Age-Related Changes in Synaptic Plasticity Associated with Mossy Fiber Terminal Integration during Adult Neurogenesis

Adult-born neuron maturation in Gli1-CreER^T2;mGFP mice

In order to investigate neurogenesis in adult hippocampus, we used a transgenic reporter mouse line Gli1-CreER^T2;Tau^mGFP (GliCreGFP), in which membrane bound enhanced GFP (mGFP) is conditionally expressed under control of the Tau promoter in neural stem cells following administration of the prodrug TM (Ahn and Joyner, 2005; Fig. 1). In Gli1-CreER^T2 mice, we observed labeling in neurons within adult hippocampus as well as astrocytes throughout brain including hippocampus, neocortex and dorsal thalamus (Fig. 1). Because astrocyte labeling seemed to occur regardless of age and/or time post-TM, we used this as an indication that TM administration was successful (see Materials and Methods). We examined MFB maturation by labeled newborn cells and their processes at different times post-TM injection in both young adult and aged animals (Fig. 1). Compared with other model systems which displayed limited utility in aging brains, TM administration in GliCreGFP mice efficiently labeled newborn GCs and their processes in young adult and aged brains (Fig. 1).

In general, the spatiotemporal pattern of newborn GC development in GliCreGFP mice was qualitatively similar to previous reports (Nicola et al., 2015; Wu et al., 2015; Toni and Schinder, 2016). In both adult and aged brain, labeled newborn GCs displayed a cell body contained within the GCL, a primary apical dendrite that branched near the junction of the molecular layer and continued to the hippocampal fissure and the presence of spines along dendrites (Fig. 1A–F). In addition, in both adult and aged brains, an axon was clearly visible emanating from the cell body and coursing through the CA3sl. These axons were contained within the mossy fiber bundle. At least three different morphologically distinct cell types were labeled post-TM injection. These included mature postmitotic GCs (Fig. 1G), astrocytes (Fig. 1H), and type 1 progenitors referred to as radial glial-like cells (RGLCs; Fig. 1I). In the dentate gyrus, the majority of labeled cells within the GCL were mature GCs or RGLCs. Very few astrocytes were observed within the GCL.

Confirmation of cell type was also performed using cellular marker analysis (Fig. 1J–M), which showed a characteristic change in composition of cell type from 4 W post-TM injection (wpi) to 16 wpi. At 4 wpi, newborn GCs appeared morphologically mature and expressed the mature neuronal marker NeuN (Fig. 1K). Although not all comparisons were statistically significant, the number of mature GCs increased at 8 wpi (Fig. 1M) and remained at this level by 16 wpi. There was a concomitant decrease in the number of cells co-immunolabeled with DCX, a marker of immature neurons, suggesting that newborn GCs were continuing to mature through 8 wpi in the GliCreGFP model (Fig. 1J–M).

MFB maturation in Gli1-CreER^T2;mGFP mice

To measure changes in MFB maturation, we performed a quantitative morphometric analysis along newborn mossy fiber axons within CA3 region at light microscopic level. These boutons were heterogenous in shape and size and could be grouped into three categories: large complex boutons (Fig. 2A), lollipop terminals (Fig. 2B), and small en passant swellings (Fig. 2C; for definitions of these categories, see Materials and Methods). Since the significance of the lollipop synapses and small swellings are not clear and our preliminary analysis indicates that these two categories do not show significant changes throughout maturation at various ages (Fig. 2I,J), we focused on large complex boutons.

Tissue sections processed for immunohistochemical labeling of GFP were used for population density MFB measures and axonal reconstructions using Neurolucida software (Fig. 2D–E). In order to assess the ability to form adult-born MFBs at various ages, we first estimated the overall population density of MFBs in stratum lucidum in 3-M-, 6-M-, 12-M-, and 18-M-old animals at 8 wpi (Fig. 2F). Bouton counts were made in 100-μm stretches of stratum lucidum. A total of 1162 MFBs were counted from between three and five animals at each time point. In order to control for differences in efficacy of TM administration, for each animal, the number of labeled GC bodies in the dentate was also counted and total MFB counts were divided by this number to normalized MFB measures between animals. Surprisingly, we found a peak increase in labeled MFBs within CA3 stratum lucidum at 6 M of age (Fig. 2F). A significantly larger number of surviving terminals were counted when injections were made at 6 M compared with 3 M (190% increase, p < 0.01) or 18 M (78% increase, p < 0.01). Increases in MFB terminal number, but to a lesser extent, were also observed post-TM injections in 12-M-old animals.

