Postnatal development of serotonergic innervation, 5-HT(1A)receptor 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 after hyperpolarization (AHP) that was associated with an increase in the minimal repetitive firing frequency (F(min)). Previous work has shown that this effect of 5-HT was mediated by the 5-HT(1A) 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 (ω- Conotoxin-GVIA and ω-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 GTPγS induced an agonist-independent increase in F(min) similar to that seen in neonates in the presence of 5-HT. Together, these results suggested that intracellular mechanisms downstream of the 5-HT(1A) 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-HT(1A) receptor. To this end, we performed ligand- binding autoradiography using [3H]8-OH-DPAT, a 5-HT(1A) agonist, and in situ hybridization using radiolabeled oligonucleotide probes specific for the 5- HT(1A) receptor. The two approaches gave remarkably similar results. The highest levels of 5-HT(1A) 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- HT(1A) 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-HT(1A) receptors in HMs during the early postnatal period, resulting in markedly different effects of 5-HT on firing behavior in neonatal and juvenile HMs.