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. Author manuscript; available in PMC: 2014 Sep 18.
Published in final edited form as: Neuroscience. 2006 Nov 13;144(2):665–674. doi: 10.1016/j.neuroscience.2006.09.053

TWO TYPES OF NEUROTRANSMITTER RELEASE PATTERNS IN IB4-POSITIVE AND NEGATIVE TRIGEMINAL GANGLION NEURONS

Yoshizo Matsuka 1,#, Brian Edmonds 2,&, Somsak Mitrirattanakul 1,*, Felix E Schweizer 2,3, Igor Spigelman 1,3,4
PMCID: PMC4166549  NIHMSID: NIHMS15966  PMID: 17101230

Abstract

Mammalian nociceptors have been classified into subclasses based on differential neurotrophin sensitivity and binding of the plant isolectin B4 (IB4). Most of the nerve growth factor-responsive IB4-negative (IB4 (-)) nociceptors contain neuropeptides such as substance P and calcitonin gene-related peptide, whereas the glial-derived neurotrophic factor-responsive IB4-positive (IB4 (+)) neurons predominantly lack such neuropeptides. We hypothesized that the differences in neuropeptide content between IB4 (+) and (-) neurons might be reflected in differences in stimulated exocytosis and/or endocytosis. To address this, we monitored the secretory activity of acutely dissociated neurons from adult rat trigeminal ganglia (TRG) using cell membrane capacitance (Cm) measurements and the fluorescent membrane-uptake marker FM4-64. Cm measurements were performed under whole-cell voltage clamp and neurons were depolarized from −75 mV to +10 mV to elicit exocytosis. Both types of TRG neurons showed similarly-sized, calcium-dependent increases in Cm, demonstrating that both IB4 (+) and (-) TRG neurons are capable of stimulated exocytosis. However, the peak Cm of IB4 (+) neurons decayed faster towards baseline than that of IB4 (-) neurons. Also, IB4 (+) neurons had stable Cm responses to repeated stimuli whereas IB4 (-) neurons loss their secretory response during repeated stimulation. These data suggested that the IB4 (+) neurons possess faster rate of endocytosis and vesicle replenishment than IB4 (-) neurons. To test this, we measured vesicle trafficking with the fluorescent membrane dye FM4-64. FM4-64 staining showed that IB4 (-) neurons exhibit a larger pool of endocytosed vesicles than IB4 (+) neurons because the peak fluorescence increases in IB4 (-) neurons were larger but slower than in IB4 (+) neurons. However, the recycled vesicles were released faster in IB4 (+) compared to IB4 (-) neurons. Taken together these data suggest that the IB4 (+) TRG neurons have faster exocytosis and endocytosis than the IB4 (-) neurons.

Keywords: exocytosis, endocytosis, sensory neurons, fluorescent dye, nociceptor, pain signaling

INTRODUCTION

The virtual lack of synapses and the offstream position of the neuronal somata in sensory ganglia with respect to the axonal processes that transmit sensory information from the periphery to the central nervous system (CNS) supported the view that somata are of limited significance in terms of an electrophysiological role in the intact animal (Lieberman 1976). This traditional view had been challenged by the demonstration that repetitive activation of sensory neurons is capable of increasing excitability of adjacent neurons (Devor and Wall 1990). Later studies suggested that this intraganglionic dialog (named cross-excitation) may be mediated by a yet to be identified activity-dependent diffusible substance(s) released from neuronal somata and/or adjacent axons, and detected by neighboring cell somata and/or axons (Amir and Devor 1996). The somata of sensory neurons were subsequently demonstrated to be capable of transmitter release in vitro (Huang and Neher 1996; Ulrich-Lai et al., 2001) and in vivo (Neubert et al., 2000; Matsuka et al., 2001). Both calcium-dependent and calcium-independent cross-excitation of vagal sensory neurons was also demonstrated (Oh and Weinreich 2002). Additional evidence for a more active role of somata in sensory signal transmission, especially in pathological states, had come from the demonstrated increases in excitability of sensory neurons and the development of spontaneous ectopic discharge which originated within sensory ganglia after peripheral nerve injury (Kirk 1974; Kajander et al., 1992; Sheen and Chung 1993; Xie et al., 1995; Zhang et al., 1997). Thus, improved understanding of the mechanism of neurotransmitter release from sensory neuron somata is needed to determine its physiological and pathological roles in sensory signal transmission.

