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

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.