Sequential development of synapses in dendritic domains during adult neurogenesis

Abstract

During the process of integration into brain circuits, new neurons develop both input and output synapses with their appropriate targets. The vast majority of neurons in the mammalian brain are generated before birth and integrate into immature circuits while these are being assembled. In contrast, adult-generated neurons face an additional challenge as they integrate into a mature, fully functional circuit. Here, we examined how synapses of a single neuronal type, the granule cell in the olfactory bulb, develop during their integration into the immature circuit of the newborn and the fully mature circuit of the adult rat. We used a genetic method to label pre and postsynaptic sites in granule neurons and observed a stereotypical development of synapses in specific dendritic domains. In adult-generated neurons, synapses appeared sequentially in different dendritic domains with glutamatergic input synapses that developed first at the proximal dendritic domain, followed several days later by the development of input-output synapses in the distal domain and additional input synapses in the basal domain. In contrast, for neurons generated in neonatal animals, input and input-output synapses appeared simultaneously in the proximal and distal domains, respectively, followed by the later appearance of input synapses to the basal domain. The sequential formation of synapses in adult-born neurons, with input synapses appearing before output synapses, may represent a cellular mechanism to minimize the disruption caused by the integration of new neurons into a mature circuit in the adult brain.

Fig. 1.

Glutamatergic input to dendritic domains and PSD-95:GFP positive clusters (PSD⁺Cs) in vivo. (A) GCs have different dendritic domains: a basal domain (= basal dendrite) and different domains in the apical dendrite. The apical dendrite divides into an unbranched segment emerging from the soma followed by a more distal branched segment (= distal domain). Because we observed a high density of glutamatergic input synapses in the proximal 15% of the unbranched apical dendrite, we defined it as the proximal domain. (B) Branched spines with several spine heads and filopodia-like protrusions in a GFP labeled adult-generated GC 28 d.p.i. (Scale bar, 5 μm.) (C) Confocal 3D image shows a PSD⁺C in a new GC that is contacted by the presynaptic marker, bassoon (bar, 1 μm.) (D) In GCs expressing PSD-95:GFP, PSD⁺C could be detected by direct intrinsic fluorescence as green clusters. The dendritic morphology of the GC was revealed by amplification the low levels of PSD-95:GFP in the cytoplasm (that could not be detected by intrinsic fluorescence) with immunofluorescence against GFP (red). The merged images of PSD⁺C (intrinsic fluorescence in green) and dendritic morphology (immunofluorescence against GFP in red) allowed attributing PSD⁺Cs to specific dendritic domains of identified GCs (Scale bar, 5 μm.)

Fig. 2.

Development of PSD⁺Cs during maturation of adult- and neonatal-generated GCs. (A) In adult-generated superficial GCs, no PSD⁺Cs were detectable during migration (7 d.p.i.) and the initial extension of the dendritic arbor (10 d.p.i.) (Scale bar, 10 μm.) B and C show the development of PSD⁺Cs at different d.p.i. The Upper row shows the distal domain and the Lower row the basal domain and the unbranched segment of the apical dendrite with the proximal domain of a GC. (B) Only at later time points (14, 17, and 28 d.p.i.) PSD⁺Cs developed during maturation of adult-generated GCs. PSD⁺Cs were first observed in high density in the proximal domain followed by PSD⁺Cs at the distal domain (Scale bar, 10 μm.) (C) In neonatal-generated superficial GCs PSD⁺Cs developed simultaneously in the proximal and distal domains during maturation of neonatal-generated GCs at 10, 14, 17, 28 d.p.i. (Scale bar, 10 μm.)

Fig. 3.

Domain-specific development of PSD⁺Cs during maturation of adult- and neonatal-generated GCs (with branching in the superficial external plexiform layer). (A) Mean PSD⁺C density at different stages (d.p.i.) during the maturation of new GCs generated in adult animals. The dendritic domains are indicated in the graph: basal (blue), proximal (green), and distal domain (red line) as well as the entire unbranched apical dendrite (gray). (B) The diagram illustrates the developmental pattern of PSD⁺Cs during maturation of adult-generated GCs. (C) Mean PSD⁺C density at different stages (d.p.i.) during the maturation of new GCs generated in newborn animals. (D) The diagram illustrates the developmental pattern of PSD⁺Cs during maturation of neonatal-generated GCs.

Fig. 4.

Development of synaptophysin:GFP-positive clusters (Syp⁺Cs) during maturation of adult- and neonatal-generated GCs (with branching in the superficial external plexiform layer). (A) In adult-generated GCs, Syp⁺Cs developed during maturation of adult-generated GCs in the distal domain (14, 17, 28 d.p.i.; Scale bar, 10 μm.) (B) Mean Syp⁺C density at different stages (d.p.i.) during the maturation of new GCs generated in adult animals. (C) The diagram illustrates the developmental pattern of Syp⁺Cs during maturation of adult-generated GCs. (D) In neonatal-generated GCs Syp⁺Cs developed earlier than in adult-generated GCs (A) in the distal domain (14, 17, and 28 d.p.i.; Scale bar, 10 μm.) (E) Mean Syp⁺C density at different stages (d.p.i.) during the maturation of new GCs generated in neonatal animals. (F) The diagram illustrates the developmental pattern of Syp⁺Cs during maturation of neonatal-generated GCs.