Postnatal Development of Serotonergic Innervation, 5-HT1AReceptor Expression, and 5-HT Responses in Rat Motoneurons

Abstract

We compared the electrophysiological responses to serotonin (5-HT) of neonatal and juvenile rat hypoglossal motoneurons (HMs) by using intracellular recording techniques in a brainstem slice preparation. In neonatal HMs (</=P8), 5-HT caused a substantial decrease in the amplitude of spike afterhyperpolarization (AHP) that was associated with an increase in the minimal repetitive firing frequency (Fmin). Previous work has shown that this effect of 5-HT was mediated by the 5-HT1A receptor and may be secondary to inhibition of N- and P/Q-type calcium channels. In contrast to results from neonates, we found that 5-HT did not inhibit the AHP in juvenile HMs (>/= P20). Application of a cocktail of calcium channel toxins (omega-Conotoxin-GVIA and omega-Agatoxin-IVA) to juvenile HMs substantially inhibited the AHP, indicating that calcium entry through N- and P/Q-type channels supports the AHP in juvenile HMs, as it does in neonates. In addition, intracellular injection of the long-lasting GTP analog GTPgammaS induced an agonist-independent increase in Fmin similar to that seen in neonates in the presence of 5-HT. Together, these results suggested that intracellular mechanisms downstream of the 5-HT1A receptor capable of inhibiting the AHP were intact in juvenile HMs. Therefore, we investigated the possibility that age-related changes in effects of 5-HT on the AHP resulted from altered expression of the 5-HT1A receptor. To this end, we performed ligand-binding autoradiography using [3H]8-OH-DPAT, a 5-HT1A agonist, and in situ hybridization using radiolabeled oligonucleotide probes specific for the 5-HT1A receptor. The two approaches gave remarkably similar results. The highest levels of 5-HT1A receptor expression were found in neonatal HMs, with maximal binding and hybridization at approximately postnatal day 7 (P7) and only low levels of receptor expression by P28. Finally, immunohistochemistry for 5-HT revealed that these developmental changes in 5-HT1A receptor expression occurred coincident with a postnatal increase in serotonergic innervation of the hypoglossal nucleus (nXII). Together, these findings indicate that developmental changes occur in the serotonergic innervation of nXII and in the expression of 5-HT1A receptors in HMs during the early postnatal period, resulting in markedly different effects of 5-HT on firing behavior in neonatal and juvenile HMs.