Mammalian nociceptive neurons can be identified on the basis of their small soma size (diameter < 25 μm) and electrophysiological properties (Harper and Lawson 1985) and have been further subdivided into two subgroups based on binding of the isolectin B4 (IB4) from the plant Griffonia simplicifolia (Silverman and Kruger 1990). In addition, the two subgroups differ in their neurotrophin requirements and neuropeptide content. Most of the nerve growth factor (NGF)-responsive IB4-negative (IB4 (-)) nociceptors contain neuropeptides such as substance P and CGRP, whereas the glial cell line derived neurotrophic factor (GDNF)-responsive IB4-positive (IB4 (+)) neurons predominantly lack such neuropeptides (Nagy and Hunt 1982; Silverman and Kruger 1990; Bennett et al., 1996; Molliver et al., 1997; Gerke and Plenderleith 2001). Their anatomical distributions also differ in that IB4 (+) neurons innervate the epidermis whereas IB4 (-) neurons innervate subcutaneous, joint and visceral structures (Lu et al., 2001; Ivanavicius et al., 2004; Aoki et al., 2005). In the spinal cord, IB4 (+) neurons terminate primarily in the inner lamina II, whereas the IB4 (-) peptidergic nociceptors terminate in lamina I, and outer lamina II (Coimbra et al., 1974; Carlton and Hayes 1989; Silverman and Kruger 1990; Gerke and Plenderleith 2004; Sanderson Nydahl et al., 2004).

The separation between the IB4 (+) and (-) neurons is not always clear-cut. First, not all small diameter neurons are nociceptors; some are low-threshold mechanoreceptors (Fang et al., 2006). Also, there is considerable overlap between IB4-labeling, neurotrophin responsiveness, and neuropeptide content of small diameter sensory neuron subpopulations (Ambalavanar and Morris 1992; Wang et al., 1994; Fang et al., 2006). To some extent, such overlap and the variability in expression of specific receptors has been attributed to species differences and to differences between neurons in cranial (trigeminal) versus dorsal root ganglia (Ambalavanar and Morris 1992; Elcock et al., 2001; Ambalavanar et al., 2005), although recent studies indicate the existence of an IB4 (+)/NGF-responsive subpopulation of nociceptors (Fang et al., 2006). Nevertheless, overall evidence suggests that the majority of IB4 (+) and (-) nociceptor populations are functionally distinct, based on factors such as the predominant localization of P2X3 receptors on IB4 (+) neurons (Vulchanova et al., 1997; Vulchanova et al., 1998; Bradbury et al., 1998), as well as differences in the membrane and action potentials, magnitudes of TTX-resistant Na+-currents, Ca2+ currents, K+ currents, fiber conduction velocities, and responses to heat, proton or capsaicin stimuli (Stucky and Lewin 1999; Dirajlal et al., 2003; Liu et al., 2004; Wu and Pan 2004; Vydyanathan et al., 2005; Fang et al., 2006).

Based on the potential link between transmitter release and cross-excitation of neurons within sensory ganglia we studied transmitter release in single sensory neurons. We hypothesized that differences in the innervation patterns and neuropeptide content of two nociceptor subclasses will be reflected in their transmitter release patterns. One way to study neurotransmitter release is via recording of cell surface area changes that occur as a result of secretory vesicle fusion with cell membrane during transmitter release using cell membrane capacitance measurements (Jaffe et al., 1978; Neher and Marty 1982; Gillis 1995). Alternatively, vesicle recycling can be quantified using fluorescent marker dyes such as the styryl FM dyes (Kirk 1974; Kajander et al., 1992; Sheen and Chung 1993; Ryan et al., 1993; Vida and Emr 1995; Zhang et al., 2004). Thus, we studied the differences in exocytosis and endocytosis between acutely dissociated IB4 (+) and (-) neurons from adult rat trigeminal ganglia by monitoring cell surface area using cell membrane capacitance measurements and by monitoring vesicle recycling using the fluorescent dye FM4-64.

