Two types of neurotransmitter release patterns in isolectin B4-positive and negative trigeminal ganglion neurons

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 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (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 toward 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 a 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 with IB4 (-) neurons. Taken together these data suggest that the IB4 (+) TRG neurons have faster exocytosis and endocytosis than the IB4 (-) neurons.

Regulation of N-type voltage-gated calcium channels (Cav2.2) and transmitter release by collapsin response mediator protein-2 (CRMP-2) in sensory neurons

“…For these reasons, we suggest that the stimulus-evoked release of CGRP occurs in a calcium-dependant manner from areas of LDCV exocytosis in a regulated fashion. This is consistent with electron microscopy studies (Alvarez et al, 1993;Hayes and Carlton, 1992) and functional investigations in the literature (Hingtgen and Vasko, 1994a;Kitamura et al, 2009;Matsuka et al, 2007). Because synaptic vesicles and LDCVs share similarities in the core aspects of the machinery involved in transmitter release (Edwards, 1998), our results showing the synaptic localization of CRMP-2 suggests that a CRMP-2-mediated increase in neurosecretion might occur via both classical and non-classical mechanisms.…”

“…Whereas SVs might be located at a distance of 20 nm from the calcium channel, LDCVs might reside as far as 300 nm away from the channels (Park and Kim, 2009), a spatial separation suggestive of differences in the calcium-dependence of release. However, FM4-64 has been used to examine cycling of LDCVs in trigeminal ganglia, in which CGRP is a major peptide transmitter and where the physiology is similar to the DRG (Matsuka et al, 2007). In addition, the stimulus-evoked release of CGRP from isolated sensory neurons occurs in a calciumdependent manner (Hingtgen and Vasko, 1994a).…”

Time‐series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy

“…In Figure 2B, the vesicle membrane is shown by red fluorescence, while the nuclei are shown by blue fluorescence. Many ring-like structures of red fluorescence clustering together around the cell's margin (see the white arrows) are believed to be the large vesicles according to previous reports (Matsuka et al, 2007;Vida and Emr, 1995). The small and unattached vesicles may present a dot-like morphology in the cytoplasm (see the white cycle region) due to the resolution limit of optical microscopy.…”

Basic research and clinical investigations of the neural basis of orofacial pain

“…These data suggested that IB4(þ) TG neurons have faster exocytosis and endocytosis than IB4(À ) neurons [16].…”