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Mast cells are involved in allergic reactions where they release numerous vasoactive and other mediators in response to IgE and antigen. They are also activated by neuropeptides and are found in close contact with neurons. Mast cell heterogeneity has now been documented for mucosal mast cells and connective tissue mast cells. Rat brain mast cells were studied in a perfusion system and were shown to release serotonin in response to the mast cell secretagogue compound 48/80 (C48/80). High-potassium neuronal depolarization also released serotonin, but this was calcium dependent, not associated with β-hexosaminidase, and was unaffected by prior treatment with C48/80. Neuronal depolarization, however, was associated with somatostatin secretion and substantially reduced subsequent C48/80 stimulation, an effect abolished by neonatal treatment of the animals with capsaicin. Perfusion with somatostatin and substance P also induced brain mast cell serotonin release. C48/80 stimulation of combined thalamic and hypothalamic slices after neuronal depolarization substantially reduced the C48/80 effect, suggesting the possible presence of endogenous inhibitors released from the hypothalamus. Finally, the α₂-receptor agonist clonidine had a slight stimulatory effect. These results indicate that brain mast cell serotonin release may be regulated by endogenous neurotransmitters and/or neuro-modulators.