Experimental Procedures

Acute dissociation of trigeminal ganglia neurons (TRG)

Following the protocol approved by the UCLA Institutional Animal Care and Use Committee (IACUC), male rats were deeply anesthetized (60 mg/kg pentobarbital, i.p). The TRGs were harvested and cut in small pieces in ice-cold Hank's Balanced Salt Solution (HBSS, Sigma, St. Louis, MO) supplemented with 20% fetal bovine serum (FBS, HyClone, Logan, UT), and incubated for 30min at 37°C in 5ml of minimal essential medium (MEM, Gibco, Carlsbad, CA) with 10% FBS, 2mM glutamine, 24mg/L insulin, 0.125% collagenase P (Roche, Indianapolis, IN) and 0.02% DNase (Sigma). TRGs were transferred for 5min at 37°C to 2ml of Ca2+- and Mg2+-free HBSS (Sigma) containing 0.25% trypsin (Sigma) and 0.05% DNase. Cells were dissociated by tissue trituration with a series of fire-polished Pasteur pipettes in 5ml HBSS containing 0.295% MgSO4 and 0.02% DNase.

Patch-clamp

Dissociated cells were grown for 4-24 hours on cover slips previously coated with 50μl of matrigel (1:100 dilution, Becton Dickinson, Franklin Lakes, NJ) and incubated for 10min with 10μg/ml of fluorescein-conjugated lectin IB4 (Vector Laboratories, Burlingame, CA). Neurons were judged to be IB4 (+) if they exhibited circular staining and fluorescence intensity several fold higher than their non-stained counterparts. Cells with ambiguous/intermediate staining intensity were not included in the study. For electrophysiological recordings, the coverslips were mounted in a perfusion chamber (Warner Instruments, Hamden, CT) on the stage of a Zeiss Axiovert 100 microscope equipped with epifluorescence (Carl Zeiss, Germany). External solution contained (in mM): NaCl 135; glucose 20; HEPES 20; CaCl2 5; MgCl2 1.5; TEA 10 with TTX 100nM, adjusted to pH 7.2 with NaOH and 310 mmol/kg with sucrose. Patch clamp recordings were made using pipettes with a tip resistance of ~3MΩ pulled from borosilicate glass (1.5 mm O.D., World Precision Instruments, Sarasota, FL) on a P97 pipette puller (Sutter Insturments, Novato, CA).

Pipettes were filled with an internal solution containing (in mM): Cs-methanesulfonate 145; NaCl 10; glucose 10; HEPES 20; EGTA 0.5; Mg-ATP 4; Na-GTP 0.6; adjusted to pH 7.2 with CsOH and 290 mmol/kg osmolality. In some experiments EGTA concentration was changed to 0 or 5 mM. Recordings were made with an Axopatch 200 patch clamp amplifier (Axon Instruments, Union City, CA) or Optopatch (Cairn Research, Faversham UK). The data were filtered at 5kHz before being sampled at 20kHz using a 6052E data-acquisition board (National Instruments, Austin, TX) and jClamp software (Scisoft Co, New Haven, CT) or software written in LabView (National Instruments, Austin, TX) running on a Pentium-chip based personal computer.

FM4-64 dye staining, confocal imaging and analysis

Dissociated cells were tested with FM4-64 dye (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide; Molecular Probes, Eugene, OR) 4-8 hours after the dissociation. FM4-64 dye (10 μM) was added in control external solution (control solution) (mM) NaCl 119; KCl 5; glucose 30; HEPES 25; CaCl2 2; MgCl2 2 adjusted to pH 7.2 with NaOH and 310 mmol/kg, or in high KCl external solution (high K+ solution) (mM) NaCl 49; KCl 75; glucose 30; HEPES 25; CaCl2 2; MgCl2 2 adjusted to pH 7.2 with NaOH and 310 mmol/kg. Since membrane-associated dye is much more fluorescent than dye in solution, we could quantify the intensity of FM4-64 fluorescence as a measure of dye-uptake. For dye-uptake experiments, neurons were imaged in the presence of the dye and stimulated by high K+ solution with FM dye. Then the dye was washed out with control solution. For dye release experiments, neurons were stimulated with the high K+ solution containing 10 μM FM4-64 for 3 minutes. Cells were washed three times with control solution to remove not-endocytosed FM dye bound to the plasma membrane and subsequently stimulated with high K+ solution without FM dye. Fluorescein-conjugated IB4 (10μg/ml) was added to the chamber for 10 min at the end of each FM4-64 experiment, washed out for 10 min and imaged. The criteria for IB4 labeling were as in patch-clamp recordings.

