Reply from M. M. Bosma

We appreciate the opportunity to respond to this letter. Although the writer's criticism of the finer details of the pharmacology has merit, he poses no alternative to the parsimonious conclusion which we have drawn from our data – that 5-HT neurones drive spontaneous activity (Hunt et al. 2005). This conclusion is not solely based on pharmacological experiments, but on physiological experiments to pinpoint the midline source of activity, surgical manipulations to show that the midline drives lateral regions, and immunocytochemical and neurone counting experiments to identify candidate cells within the initiating region. Our evidence is: that a region of the midline initiates and drives spontaneous activity in the E11.5 hindbrain; that newly differentiated 5-HT-positive neurones (by immunocytochemistry) are found in and comprise 80% of the neurones within the narrow band, centred on the midline, in which activity originates; that pharmacological blockade of 5-HT2A receptors reversibly abolishes activity; and that the 5HT2A receptor is expressed (by immunocytochemistry) in the appropriate position to mediate propagation of activity. In addition, our recent experiments (most mentioned in the paper, Hunt et al. 2005) show that antagonists to GABAA, GABAB, glycine, metabotropic and ionotropic glutamate, nicotinic and muscarinic acetylcholine, purine, noradrenaline, and substance P receptors do not abolish activity. We also have shown that blockers of D1 and D2 (and related) dopamine receptors, applied separately or in combination, do not abolish activity (sulpiride, 10 µm; SCH 23390, 10 µm; unpublished data). This large body of consistent data led us to the conclusion that the 5-HT neurones mediate spontaneous activity, probably via the 5-HT2A receptor (although we did not examine expression of the 5-HT2C receptor). Indeed, had we presented this evidence, and concluded that the 5-HT neurotransmitter system was not involved, our conclusions would not have been accepted. Although the results of pharmacological manipulation may not be as clear-cut as those observed in other systems, these were performed in a biological preparation in which receptor properties are somewhat less clearly defined than in the adult mammalian brain, which is itself less defined than the recombinant heterologous systems in which many of the pharmacological assessments cited by the writer have been made. Given that the mechanisms of excitability within immature nervous systems are often different from those in the mature counterpart of the same brain structure, it would not be surprising that in the immature animal, details of the pharmacological profile of specific receptor subtypes may be different from that seen in mature tissues. It is also possible that the similar pKi values for ketanserin and spiperone on the recombinant 5-HT2A receptor in an expression system may not reflect exactly their relative efficacies in the embryonic hindbrain in vivo. With regards to the inconsistent effects of agonist addition, there have been suggestions that some 5-HT receptors can be constitutively active (although not shown in vivo). However, it is also possible that the amount of transmitter present in the tissue is enough to chronically activate the receptors; thus, addition of exogenous agonists to the 5-HT receptors may have no additional effect. However, we share the concerns of the writer about the inconsistencies of our pharmacological data with those seen in the literature. We will continue to explore these issues, investigating: the expression of the 5-HT2C receptor; dopamine-containing neurones and receptors; the presence of a different, as yet unidentified, population of neurones in the midline; and the small possibility that there is a redundant pacemaker system mediated by other mechanisms that is clouding the pharmacological waters. In summary, the pharmacological profile of spontaneous activity in embryonic hindbrain does not absolutely fit that of mature 5-HT2A receptors. However, our data ruling out many other transmitter systems and the physiological and immunocytochemical evidence cited above make the 5-HT-positive neurones by far the most likely candidates for driving spontaneous activity in this developing structure.