Characterization of serotonergic neurons using concurrent fluorescent retrograde axonal tracing and immunohistochemistry.

The recent introduction ofa number offluorescent retrograde axonal tracers that have different emission spectra (1,2,9,10,25,26) has permitted the investigation of possible collateral projections of senotonengic neurons. Extensive collateralization from single neurons in the raphe nuclei was expected given the widespread distribution ofserotonergic nerve terminals in the central nervous system ( 1 1 , 1 7 ). However, only recently has divergent axonal branching been documented unequivocally in the ascending projections ofthe midbrain raphe (21,22,25)and descending projections ofthe medullany raphe (8) systems using multiple fluorescent retrograde tracing methods. Moreover, this technique has also revealed some very crude topography in the projections of the raphe nuclei. For example, although dorsal naphe neurons give off axon collaterals both within (2 1) and between (22,25) forebrain nuclei, it is also clean that there exists a gross inverted dorsal-ventral topography in these forebrain projections. Cell bodies situated ventrally within the dorsal raphe nucleus are more likely to project to the cortex (23), whereas those situated dorsally within the nucleus are more likely to project to the substantia nigna and/on ventral midbrain (23). In addition, neurons in the unpaired, midline-situated dorsal naphe nucleus were shown to project either ipsilaterally or contralaterally, but never bilaterally, to the forebrain (23). In spite of the detailed analysis of raphe projections that was permitted by the advent of multiple fluorescent tracing, it was not completely clean that all of the netnogradely labeled raphe neurons were senotonergic. Indeed, detailed studies of the dorsal naphe nucleus have sug.j ested that a number of nonserotonergic neunonal cell bodies are present in the nucleus (18). Also present are y-aminobutynic acid (GABA)-ergic cells that are probably separate from the serotonin neurons (6,13),