TRG neurons were examined by confocal microscopy during FM4-64 experiments. Confocal optical sections were scanned using a Zeiss LSM 410 laser scanning microscope (Carl Zeiss) with 10-fold attenuated laser excitation at 568nm, long-pass emission filter with a cut-off of 590nm, objective: X100, 1.4 NA oil-immersion lens, pinhole size: 60 ayres, and constant contrast of 300 for FM dye staining and 400 for FM dye destaining. Fluorescence intensities of neurons were analyzed using ImageJ (http://rsb.info.nih.gov/ij). Background fluorescence obtained from 20 μm diameter circular fields near the imaged cells was subtracted from the cell fluorescence values.

RESULTS

We recorded cell membrane capacitance (Cm) from small, voltage-clamped TRG neurons to compare the secretory responses between IB4 (+) and (-) neurons. Fig. 1 shows a recording from an IB4 (+) neuron. The neuron was voltage clamped at -75 mV and a sine wave voltage command (amplitude: 13 mV rms, 800 Hz) was superimposed onto the holding potential to measure series resistance (Rs; 6 MΩ), membrane resistance (Rm, 300 MΩ) and cell membrane capacitance (Cm, 28 pF; see methods). A depolarization to +10 mV elicited an inward current due to the activation of voltage-dependent calcium channels. No accurate capacitance measurements can be made during large changes in membrane conductance, but when the measurements were resumed, Cm had increased by 456 ± 84 fF (p < 0.001) and Rm had decreased by 109 ± 28 MΩ (p = 0.002) with no change in Rs (0.055 ± 0.019 MΩ). The decrease in Rm might result from the insertion of vesicular channels into the plasma membrane during exocytosis. Assuming a specific membrane Cm of 1μF/cm2 and a vesicle diameter of 45-60 nm (Alvarez et al., 1993; Bao et al., 2003), a single vesicle has a membrane capacitance of 60-110 aF. The 450 fF increase in Cm in Figure 1 thus corresponds to the fusion of 4000-7500 vesicles.

Fig. 1. Representative recording of membrane parameters.

Fig. 1

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Depolarization pulse induces an inward Ca2+ current and membrane capacitance jump. Thick line in Vm represents the sine wave.

Both IB4 (+) and (-) neurons responded to depolarizations with rapid Cm increases of similar size (Fig. 2), indicating that they both are capable of fast exocytosis and suggesting that they might have a similar number of releasable vesicles. However, closer inspection of individual examples (Fig. 2A) and averaged data of Cm increases (Fig. 2B) revealed important differences in the response of these neurons to depolarization. In 12 of 14 IB4 (+) neurons, the evoked Cm peaked within 100 ms after the depolarization followed by a relatively fast decay, most likely due to relatively rapid endocytosis. By contrast, in 10 of 11 IB4 (-) neurons, the initial depolarization-evoked Cm increase was followed by a slower additional Cm increase. Since the secondary increases in Cm occurred at different times after the stimulus, the averaged traces appeared relatively flat (Fig. 2B). A single exponential fit to the Cm decay in individual neurons revealed that the decay time constant in IB4 (+) neurons (0.8 ± 0.3 s, n = 12) was significantly different (p < 0.05, t-test) than the decay time constant in IB4 (-ve) neurons (2.8 ± 1.0 s, n = 8). In 2 IB4 (+) and 3 IB4 (-ve) neurons the Cm continued to rise throughout the recording period, precluding decay time constant measurements. The differences in Cm decay suggested that the IB4 (+) and (-) neurons may possess two different rates of exocytosis and endocytosis. To test whether we missed a fast phase of membrane retrieval in IB4 (-) neurons, we shortened the depolarization to 10 ms. However, while Cm increases were still observed even in response to such short stimuli, there still was no obvious fast phase of endocytosis in IB4 (-) TRG neurons (Fig. 2C). Moreover, the relationship between the initial Cm jump and duration of the depolarization was similar for IB4 (+) and (-) neurons (Fig. 2D).