The same labeled tissues were used to perform single axon analyses. Axons were traced within a region from the medial border of CA3 through stratum lucidum as far as possible until a break in the axon was detected. Faulkner et al. (2008) previously showed in young adult animals that it takes eight weeks for an MFB to be morphologically mature. We therefore chose 4, 8, and 16 wpi to study MFB changes in presynaptic and postsynaptic structural elements during this time. Previous work suggested interbouton density was a characteristic feature of mature en passant MFBs (Amaral and Dent, 1981). We measured the MFB density (Fig. 2G) and interbouton distance (Fig. 2H) along single axons to access the changes of MFB formation on single axons during AHN. In young adult animals (3 M), there was a dramatic decrease in MFB density from 4 to 8 wpi (4 wpi, 32.7 × 10⁻³/μm, 8 wpi, 13.5 × 10⁻³/μm, p < 0.005; Fig. 2G). The decrease was almost threefold at 16 wpi (4 wpi, 32.7 × 10⁻³/μm, 16 wpi, 11.6 × 10⁻³/μm, p < 0.005). Consistent with this observation, the interbouton distance increased from 4 to 8 wpi in young adult mice and reached ∼75 μm at 16 wpi (4 wpi, 43.9 μm, 16 wpi 74.6 μm, p < 0.05; Fig. 2H).

Previous EM analysis of newborn MFB development in young adult brain found MFBs can reach a mature perimeter size at 4W, but it takes approximately 8W for the newborn MFBs to have a mature synapse density (Faulkner et al., 2008). To better quantitatively understand the size distribution of MFBs during AHN, we measured the diameter of each newborn MFB at 4, 8, and 16 wpi and plotted the accumulative distributions of the MFB diameters (Fig. 3A–C). In young adult brain (3 M), we observed a small increase in mossy fiber diameter from 4 to 8 wpi, but no further significant changes were observed between 8 and 16 wpi (Fig. 3A). This quantitative analysis is consistent with prior observations that in young adult brain, newly formed MFBs reach mature size 4W after they are born.

Age-related changes in MFB maturation during AHN

Considerable evidence suggests that adult neurogenesis is severely reduced in aging brains. However, the integration of new neurons into existing hippocampal circuitry and the functional plasticity to hippocampal circuitry in aged brain have not been addressed. To determine whether newborn neurons in aged brain underwent a maturation process similar to young adult we measured MFB terminal densities along newborn mossy fiber axons (Fig. 2G). At 4 wpi, the MFB densities were significantly lower in middle age (12 M) and aged (18 M) mice compared with young (3 M) adults (3M4 wpi, 32.7 × 10⁻³/μm, 12M4 wpi, 8.7 × 10⁻³/μm, 18M4 wpi, 10.9 × 10⁻³/μm, p < 0.005). In sharp contrast to young adult animals, the MFB densities were gradually increased from 4 to 16 wpi in 12 M and 18-M-old mice. While such an increase in 12 M middle age mice was not dramatic, there was nearly a 2.5-fold increase in MFB density in 18 M aged mice from 4 to 16 wpi (18M4 wpi, 10.9 × 10⁻³/μm, 18M16 wpi, 23.3 × 10⁻³/μm, p < 0.01). As a consequence, at 16 wpi, MFB density was twofold higher in 18 M aged mice compared with 3 M young adults (3M16 wpi, 11.6 × 10⁻³/μm, 18M16 wpi, 23.3 × 10⁻³/μm, p < 0.01). Interbouton densities also showed differences between young adult and aged mice (Fig. 2H). While interbouton density increased with maturation in young adult animals (3 M), interbouton distances decreased in middle age (12 M) and aged (18 M) mice. In line with MFB density analysis, interbouton spacing was significantly lower in 18 M animals after 16 wpi (18M4 wpi, 110.4 μm, 18M16 wpi, 48.2 μm, p < 0.05; Fig. 2H). Together, these observations indicate that there are significant differences in the dynamics of new-born MFB development in young and aged brain: in young adult brain, numerous MFBs are formed at 4 wpi, but many of them disappear later at 8 and 16 wpi. In aged brain, however, fewer form initially at 4 wpi, but MFBs are continuously added and their density can be even higher than young brain at 16 wpi.

