The whole central nervous system (CNS) of vertebrates is reached and profusely innervated by serotonin (5-hydroxytryptamine; 5-HT) releasing fibers. Such a diffuse distribution of serotonergic axons arises entirely from a relatively small number of somata (approximately 28,000 in the mouse; Ishimura et al., 1988) that are confined in the brainstem and clustered in B1 to B9 raphe nuclei. Serotonergic neurons represent one of the first neuronal systems to be specified during development (Levitt and Rakic, 1982). In the mouse, as early as embryonic day 11.5 (E11.5) the newly specified neurons start to elongate their axons rostrally in the medial forebrain bundle. Starting from E13.5, serotonergic fibers are driven along the main brain trajectories, reaching their targets by the end of gestation (Lidov and Molliver, 1982; Gaspar et al., 2003). The subsequent post-natal terminal field development has been described as a gradual colonization of the target with a region-specific timing (Lidov and Molliver, 1982). In the adult, 5-HT fibers display at least two different morphologies, originating from Dorsal (D-fibers) or Median (M-fibers) raphe nuclei. D-fibers appear thin with fusiform homogeneous varicosities and are more abundant than M-fibers, which show larger and oval varicosities along thin axons (Kosofsky and Molliver, 1987; Mamounas and Molliver, 1988; Wilson et al., 1989; Törk, 1990; Bang et al., 2012).

Classically, 5-HT immunolabeling has been used to study serotonergic fiber morphology (Lidov and Molliver, 1982; Azmitia and Gannon, 1983; D’Amato et al., 1987; Törk, 1990; Nyakas et al., 1994). However, as 5-HT is released and rapidly metabolized, reliable 5-HT immunostaining is not easily achievable thus requiring L-tryptophan and MAO-A inhibitors administration before tissue harvesting (Lidov and Molliver, 1982; Azmitia and Gannon, 1983; D’Amato et al., 1987; Nyakas et al., 1994). Alternatively, 5-HT transporter (SERT) immunohistochemistry has been used (Belmer et al., 2017). However, in the adult brain SERT immunostaining partially overlaps with serotonergic fibers since SERT is not present on their whole extent (Brown and Molliver, 2000; Amilhon et al., 2010; Descarries et al., 2010). Furthermore, SERT is transiently expressed in non-serotonergic neurons in the developing brain (Lebrand et al., 1998; Narboux-Nême et al., 2008).

Here we used the Tph2^GFP knock-in mouse line in which Tph2, the rate-limiting enzyme of 5-HT synthesis, has been replaced by the enhanced GFP reporter that accumulates and freely diffuses within the cytoplasm of all 5-HT synthetizing neurons, without affecting brain 5-HT levels or serotonergic system development and organization (Migliarini et al., 2013). This ensures an easy and high fidelity visualization of the fine-grain anatomy of the whole 5-HT system, which already allowed us to study different aspects of 5-HT fiber dynamics, both in vitro and in vivo, as well as during mouse development and in adulthood (Migliarini et al., 2013; Pelosi et al., 2015; Pacini et al., 2017; Pratelli et al., 2017). We combined GFP immunofluorescence with confocal microscope imaging and 3D-reconstruction to quantitatively characterize the post-natal development of serotonergic fibers in selected brain regions. Our approach allowed the characterization of previously unreported changes in regional density and morphology of 5-HT axons that occur in a time- and region-specific manner.

The possibility to use a knock-in replacement strategy in the mouse allows the labeling of specific neuronal populations together with their axonal projections and the analysis of their dynamic processes during development (Pasqualetti et al., 2002). Here we have successfully used the Tph2^GFP knock-in mouse line (Migliarini et al., 2013) to label and study the region-specific organization of serotonergic fibers in the developing post-natal mouse brain. We measured discrete parameters including the occupancy index (% of volume occupied by serotonergic fibers; Figure 1A), the total length of fibers passing in the analyzed block of brain tissue and the mean diameter of 5-HT axons (Figure 1B). Moreover, as serotonergic fibers are characterized by alternating thin and thick segments, the diameter of each edge, 0.2 μm long units generated by IMARIS to build 3D fibers (Figures 1B–C), was used to draw an ED distribution curve (Figure 1D).