Fig 2. Differences in Cm decay between IB4 (+) and IB4 (-) neurons.

Fig 2

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A: recordings from representative neurons with a 300 ms depolarization pulse. B: averaged traces from IB4 (+; n = 14) and IB4 (-; n = 11) neurons. IB4 (+) neurons showed a fast Cm jump and a quick decay after the depolarization pulse. IB4 (-) neurons showed a slow-peaking increase followed by a slow decay. C: recordings from representative neurons with a 10 ms depolarization pulse. Note the difference in scale compared to A and B. D: relationship between stimulation length and the initial ΔCm. The initial Cm increase after stimulation is similar between IB4 (+) and (-) neurons (n = 6 each).

We next tested whether the absence of a fast retrieval mechanism might compromise the ability of IB4 (-) neurons to respond to repetitive stimuli. For this, we stimulated the neurons with short, 10 ms depolarizations every 90 s and measured the amplitude of the Cm increase as well as the amplitude of the Ca2+ current. In both IB4 (+) and (-) groups, the amplitude of the Ca2+ current became smaller with each depolarization (Fig. 3B), probably due to wash-out of an endogenous factor(s) during whole-cell recording. The amplitude of Cm increases also declined with repetitive stimulation (Fig. 3A). However, the decrement in the Cm amplitude with repetitive stimulation was much smaller in IB4 (+) neurons compared to IB4 (-) neurons (Fig. 3A). Thus, the relation between the Cm amplitude and Ca2+ current was unchanged between the first and seventh stimulus (Fig. 3C). By contrast, Cm increases could not be evoked in IB4 (-) neurons by the seventh stimulus. The baseline Cm changed less than 5% in both neuron types during repetitive stimulation indicating that there was no net accumulation of membrane at the cell surface. These data support or are at least consistent with the hypothesis that slow endocytosis and/or recycling/replenishment in IB4 (-) neurons can deplete a releasable pool of vesicles during repetitive stimulation. IB4 (+) neurons that showed a fast endocytosis phase (Fig. 2B) were able to maintain responses albeit at a depressed level.

Fig. 3. Loss of depolarization-evoked Cm transient with repetitive stimulation in IB4 (-) neurons.

Fig. 3

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A: Cm transients were evoked at 90 s intervals with short (10 ms) depolarizing pulses. Note the almost complete loss of Cm response in IB4 (-) neurons (n = 5) by 7.5 min and the sustained responses in IB4 (+) neurons (n = 5) even at 9 minutes. Data are expressed as mean ± SEM. *, (p < 0.05, two-way RM ANOVA post hoc test). B: peak Ca2+ currents were similarly diminished in IB4 (+) and (-) neurons (n = 5 each) at the 7th stimulation point. C: Cm was normalized by ICa using the data from panels A and B. Note; there is no difference between IB4 (+) and (-) neurons at the 1st stimulation point (0 min). While ΔCm/-ICa did not decrease in IB4 (+) neurons by the 7th stimulus (9 min), it was eliminated in IB4 (-) neurons. *, (p < 0.05, t-test).

To monitor endocytosis more directly, we used the fluorescent styryl-dye FM4-64 (Vida and Emr 1995). The fluorescence of this water-soluble dye increases by many orders of magnitude when it partitions into biological membranes. Although the dye binds to membranes with kinetics that are diffusion-limited, the observed fluorescence increase upon dye addition in the absence of stimulation was quite slow for both IB4 (+) and (-) neurons (Fig. 4A, B). Such slow fluorescence increases are consistent with spontaneous insertion (and retrieval) of vesicle membranes in the absence of stimulation. Fluorescence intensity increased more slowly in IB4 (-) neurons than in IB4 (+) neurons (Fig. 4C) but a suggestive trend of greater staining intensity in IB4 (-) neurons did not reach statistical significance (Fig. 4B). To further stimulate the exocytosis-endocytosis cycle, we depolarized the neurons by applying a high potassium solution (Fig. 4B). Only a moderate additional increase in fluorescence was observed in either neuron but the greater dye-uptake in IB4 (-) neurons now reached statistical significance. This depolarization-dependent increase might be due to a relatively small, depolarization-dependent component of the exocytosis-endocytosis cycle or due to a saturation of the process during dye application in the absence of stimulation. Subsequent washing of the neurons in control solution without added dye led to a slow decrease in fluorescence intensity in the absence of stimulation (Fig. 4B, 5).