A different pattern of MFB terminal size maturation was also observed. In aged mice (18 M), MFB diameter decreased over time (Fig. 3B,D,E). At 4 wpi, the population of MFB were on average relatively large. From 4 to 16 wpi, however, their average size decreased. When MFB sizes were compared between ages at 4 wpi it is apparent that middle age (12 M) and aged mice (18 M) have a significantly higher population of large MFBs compared with young adults (3 M; p < 0.005, Kolmogorov–Smirnov test for distribution difference; Fig. 3C). The trajectory of MFB size changes during maturation are strikingly different between age groups (Fig. 3D,E): while the average MFB size in 3-M-old mice increases from 4 to 16 wpi, the average size in 18-M-old mice decreases. What is also interesting is that by 16 wpi the average MFB size is similar in adult (3 M) and aged (18 M) animals (Fig. 3E). These data indicate that in young adult brain a pruning mechanism refines MFB development, while in aged brain a progressive accumulation of MFBs from larger to smaller sized overall occurs during maturation.

Ultrastructural analysis of MFB integration in young adult and aged brain

Apparent differences in MFB number and size during maturation between young adult and aged animals suggests the developmental plasticity of the integration of these newborn MFBs is altered with aging. In order to investigate in high resolution morphologic changes in MFB maturation, we performed quantitative immuno-EM at 4, 8, and 16 wpi in young adult (3 M) and aged (18 M) GliCreGFP transgenic mice. We used a serial immuno-EM approach to reconstruct and quantify the development of newborn MFBs labeled in the GliCreGFP mice focusing on larger MFBs that terminate with TEs on the proximal region of CA3 pyramidal neurons to concentrate our analysis on the more developed newborn MFBs at each time point.

A total of 19 labeled large MFBs at 4, 8, and 16 wpi from 3 M young adult and 18 M aged brains were reconstructed and analyzed. Three MFBs at each time point were analyzed except at 3M4 wpi, where four MFBs were analyzed. Each MFB was reconstructed from on average 32 serial thin sections (ranging from 16 to 45 sections). Consistent with observations at the light microscopic level, in EM preparations we found that MFB densities were significantly reduced from 4 to 16 wpi in 3 M mice, while densities gradually increased, but were not statistically different, along the same timeline in 18 M mice (3 M: 32 × 10⁻³/μm, 18W: 12 × 10⁻³/μm, p < 0.05; n = 50–100 boutons at each time point; Fig. 4A).

It has been previously reported that in 2-M-old young adult mice adult-born MFBs initial form synaptic contacts on dendritic shafts of CA3 pyramidal cells by two weeks (Faulkner et al., 2008). By four weeks, many of these new boutons reached a mature size and contained a mature number of invading TEs. The number of synaptic contacts within each bouton continued to increase until eight weeks when the new MFBs reach morphologic maturity and remain stable through at least 16 weeks (Faulkner et al., 2008). Consistent with this timeline, we found that MFBs at 4 wpi in 3-M-old mice had already exhibited invaginating TEs with multiple spine heads on each TE. To further analyze the maturation of MFBs from 4 to 16 wpi, we quantified the development of spine heads on TEs (Fig. 4B). The normalized spine head counts (see Materials and Methods) in the 4 wpi samples from 3-M-old mice were low, but doubled by 8 wpi and remained the same at 16 wpi, indicating that the postsynaptic components of adult-born MFBs in young adult brain also undergo a temporal maturation process from four to eight weeks (3M4 wpi: 8 × 10⁻² normalized spines/MFB, 3M8 wpi: 18 × 10⁻² normalized spines/MFB, p < 0.05; n = 3–4 reconstructed boutons at each time point). By contrast, the samples from 18 M aged brain showed a very different pattern: normalized spine head counts remained stable from 4 wpi to 16 wpi (18M4 wpi: 20.6 × 10⁻² normalized spines/MFB, 18M8 wpi: 20.3 × 10⁻² normalized spines/MFB) and at numbers similar to mature 3 M, 16 wpi animals (3M16 wpi: 18 × 10⁻², 18M16 wpi: 21 × 10⁻² normalized spines/MFB, p = 0.19; Fig. 4B). This surprising finding suggests that newborn MFBs in aged animals are complex from earliest stages of maturation and remain so over time.

