We initially evaluated tissues from each of the three different genotypes of mice to confirm that the distribution of identified serotonin neurons () was consistent with previous analyses using a different indicator allele, where serotonin neuron subtypes were detected by expression of β-galactosidase (Jensen et al., 2008
). As found in the previous study, r1-Pet1
neurons populated the DR and a portion of the MR and B9 area while r2-Pet1
neurons resided exclusively within the MR and B9. r3 / 5-Pet1
neurons populated MR and B9, probably corresponding to r3-derived neurons, as well as the rostral pole of the RMg, probably associated with r5 as the intervening r4 does not produce serotonin neurons and thus creates a gap separating the rostral and caudal serotonin systems.
Fig. 1 The distribution of serotonin neurons with developmental origin in rhombomere (r) 1, 2 or either 3 or 5 identified by eGFP expression was consistent with prior fate mapping (Jensen et al., 2008). (A–C) Darkfield illumination of immunoperoxidase (more ...)
Major efferent projections
Consistent with the observation that r1-Pet1
neurons account for many serotonin neurons, their axons were widespread and detected in several brain areas. r1-Pet1
neurons were the major source of cortical innervation, where they were found in all regions and through all lamina (). r1-Pet1
axons were typically fine with periodic undulations in diameter along their course or had small (< 1 µm in diameter) varicosities, consistent with their majority origin in the DR (Kosofsky & Molliver, 1987
; Jensen et al., 2008
axons innervated superficial layers of cortex where their axons typically had small varicosities (). Larger varicose axons from r1-Pet1
neurons were found in the lateral entorhinal cortex.
Fig. 2 Projection patterns of r1-Pet1 neurons (left column), r2-Pet1 neurons (center column) and r3 / 5-Pet1 neurons (right column) in the prefrontal and parietal cortices. (A–A″) In the prelimbic cortex, parallel lines designate the layer V (more ...)
r1-Pet1 axons also provided a major innervation to the hippocampal formation, where axons with both small and large varicosities were found (). r1-Pet1 axons with large beaded morphology were particularly concentrated in the base of the granule cell layer in the dendate gyrus, in stratum radiatum of CA3 and at the border between strata lacunosum-moleculare and radiatum in CA1. Fine axons were found more abundantly in the molecular layer of the dentate gyrus and stratum radiatum of CA1.
Fig. 3 Hippocampal projections from r1-Pet1 neurons (left column), r2-Pet1 neurons (center column) and r3 / 5-Pet1 neurons (right column). (A–A″) Innervation of the dentate gyrus; boxed areas shown at higher magnification in B through B″. (more ...)
Subcortically, r1-Pet1 axons were highly abundant in the septum, shell of the nucleus accumbens, basolateral amygdala, lateral globus pallidus, substantia innominata, ventral pallidum, hypothalamus, ventral tegmental area and substantia nigra. Dense tracts of axons were also visible passing through the lateral hypothalamus, lateral preoptic area, nucleus of the diagonal band and medial septum ( and ).
Fig. 4 Subcortical projection pattern of r1-Pet1 neurons, r2-Pet1 neurons and r3 / 5-Pet1 neurons. (A–C) Boxed areas within line drawings adapted from Paxinos mouse atlas plates (Paxinos & Franklin, 2001) indicate the location of the photomicrographs (more ...)
Fig. 5 Schematic mapping, based on the flat maps of Swanson (2004), showing areas innervated by r1-Pet1 neurons (A), r2-Pet1 neurons (B) or r3 / 5-Pet1 neurons (C). (A) r1-Pet1 neurons have widespread axons including all of the targets of the DR as well as some (more ...)
In contrast to the widespread projections of r1-Pet1 neurons, r2-Pet1 neurons provided selective and often dense innervation of a handful of regions. In the cortex, r2-Pet1 axons were identified in the medial prefrontal, the parietal cortex and, to a lesser extent, the perirhinal cortex ( and ). In cortical areas, r2-Pet1 axons were found more often in layers III–VI than in superficial layers I / II. The morphology of r2-Pet1-derived axons was more consistently that of large beaded axons. In the hippocampus, large beaded axons were found at the base of the granule cell layer and within the hilus of the dentate gyrus, as well as in the stratum lacunosum-moleculare / radiatum border of area CA1, and to a lesser extent the stratum radiatum of CA3 ().
In subcortical regions, r2-Pet1 axons were detected in the medial septum, suprachiasmatic nucleus and the periventricular nucleus of the thalamus ( and ). Additional but sparse r2-Pet1 axons were identified in the basolateral amygdala (BLA), and generally throughout the hypothalamus.
r3 / 5-Pet1 neurons had distinct sets of axon projections in a unique pattern. In frontal and parietal cortices, only rare individual fibers were visible from r3 / 5-Pet1 neurons (). However, r3 / 5-Pet1 axons were detected in the piriform and amygdalar corticies as well as in the hippocampal formation (). Within the hippocampus, r3 / 5-Pet1 axons were distinctly beaded and found in similar areas as beaded axons arising from r1 or r2, i.e. within the sub-granule cell layer and hilus of the dentate gyrus and the border between strata lacunosum-moleculare and radiatum in area CA1 ().
r3 / 5-Pet1 axons were rare in some subcortical areas heavily innervated by r1-Pet1 and r2-Pet1 axons such as the periventricular nucleus of the thalamus and the suprachiasmatic nucleus (). Sparse fibers from r3 / 5-Pet1 neurons were detected within midline thalamic areas as well as throughout the hypothalamus. More caudally, r3 / 5-Pet1 axons had all but exclusive innervation of the ventral, anterior and dorsal tegmental nuclei, also known as the tegmental nuclei of Gudden, as well as rich preference for the rostral–dorsolateral medulla including locus coeruleus and zones within the dorsal lateral parabrachial nucleus ( and ). These areas also received some innervation from r1-Pet1 axons (), but were poorly innervated by r2-Pet1 axons ().