Fig. 4. FM4-64 is taken up preferentially by IB4 (-) neurons.

Fig. 4

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A: images of a rat TRG neuron after FM4-64 loading. The images from panel B. FM4-64 (10 μM) was kept in chamber for 100 sec without perfusion and FM4-64 with KCl (75 mM). Then neurons were perfused with control solution. B: differences in FM4-64 (10 μM) loading between IB4 (+) and (-) neurons. Data are expressed as mean ± SEM. There is significant difference (*, p < 0.001, two way RM ANOVA) between IB4 (+) and (-) neurons (n = 8 each) after KCl application. C: the data were normalized with the point just before FM4-64 and KCl 75 mM stimulation at panel B. Arrow shows that the FM4-64 dye was added at 40 s. Data were expressed as mean ± SEM. *, p < 0.001 with two way RM ANOVA.

Fig. 5. FM4-64 is released faster by IB4 (+) neurons.

Fig. 5

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A: the signal decay rate without stimulation is increased as laser exposure frequency increases. Shorter exposure intervals showed a steeper decline, suggesting that signal bleaching occurred. B: the % decrease in signal intensity at 10 s intervals between exposures was bigger than at 2 s or 0.5 s intervals when plotted against response number, suggesting that TRG neurons have a background vesicular release. Here, IB4 (+) and (-) data were combined. C: The % decrease of the FM signal intensity of IB4 (+) and (-) neurons was similar. D: IB4 (+) and (-) neurons exhibit differences in the FM4-64 signal decreases after KCl (75 mM) stimulation. The signal reduction is faster in IB4 (+) neurons (n = 10) than in IB4 (-) neurons (n = 12) (p<0.001, two way RM ANOVA). Asterisks show difference between IB4 (+) and (-) with post hoc test (* : p<0.05, ** : p<0.01). E: Background release in the presence of reduced extracellular Ca2+ (0.1 mM) is not different than in 2 mM Ca2+.

To determine whether fluorescence loss in the absence of stimulation was due to ‘background’ release or the result of photobleaching due to repetitive exposure to the excitation laser beam we measured the fluorescence intensity in images taken every 10, 2 or 0.5 seconds (Fig. 5A). When plotting intensity against time, a steeper decline can be observed at shorter exposure intervals (Fig. 5A) consistent with photobleaching of the dye. However, when plotting fluorescence intensity against exposure number, a much larger intensity decrease per exposure could be observed at the longer interval of 10 s than at the shorter intervals (Fig. 5B, C). This suggests that the fluorescence loss during the 10 s interval cannot solely be accounted for by photobleaching and lends further support to the hypothesis that TRG neurons exhibit robust vesicle turnover in the absence of ‘explicit’ stimulation. The background release was little changed in low extracellular [Ca2+] (Fig. 5E). We next tested whether dye release could be stimulated by K+-induced depolarization. As shown in Fig. 5D, application of a high K+ solution (filled symbols) increased the rate of dye release from both IB4 (+) and (-) neurons relative to non-stimulated controls (open symbols). Consistent with the larger stimulation induced Cm increase (Fig. 2A, B, D), IB4 (+) neurons also showed a more rapid dye release in response to stimulation compared to IB4 (-) neurons (Fig. 5D).

DISCUSSION

In this study we found that: 1) IB4 (+) trigeminal neurons exhibit a faster decrease of peak Cm after a depolarizing stimulus as compared to IB4 (-) neurons; 2) Cm responses to repetitive stimulation could not be sustained in IB4 (-) neurons; 3) IB4 (-) neurons exhibit a larger capacity for FM4-64 dye uptake than IB4 (+) neurons; and 4) high K+-induced FM4-64 dye release is faster in IB4 (+) neurons compared to IB4 (-) neurons.