Sharing of newly formed MFBs with old MFBs in young adult brain is common even after the new MFBs are morphologically mature

Previous studies have suggested that newborn MFBs in adult brain compete with preexisting MFB for terminal space on postsynaptic TEs (Toni et al., 2008; Toni and Schinder, 2016). That is, the labeled newborn MFBs are shown to “share” a TE with an unlabeled more mature MFB. It is suggested that over time the newborn MFBs progressively occupy more of the TE and eventually outcompete for sole occupancy of the TE. In order to understand the dynamic of sharing during the maturation of adult-born MFBs, we focused our analysis on whether and how each reconstructed MFB from various timepoints exhibited sharing with neighboring mature MFBs.

In 3 M young adult samples, we clearly saw sharing at four weeks, the earliest stage analyzed. As shown in Figure 5A–C, a representative four-week-old MFB was just starting to invade an old mature MFB by pushing away the surrounding astrocytic processes and sharing a TE with the old MFB. More robust sharing was also observed in eight-week-old MFBs (Fig. 5D–G). Unexpectedly, we also observed sharing in the 16-week-old MFBs (Fig. 5H–K), although by this time these MFBs should be fully mature. When we quantified the fraction of each shared TE that was in contact with a newborn MFB we observed 39% at 4 wpi, and this remained fairly constant through 16 wpi, 44% of shared TE per MFB (Fig. 4C; n = 8–15 reconstructed TEs for each time point) These results suggest that sharing of newborn MFBs with old MFBs is very common in young adult brain, even after the MFBs are morphologically fully mature.

The four 4 wpi MFBs from 3 M young adult animal were classified as stage 3a based on previously published classifications (Faulkner et al., 2008). In order to understand more the sharing dynamic, we reconstructed an additional pair of stage 2 small MFBs at 4 wpi that were on a segment of newborn mossy fiber 40 μm apart (Fig. 6). This segment of mossy fiber seemed to have contacts with dendritic shaft and spine heads from a postsynaptic CA3 pyramidal cell. A clear sharing with a spine head that was mostly encircled by a mature MFB was observed in one of the new MFB. Thus, based on previous results (Faulkner et al., 2008) and the current study, we conclude that in young adult brain, the integration of MFBs into existing mature neural circuit is a highly dynamic process involving both de novo formation of new synaptic contacts with postsynaptic dendritic shaft and takeover of preexisting mature MFB by invasion and sharing.

Sharing and replacing preexisting old MFB as a major mechanism for integration of newborn MFBs in aged brain

Sharing also occurred in the samples from 18 M aged brain. Quantification of the percentage of each shared TE that was in contact with a newborn MFB (Fig. 4C) showed that 39% was observed at 4 wpi, but this dropped dramatically to only 11% at 8 wpi, and no sharing was observed from 16 wpi samples (Fig. 4C). An example of sharing at 8 wpi is shown in Figure 7A–C, in which the invading new MFB almost replaced the old MFB. At 16 wpi (Fig. 7D–I), the MFB was mature and was morphologically no different from the neighboring preexisting MFBs. In addition, no evidence of de novo synapse formation was observed in all the 18-M-old samples we analyzed. It is important to note that in this current study an average 32 serial thin sections were used for reconstructing MFBs. Each thin section for EM was 70 nm. The thickness analyzed for each MFB is therefore at least 2 μm which is sufficient enough to cover large portion of MFB and its associated TEs. This explains why we were able to detect sharing in many MFBs at various timepoints. Thus, the fact that we observed no de novo synapse formation in all samples and no sharing at 16 wpi sample in 18 M aged brain strongly suggests a limited integration capacity of newborn MFBs in aged brain.

The lack of sharing in 16 wpi and no evidence of de novo synaptic formation in 18 M aged brain were surprising. However, these findings were consistent with the finding that postsynaptic TEs with spine heads were morphologically mature throughout the 4 to 16 wpi period in 18-M-old brains (Fig. 4B) and suggests the major mechanism for newborn MFB integration in aged brain is through replacement of preexisting MFBs. Unlike young adult brain, sharing in the aged brain is relatively transient. Once mature, new MFBs completely replace old MFBs without further sharing in later stage.