Projections from r1-Pet1 neurons to remaining serotonin cell groups
We specifically examined how each of these genetically defined and eGFP-containing groups of serotonin neurons innervated the remaining unmarked serotonin neurons in the pons and medulla. Such projections between different serotonin neuron subtypes have the capacity to engage feedback inhibitory regulation of serotonin cell activity through serotonin release and subsequent signaling via the 5-HT1A autoreceptor (reviewed by Barnes & Sharp, 1999
). To assess this inter-system connectivity, we examined tissue from mice of each of the three different genotypes [En1::cre
; and Egr2::cre
/ 5), ePet1::Flpe
] where immunofluorescence labeling for eGFP identified the particular genetic subgroup of serotonin neurons, while labeling for TPH2 identified all remaining serotonin neurons. In this analysis, no assumptions were made regarding the rhombomeric origin or molecular signature of serotonin neurons lacking eGFP.
r1-Pet1 axons (GFP+) innervated neurons immunolabeled with TPH2 but which were GFP-negative and thus not derived from r1. The densest ramification of such axons was visible in those portions of the MR populated by non-r1-derived serotonin neurons (). Strings of r1-Pet1 axons were often detected either overlapping with or in close proximity to non-r1-derived serotonin cell bodies or dendrites in the MR (). r1-Pet1 axons were also detected in both RMg () and ROb (). Most of the r1-Pet1 axons in the medulla were visible as short segments aligned along the dorsoventral axis overlying RMg and ROb. In contrast to these areas, very few r1-Pet1 axons were detected in the RPa (). The Ppy and LPGi areas received a few more axons than RPa ().
Fig. 6 Projections of r1-Pet1 neurons (green) to other groups of serotonin neurons, identified by immunolabeling for TPH2 (red). (A) r1-Pet1 axons, detected by immunolabeling for eGFP (green), are visible coursing dorsal–ventral (arrows) overlying non-r1-derived (more ...)
Regionally selective innervation patterns of r1-Pet1 axons were statistically demonstrable using anova (), which revealed a significant effect of REGION (F5,28 = 9.84, P < 0.001) on the density of r1-Pet1 axons. Post-hoc analyses with LSD tests indicated that r1-derived serotonin axon densities in MR are greater than those in any other regions of interest in the raphe (P < 0.05; ). The axon densities within RMg are significantly greater than those in RPa and LPGi (P < 0.05), but were not significantly different from the axon densities detected in ROb and Ppy. The ROb has lower axon densities than MR, and greater densities than RPa, Ppy and LPGi (P < 0.05). The axon densities within Ppy and LPGi were not significantly different (P > 0.05).
r1-Pet1 axons have a differing propensity to innervate other serotonin cell groups. Asterisks denote significant differences between groups.
Projections from r2-Pet1 neurons to remaining serotonin cell groups
r2-Pet1 axons were detected in the vicinity of non-r2-derived serotonin neurons nearby within the MR (). Notably, r2-Pet1 axons were largely undetected in any other raphe nucleus, except for an individual truncated fiber occasionally detected in darkfield images (). Thus, r2-Pet1 projections are highly restricted within the MR, proximal to the location of their cell bodies.
Fig. 8 Maximal projections of Z-stack images showing axons of r2-Pet1 neurons abundant only in the MR, proximal to the location of r2-Pet1 cell bodies. (A) MR neurons rostral and dorsal to r2-Pet1-derived neurons intermix with axons from r2-Pet1 neurons, detected (more ...)
Fig. 9 Comparison of projections to the DR by r2-Pet1 (A, A′)- and r3 / 5-Pet1 (B, B′)- neurons. (A) Using darkfield illumination, r2-Pet1 neurons are visible in the MR (arrows) ventral to the DR (bracketed region) at the base of the aqueduct (more ...)
Projections from r3 / r5-Pet1 neurons to remaining serotonin cell groups
In contrast to r2-Pet1 axons, the r3 / r5-Pet1 axons provided innervation to other anatomically defined groups of serotonin neurons. In particular, r3 / 5-Pet1 axons were clearly distributed throughout the rostral–caudal and dorsal–ventral extent of the DR (). Within the DR, there was a mild regional bias such that the axons appeared slightly more concentrated in the dorsal portion at the rostral level, and in the lateral parts at the mid-rostrocaudal level of the DR. Strings of double-labeled axons with varicosities overlying serotonin neurons were detected in the DR () as well as the MR ().
Fig. 10 Projections of r3 / 5-Pet1 neurons to other serotonin neuron groups. (A) In the DR, axons of r3 / 5-Pet1 neurons (arrow) intermingle with serotonin neurons. Scale bar = 50 µm. (B) Another section of the DR at higher magnification (scale bar = (more ...)
In the caudal raphe nuclei, r3 / 5-Pet1 axons were also detected in the lateral medulla including the Ppy and LPGi where they intermingled with non-r3 / r5 derived serotonin neurons (). However, the appearance of r3 / 5-Pet1 axons was negligible in the ROb and RPa ().
Thus overall while r2-Pet1 neurons did not substantively innervate other groups of serotonin neurons, axons arising from either r1-Pet1 or r3 / r5-Pet1 neurons selectively innervated distinct subsets of serotonin neurons.