Differences in vesicular content and release

Fusion of large dense-cored vesicles (LDCV) and small synaptic vesicles (SSV) might both contribute to exocytosis in TRG neurons. The majority of small diameter IB4 (-) neurons are peptidergic nociceptors (Nagy and Hunt 1982)(Silverman and Kruger 1990; Fang et al., 2006). Neuropeptides such as substance P and CGRP are often stored in the same large (60-100 nm) dense core vesicles (LDCVs) in dorsal root ganglia (Merighi et al., 1988) and in the central terminals of primary afferent neurons (Merighi et al., 1989; Plenderleith et al., 1990). By contrast, LDCVs are quite rarely associated with IB4 (+) synaptic terminals in the rat spinal cord (Gerke and Plenderleith 2004). Virtually all exocytotic profiles of LDCVs are observed at structurally unspecialized sites while small synaptic vesicles (SSVs) are usually observed at active synaptic zones in the spinal cord (Zhu et al., 1986). This relationship does not hold for SSVs in sensory neuron somata, which undergo vesicular release in the absence of any synaptic specializations. Release of LDCVs was demonstrated not only as a mechanism of neuropeptide release but also as a mechanism for insertion of receptors into the plasma membrane of sensory neurons (Bao et al., 2003).

The faster decay of evoked IB4 (+) TRG neurons is suggestive of faster endocytosis compared to IB4 (-) neurons. Since Cm increases in in IB4 (-) neurons are not sustained during repetitive stimulation , this also suggested that slow endocytosis and/or recycling/replenishment in IB4 (-) neurons can deplete a releasable pool of vesicles during repetitive stimulation. Evidence from the leech serotonergic Retzius neurons suggests that bulk release from LDCVs is slower than release from SSVs (Bruns et al., 2000). Slower release from LDCVs could also explain the secondary increases in peak Cm observed in individual recordings from IB4 (-) TRG neurons. These secondary increases in Cm occurred at different times after the depolarizing stimulus. As a result, the averaged record from different neurons appeared as a relatively flat peak response with little decay during the 3 sec recording period (Fig. 2A, B). Since the fluorescein-conjugated IB4 was applied prior to patch-clamp recordings, we cannot exclude the possibility that binding of fluorescent IB4 contributed the differences in evoked Cm changes. However, experiments with FM4-64 also demonstrated that KCl-stimulated vesicle release occurred slower in IB4 (-) than in IB4 (+) neurons (Fig. 5D); here IB4 labeling was done after the FM4-64 measurements. Also, it was demonstrated that IB4 binding does not alter action potential properties (Stucky and Lewin 1999) or capsaicin responses (Liu et al., 2004) of sensory neurons.

Background release

We observed unstimulated uptake and release of FM4-64 in our experiments and found that this unstimulated release was unchanged in low extracellular [Ca2+]. It was reported that there are two secretory pathways in neurons, the regulated and the constitutive pathway (De Camilli and Jahn 1990; Jung and Scheller 1991). The constitutive pathway is used for protein secretion by a constant exocytosis of microvesicles derived from the trans-Golgi network in the absence of secretagogues, while in the regulated pathway, the contents of LDCVs or SSVs are released upon stimulation by secretagogues. Membrane added through either mechanism is retrieved via endocytosis. We suspect that the constitutive pathway may account for the unstimulated release that we observed in this study. Alternatively, unstimulated release may result from the Ca2+-independent regulated pathway of vesicular release which has also been demonstrated in mammalian sensory neurons (Zhang and Zhou 2002; Zhang et al., 2004).

