Developmental regulation of neurotransmitter phenotype through tetrahydrobiopterin

During development, sympathetic neurons innervating rodent sweat glands undergo a target-induced change in neurotransmitter phenotype from noradrenergic to cholinergic. Although the sweat gland innervation in the adult mouse is cholinergic and catecholamines are absent, these neurons continue to express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The developmental suppression of noradrenergic function in these mouse sympathetic neurons is not well understood. We investigated whether the downregulation of the enzyme aromatic l-amino acid decarboxylase (AADC) or the TH cofactor tetrahydrobiopterin (BH4) could account for the loss of catecholamines in these neurons. AADC levels did not decrease during development, and adult cholinergic sympathetic neurons were strongly immunoreactive for AADC. In contrast, BH4 levels dropped significantly in murine sweat gland-containing footpads during the time period when the gland innervation was switching from making norepinephrine to acetylcholine. Immunoreactivity for the rate-limiting BH4 synthetic enzyme GTP cyclohydrolase (GCH) became undetectable in the sweat gland neurons during this phenotypic conversion, suggesting that sweat glands reduce BH4 levels by suppressing GCH expression during development. Furthermore, extracts from sweat gland-containing footpads suppressed BH4 in cultured mouse sympathetic neurons, and addition of the BH4 precursor sepiapterin rescued catecholamine production in neurons treated with footpad extracts. Together, these results suggest that the mouse sweat gland-derived cholinergic differentiation factor functionally suppresses the noradrenergic phenotype during development by inhibiting production of the TH cofactor, BH4. These data also indicate that GCH expression, which is often coordinately regulated with TH expression, can be controlled independently of TH during development.