Analysis at PND 0, when serotonergic fibers have reached their targets in the rostral brain, revealed a marked difference in the density of GFP-immunoreactive fibers among the analyzed areas that were further highlighted by 3D-reconstruction (Figures 2A–I). In particular CPu, S1BF and SN showed the lowest occupancy index (e.g., less than 0.1%) and GP the highest (i.e., 1.15 ± 0.19%; Figure 2J). On the other hand, we observed a rather homogeneous morphology of 5-HT fibers, which were characterized by a dot shape (Figures 2B–I). This was particularly evident in mPFC, GP, Hp, BLA, DLG and SN, in which the segments connecting the dot-shaped structures were barely detectable (Figures 2B,D,F–H). As a consequence, such a discontinuous profile did not allow to perform further fiber measurements.

At PND 7, connecting segments became detectable in all the analyzed brain districts while the dot shape was maintained, and an overall increase of fiber density was observed in line with the progression of terminal field development (Figures 3A–I). This trend was confirmed by 3D-reconstruction showing an increase in the occupancy index, which was particularly evident for S1BF, Hp, BLA and SN (Figure 3J), with the exception of DLG in which a significant decrease was observed (log₂ fold change −0.66; Figure 3J). The possibility to detect the connecting segments allowed to measure the total fiber length (Figure 3K), and the mean diameter (Figure 3L). Notably, a similar distribution of all ED curves was observed (Figure 3M).

The morphology of serotonergic fibers appeared more uniform at PND 14 (Figures 4A–I). Interestingly, 3D-reconstruction revealed divergent developmental patterns among the analyzed areas. Fibers innervating CPu, DLG and SN exhibited a significant increase in occupancy index (log₂ fold change +0.53, +0.93 and +0.59, respectively) and total length (log₂ fold change +0.54, +0.65 and +0.45, respectively) as compared to PND 7. A similar trend was observed in Hp, although not statistically significant. By contrast, fibers present in mPFC, S1BF and BLA showed a reduced occupancy index (log₂ fold change −0.42, −0.29 and −0.18, respectively; Figure 4J), which can be ascribed to changes in total length (i.e., mPFC, log₂ fold change −0.30) and mean diameter (i.e., S1BF and BLA, log₂ fold change −0.08 and −0.07, respectively; Figures 4K–M). Finally, no significant changes were detected in serotonergic fibers innervating GP (Figures 4J–M).

The dot-shaped feature observed at earlier stages was lost at PND 28 when 5-HT fibers display a uniform and smooth appearance (Figures 5A–I). As compared to PND 14, fibers in GP and BLA showed a marked increase in the occupancy index (Figure 5J), total length (Figure 5K) and an increase in their mean diameter (Figures 5L,M). A similar rearrangement was also apparent in DLG, CPu and S1BF due to changes in total length (i.e., DLG) or in mean diameter (i.e., CPu and S1BF). mPFC, Hp and SN displayed subtle fluctuation of the analyzed parameters but showed no significant change in the occupancy index as compared to PND 14 (Figures 5J–L). On the whole, the regional specificity of the morphological development observed between PND 14 and PND 28 accounted for the identification of two main fiber classes with distinctive features, as highlighted by ED curves (Figure 5M). In fact, higher frequency of edges in the diameter range of 0.15–0.3 μm was found in serotonergic fibers present in mPFC, GP, BLA, HP, DLG and SN as compared to CPu and S1BF. Conversely, CPu and S1BF displayed higher frequency of edges, whose diameter was >0.6 μm.

According to the classical view, serotonergic fibers in rodents complete their terminal field development within 1 month after birth (Lidov and Molliver, 1982). In line, total fiber length measured at PND 28 in mPFC, CPu, BLA, DLG and SN was maintained up to adulthood (Figures 6A–I). However, morphology of fibers in mPFC, CPu and SN was clearly remodeled, displaying a significant reduction in mean diameter in the adult as compared to PND 28 (Figure 6L). In GP and S1BF the mean diameter was reduced as well (Figure 6L). While in GP both occupancy index and total length were significantly reduced, in S1BF the occupancy index was unchanged due to an increase in total length (Figures 6J,K). A further discrepancy from the classical view was observed in Hp where serotonergic fibers remarkably decreased in total length and became thicker (Figures 6K,L). Finally, as shown by the ED curves, the regional differences in axonal morphology observed at PND 28 were exacerbated in the adult with the exception of Hp, whose ED distribution approached that of CPu and S1BF (Figure 6M). On the whole, these data demonstrated for the first time that the establishment of target-specific heterogeneity in serotonergic fibers proceeds beyond the first month after birth, thus extending the time window of terminal field development.