Anatomical evidence of vesicular release

The central terminals of mammalian DRG neurons possess LDCVs ranging from 81-121 nm in diameter (Zhang et al., 1998), corresponding to a vesicle capacitance of 200-450 aF, while SSVs range from 45-60 nm (Alvarez et al., 1993), corresponding to 60-110 aF. A Cm increase of 300 fF in response to a depolarizing stimulus would thus correspond to the release of ~600-1500 LDCVs, ~2700-5000 SSVs or a combination thereof. Despite this and other physiological evidence for vesicular release of neurotransmitter candidates from the somata of sensory neurons (Huang and Neher 1996; Neubert et al., 2000; Ulrich-Lai et al., 2001; Zhang and Zhou 2002), there is a stark paucity of anatomical data to complement physiological findings. Published electron microscopy (EM) investigations of the cellular organelles in sensory neuron somata do not reveal presence of vesicles in proximity to the plasma membrane of the type that are commonly observed at the synaptic terminal specializations of these neurons in the sympathetic ganglia (Heym et al., 1993), the spinal cord or brainstem (Knyihar-Csillik et al., 1982; Carlton and Hayes 1989; Plenderleith et al., 1990; Valtschanoff et al., 1994), or at the reconstructed peripheral axon terminals of unmyelinated sensory neurons (Kruger et al., 2003). Only one study illustrates a single vesicle in close apposition to the somatic plasma membrane of IB4 (+) neurons (Streit et al., 1986), whereas only scattered cytoplasmic vesicles may be found at considerable distances from the plasma membrane in others (Dixon 1963; Pineda et al., 1967; Peach 1972; Lehtosalo et al., 1984). What can account for such a discrepancy between physiological and anatomical studies? It is important to consider that a TRG neuron with 30 pF cell membrane capacitance has a surface area of about 3000 μM2, thus the cumulative fusion of 3000 vesicles corresponds to the fusion of only 1 vesicle per square micron, which might easily be missed on EM. Furthermore, stimulation and subsequent depletion of vesicles in the vicinity of the plasma membrane might occur during fixation procedures in native tissue, thus leading to an underestimate of vesicles at the membrane. The absence of the protection afforded by the blood-brain barrier in neurons within sensory ganglia may be a contributing factor (Ten Tusscher et al., 1989; Allen and Kiernan 1994; Wadhwani and Rapoport 1994). If so, evidence for vesicles near the plasma membrane regions may be obtainable by fast fixation of TRG neurons after acute dissociation and subsequent examination of the cell ultrastructure.

Relevance to intra-ganglionic communication and pathological pain states

The nature of the chemical mediator(s) of interneuronal cross-excitation within sensory ganglia is unknown (Amir and Devor 1996). Our data now demonstrate that both IB4 (+) and (-) TRG neurons are capable of exocytosis and could thus serve as a source for this chemical mediator. The difference in exocytosis and endocytosis suggests that both types likely secrete different compounds. Adenosine triphosphate (ATP), substance P and CGRP are likely candidates for release from nociceptors, since their release can be induced by depolarizing stimuli and by application of capsaicin (Neubert et al., 2000; Matsuka et al., 2001; Ulrich-Lai et al., 2001), which activates receptors found almost exclusively in nociceptor populations (Del et al., 1996; Caterina et al., 1997; Tominaga et al., 1998). Both somata and processes of sensory neurons respond to applications of ATP and substance P via purinergic and neurokinin receptors, respectively (Dray and Pinnock 1982; Spigelman and Puil 1988; Spigelman and Puil 1990; Bowie et al., 1994; Carlton et al., 1996; Burnstock 1996; Cook et al., 1997; Szucs et al., 1999). Moreover, sensory neurons in culture form synapses that appear to utilize ATP as their neurotransmitter (Zarei et al., 2004). Since the majority of IB4 (+) neurons lack neuropeptides, they likely release ATP as their main transmitter, while glutamate is unlikely to be released by exocytosis from peptidergic IB4 (-) nociceptors because they appear to lack vesicular glutamate transporters (Oliveira et al., 2003; Landry et al., 2004; Morris et al., 2005).

Conclusions

In this study we showed that the patterns of vesicular release from somata of IB4 (+) and IB4 (-ve) trigeminal neurons are different. The IB4 (-) neurons release and uptake neurotransmitters slowly and to a greater extent than the IB4 (+) neurons. This adds to the growing body of evidence for separate physiological roles of the two subpopulations of sensory neurons.

ACKNOWLEDGEMENTS

This study was supported by Whitehall Foundation grant F98-34 and NIH DE14573.

Abbreviations

TRG

trigeminal ganglion

IB4

isolectin B4

CGRP

calcitonin gene related peptide

Cm

cell membrane capacitance

Footnotes

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