The New Evidence Of Histamine Acts As A Sympathetic Neurotransmitter

Background and objectiveHistamine (HA), an important auto active substance in human body, serves extensive functions in normal physiology and in various pathologic conditions. In the central nervous system, considerable evidence has proved that HA, as a neurotransmitter, is involved in the cognition and the memory, the regulation of the cycle of sleeping and waking, as well as the water intake and food intake. In the peripheral nervous system, HA exists in both classic mast cells and minor non-mast cell associated with HA pools. HA from mast cells plays an important role in regulation and modulation of immune response, but the physiologic and pathologic role of the HA from non-mast cell remains unclear. Previous studies have demonstrated that the HA from non-mast cell is present within the rat enteric nervous system, the superior cervical ganglion (SCG) and sensory cells of the carotid body, indicating that there may be some relations between HA and those neurons. Our previous study found the co-localization of histidine decarboxylase (HDC), and tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH) in the SCG of guinea pig, the coexistence of HA and norepinephrine (NE), and the co-release of HA and NE from sympathetic terminals after stimulation. In addition, the endogenous HA could inhibit self-release and the release of NE through activation of the presynaptic HA H3 receptor. Accordingly, we propose for the first time that HA may be an unrecognized sympathetic neurotransmitter, and we further propose the hypothesis of the negative feedback that HA may provide in regulating sympathetic neurotransmission. As for the questions of whether HA widely coexists with NE in the sympathetic nervous systems and the subcellular localization of HA in sympathetic neurons, and of whether the release kinetics of HA vesicles in sympathetic neurons follows the character of classical transmitters, answers to them may serve as keys to ultimately defining HA as a novel sympathetic neurotransmitter.Therefore, the present investigation was designed to examine whether HA colocalizes with NE in the sympathetic nervous system in different species by using multiple immunofluorescence histochemistry and BDA anterograde tracing technique, and also to visualize the subcellular distribution of HA in the sympathetic nerve terminals and the cultured sympathetic ganglion with pre-embedding immunoelectron microscopy. HA synaptic vesicles releasing and mobility kinetics were studied by using FM1-43 dye in the cultured sympathetic ganglion with the aim of finding some new evidence for HA as a sympathetic neurotransmitter.Results1. Colocalization of HA and NE in sympathetic nervous systems in different species Using double immunofluorescence histochemistry combining to the confocal microscope, coexistence of NE and HA was displayed in the SCG and celiac ganglion principal neurons of the mouse and dog as well as in the vas deferens, mesenteric artery axon, and varicosities of the mouse and guinea pig. In the SCG of mice and dogs, the rats of NE-like immuoreactive neuron exhibiting HA-like immunoreactivity were 70% and 40% respectively. In addition, 60% of NE immunoreactive cells showed double-labeling in the celiac ganglion of dogs. While 80% of NE-like immunoreactive cells showed double-labeling in the vas deferens and mesenteric artery of mice, 30% showed double-labeling in those two tissues of guinea pigs. Otherwise, after injection of an anterograde tracer biotinylated dextranamine (BDA) into the SCG, the results of triple immunofluorescence histochemistry showed that in the cardiac sympathetic axons and varicosities labeled by BDA, 20% BDA-filled varicosities were only HA-immunoreactive, and 10% contained both HA and NE-like immunoreactivity. It indicated that HA widely existed in the sympathetic nervous system in different species and colocalized with NE. These results provide direct cellular morphological evidence that HA can be classified as a sympathetic neurotransmitter.2. Subcellular localization of HA in sympathetic nerve endings and the cultured SCG neuronsUsing pre-embedding immunoelectron microscopy, HA-like high-density immunoreactive products were seen in the small vesicles (50 nm in diameter) of the guinea pig vas deferens and cultured guinea pig SCG neurons. Moreover, the proportion of the HA-like positive vesicles were more than 90% in all synatic vesicles, while in the large vesicles (diameter >100 nm) and very few small vesicles there were not HA-like immunoreactive products. These results suggest that HA is located in the small vesicles in sympathetic neurons and provide direct subcellular morphological evidence that HA is classified as a sympathetic neurotransmitter.3. HA synaptic vesicles cycle kinetics in the cultured sympathetic ganglion neuron boutonsBy the means of the axtivity-dependent dye FM1-43 in combination with HA immunofluorescence histochemistry in cultured guinea pig SCG neurons boutons, we studied HA synaptic vesicles releasing kinetics by destaining and measured the mobility of HA synaptic vesicles by FRAP. The results of the confocal microscope imaging and statistic analysis showed that both intensity of HA-like red fluorescence and FM1-43 green fluorescence were simultaneously decreased with the extension of dipolar stimulation period, and both of them were correlativity (r = 0.989, P < 0.01). It indicated that HA synaptic vesicles in sympathetic neurons underwent endocytosis and exocytosis like the release kinetics of the classical transmitter vesicles. By measuring the intensity of HA-like red fluorescence and FM1-43 green fluorescence in fixed cultured cell with different dipolarization time, the results showed that the intensity of HA-like red fluorescence was stronger than that of FM1-43 green fluorescence. As FM1-43 fluorescence signa mostly reflected the recycling pool under the condition of dipolarization in this study, the difference of the fluorescence intensity between HA and FM1-43 implied that HA vesicles were comprised of the readily releasable pool and the reserve pool in sympathetic neuron boutons. FRAP measurement and the results of HA immunohistochemistry demonstrated that in cultured SCG neuron boutons, the diffusion coefficient of HA vesicles was 0.09μm2/s, the other vesicles'was 0.08μm2/s and there was not significant different between the mobility of HA synaptic vesicles and other vesicles.Conclusion1. HA widely exists in different sympathetic system of different species and colocalized with NE, the classical sympathetic neurotransmitter.2. HA locates in small synaptic vesicles of sympathetic nerve.3. The release kinetics of HA vesicles in sympathetic neurons follow the way of the classical transmitter vesicles.