Serotonergic fiber development in the post-natal brain involves a massive and progressive increase in the amount of fibers within the target region that is commonly referred to as terminal field development (Lidov and Molliver, 1982). Measuring the total length of GFP-immunoreactive serotonergic fibers, we could identify two distinct developmental patterns showing either a progressive or a transient increase in fiber length from early post-natal stages up to adulthood, with a region-specific timing. Examples of the first scenario were observed in CPu, BLA, DLG and SN, where the total length of serotonergic fibers increased starting from PND 7 (BLA and SN) or PND 14 (CPu and DLG), peaked at PND 28 and remained unchanged up to adulthood. The second scenario was characterized by two distinct temporal trends. In cortical regions (mPFC, S1BF) serotonergic innervation peaked at PND 7, decreased up to PND 28 and, limited to the S1BF, increased again in the adult brain. In GP and Hp serotonergic innervation transiently increased up to PND 28 and then appeared pruned in the adult. Given the well-established role of 5-HT signaling in brain development (Teissier et al., 2017), and given that the density of serotonergic fibers could be directly linked to 5-HT levels, the presence of two distinct and region-specific patterns of serotonergic fiber development may account for different developmental roles of 5-HT. In particular, a transient increase of serotonergic fibers could reflect the requirement of higher 5-HT levels within time-windows that may be critical for the establishment and refinement of local neuronal circuitry. An example is provided by 5-HT fibers in the somatosensory cortex highlighting a transient increase in serotonergic innervation followed by a drop at PND 10 (Fujimiya et al., 1986; D’Amato et al., 1987). It has been postulated that this transient increase of serotonergic fibers, likely resulting in elevated 5-HT levels, could be linked to the correct establishment of thalamocortical pathway. Accordingly, bidirectional alterations of serotonergic signaling result in abnormal barrel field formation and a reduced cortical thickness (Miceli et al., 2013; Narboux-Nême et al., 2013). This suggests that a proper balance in serotonergic neurotransmission is required for the correct post-natal development of cortical regions. In this view, the transient increase in serotonergic innervation observed in Hp could be involved in controlling the intense post-natal developmental events (Angevine, 1965), such as the massive neurogenesis taking place during the first three post-natal weeks (reviewed in Reznikov, 1991). Similarly, the peak of 5-HT innervation observed in GP at PND 28 could play a role in the refinement of basal ganglia circuitry, as already demonstrated for dopamine levels during post-natal development (Sivam et al., 1991).

In the present study, we also provided a fine morphological characterization of the serotonergic fiber development in the postnatal mouse brain, showing that it proceeds in a comparable manner among all the regions analyzed. In fact, at birth fibers presented thick and dot-shaped enlargements with few and often barely detectable connections, in line with previous reports (D’Amato et al., 1987). Subsequently, thin connecting segments were detectable and fibers progressively became more smooth and uniform along their length up to PND 28. Eventually, additional intrinsic rearrangements resulted in the acquisition of the morphology observed in the adult. Our measurements display some discrepancies as compared to those obtained by Belmer et al. (2017), who also used a 3D-reconstruction approach to assess the morphology of SERT immunoreactive axons in the limbic system of adult mice. The use of SERT rather than GFP immunohistochemistry may likely account for the discrepancies observed as SERT not fully overlaps with serotonergic fibers (Brown and Molliver, 2000; Amilhon et al., 2010; Descarries et al., 2010). Thanks to the ED distribution analysis of GFP-immunoreactive serotonergic fiber, we were able to distinguish two main trends likely reflecting the presence of M and D fibers described by Kosofsky and Molliver (Kosofsky and Molliver, 1987; Törk, 1990). Accordingly, the first trend included serotonergic fibers present in mPFC, GP, BLA, DLG and SN that showed the lowest mean diameter, in line with their dorsal raphe origin (Muzerelle et al., 2016). However, despite the presence of fibers originating from serotonergic neurons located in the median raphe such as those innervating the Hp, the second included CPu and S1BF as well, which receive inputs from the dorsal raphe (Muzerelle et al., 2016). Nevertheless, since ED graphs depict the frequency of a given diameter describing the mean shape of fibers in each region, a specific fiber morphology (e.g., M vs. D) could not be associated to a precise curve trend. Further studies, including single fiber reconstruction approaches, are required to solve this issue.

Altogether, our data describe the general route of maturation of ascending serotonergic projections, which invade their targets with thick, dot-shaped fibers that progressively become smooth and uniform along their length, acquiring their final region specific morphology only after PND 28. Despite this common behavior in morphological rearrangements, we identified previously unreported maturation patterns that may underlie specific developmental roles for 5-HT within critical time-windows.

GM, ABe, MPr, NB, ABo, AG and SM performed experiments; analyzed data. GM, ABe, MPr and MPa interpreted data and wrote the article. MPa conceived the work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.