suggest that after exocytosis induced by short periods of stimulation, transient rises in intracellular calcium evoked by releasing intracellular calcium stores may not have a role in endocytosis in the aortic baroreceptor neurons. Preincubation of the fura 2-loaded nodose ganglion cells in 500 nM thapsigargin for 5 min blocked the caffeine-induced increases in [Ca2+]i but not the KCl-induced increases (n = 4; Fig. 5, D and F). These data confirm that the doses of caffeine and thapsigargin were sufficient to produce the required changes in intracellular calcium pools.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study are that baroreceptor terminals exhibit frequency-dependent depression of endocytosis and that group III mGluRs appear to be involved in feedback inhibition of vesicle recycling at both the low and high frequencies. At the beginning of high-frequency stimulation, the neurotransmitter release is greater, as suggested by increased postsynaptic events. However, the increased neurotransmitter release is not sustained toward the end of a prolonged stimulation period. The inability of neurons to compensate for the increased vesicle exocytosis during high-frequency stimulation by increased endocytosis has been suggested to contribute to the frequency-dependent depression of synaptic transmission in several types of central neurons (33, 38, 42, 50). In the present study, the frequencies at which depression of endocytosis occurred were similar to the input frequencies (5-10 Hz) at which 50% depression in postsynaptic responses in the NTS to sensory afferent stimulation was observed (2, 31). The data suggest that, in baroreceptor afferents, altered endocytosis could contribute to the frequency-dependent depression of synaptic transmission observed in vivo.

A number of studies have investigated the role of group III mGluRs in presynaptic modulation of synaptic transmission. In the locus coeruleus, activation of presynaptic group III mGluRs negatively modulates excitatory synaptic transmission during high-frequency stimulation (11). In vivo and in vitro studies (17, 28, 31) in the NTS have proposed involvement of presynaptic mGluRs in the frequency-dependent depression of synaptic transmission between the baroreceptor afferents and NTS neurons. Furthermore, nodose ganglia neurons isolated from adult rats have been shown to express mainly group III mGluRs, mGluR4, -7, and -8 (24). In the present study, blockade of group III mGluRs with the specific antagonist CPPG facilitated endocytosis in the aortic baroreceptor neurons at all the frequencies tested (0.5, 1, and 10 Hz). This suggests that activation of the presynaptic mGluRs by glutamate released from the presynaptic afferents in vivo may be involved in feedback inhibition of endocytosis. However, because endocytosis in presence of group III mGluR antagonist was effected at both the lower and higher frequencies, it does appear that this receptor subtype was responsible for the frequency-dependent suppression of endocytosis.

Because blockade of group III mGluRs resulted in facilitation of endocytosis, it was hypothesized that activation of mGluRs may inhibit endocytosis. However, in the present study, the effects of L-AP4 on endocytosis were heterogenous with some neurons exhibiting facilitation of endocytosis and others exhibiting inhibition. When averaged, the results suggest that group III mGluRs may have no effect on endocytosis. This result was unexpected and inconsistent with the results obtained with the group III antagonist CPPG. However, a second interpretation could be that the population of mGluRs activated by glutamate release during direct stimulation is different from the population of mGluRs activated by bath application of L-AP4. The glutamate released during stimulation is most likely to reach higher concentrations only at those microdomains near the active zone of the presynaptic terminals (8). This potentially activates a select population of group III mGluRs expressed near the active zones (45). Bath application of L-AP4 would be expected to activate all of the presynaptic mGluRs, including those close and farther away from the active zone. This may contribute to the equivocal effects of group III mGluR ligands on endocytosis in these neurons.

It is well established that calcium influx through voltage-activated Ca2+ channels is required for stimulating the fusion of synaptic vesicles at the release sites (46). Studies using fluorescent imaging of styryl dyes have also shown that calcium also regulates the subsequent retrieval of synaptic vesicles. Depending on the type of nerve preparation, increases in intracellular calcium after prolonged stimulation either inhibits or facilitates endocytosis (35, 40, 47). In the present study, the role of extracellular calcium was evaluated by removal of calcium from the extracellular bath and blockade of voltage-activated Ca2+ channels with Cd+2. Neither of these manipulations significantly altered endocytosis. Thus it appears that extracellular calcium may not be required for endocytosis. However, there may be alternate explanations. The design of the present study, which focused on endocytosis, required that normal exocytosis be induced first before calcium manipulation. In the aortic baroreceptor neurons, influx of calcium occurring during exocytosis might serve as the initiating event and be sufficient for endocytosis. This could explain the lack of effect on endocytosis in the present study, where alterations in the extracellular calcium were made subsequent to exocytosis.

Transient increases in intracellular calcium with caffeine or depletion of stores with thapsigargin also had no effect on endocytosis. However, release of calcium from the intracellular stores by calcium influx occurring during exocytosis may be sufficient to trigger vesicle retrieval, and additional increases in intracellular calcium during endocytosis would have no further effect.

In conclusion, endocytosis in aortic baroreceptor neurons does exhibit frequency-dependent depression. Endocytosis is inhibited by mGluR activation, but group III mGluRs do not seem to be responsible for the frequency-dependent depression of endocytosis.

Perspectives

The data from the present study indicate that the frequency-dependent depression of vesicle recycling observed under in vitro conditions could be one of the contributing factors to the frequency-dependent depression of synaptic transmission at baroreceptor afferents. Glutamate is the primary neurotransmitter at baroreceptor afferents and has been hypothesized to have the potential to modulate its own release via presynaptic mGluRs (17). In vitro study (21) of primary aortic baroreceptor neurons in culture suggest that presynaptic group III mGluRs are indeed involved in modulation of exocytotic and endocytotic processes that can regulate neurotransmitter release. However, the current data do not support an exclusive role for group III mGluRs in the frequency-dependent depression of endocytosis. Baroreceptor afferents have been shown to express receptors for neurotransmitters besides glutamate (1). If these cotransmitters are released along with glutamate, they may be involved in heteroreceptor-mediated inhibition of endocytosis. Because the frequency-dependent depression of endocytosis in the aortic baroreceptor neurons was observed in vitro in the absence of any postsynaptic contacts with NTS neurons, it may be inferred that the factors involved are of presynaptic origin. Future studies will address these issues.


    ACKNOWLEDGEMENTS

The authors thank Kathy Lindsley for excellent technical assistance in preparation of the cell cultures.


    FOOTNOTES

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

Address for reprint requests and other correspondence: M. Hay, Dalton Cardiovascular Research Center, Univ. of Missouri-Columbia, 134 Research Park, Columbia, MO 65211 (E-mail: HayM{at}missouri.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 October 2000; accepted in final form 6 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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