Maturation Dynamics of the Axon Initial Segment (AIS) of Newborn Dentate Granule Cells in Young Adult C57BL/6J Mice

Abstract

Newborn dentate granule cells (DGCs) are generated in the hippocampal dentate gyrus (DG) of rodents through a process called adult hippocampal neurogenesis, which is subjected to tight intrinsic and extrinsic regulation. The use of retroviruses encoding fluorescent proteins has allowed the characterization of the maturation dynamics of newborn DGCs, including their morphological development and the establishment and maturation of their afferent and efferent synaptic connections. However, the study of a crucial cellular compartment of these cells, namely, the axon initial segment (AIS), has remained unexplored to date. The AIS is not only the site of action potential initiation, but it also has a unique molecular identity that makes it one of the master regulators of neural plasticity and excitability. Here we examined the dynamics of AIS formation in newborn DGCs of young female adult C57BL/6J mice in vivo Our data reveal notable changes in AIS length and thickness throughout cell maturation under physiological conditions and show that the most remarkable structural changes coincide with periods of intense morphological and functional remodeling. Moreover, we demonstrate that AIS development can be modulated extrinsically by both neuroprotective (environmental enrichment) and detrimental (lipopolysaccharide from Escherichia coli) stimuli.SIGNIFICANCE STATEMENT The hippocampal dentate gyrus (DG) of rodents generates newborn dentate granule cells (DGCs) throughout life. This process, named adult hippocampal neurogenesis, confers a unique degree of plasticity to the hippocampal circuit, and it is crucial for learning and memory. Here we studied, for the first time, the formation of a key cellular compartment of newborn DGCs, namely, the axon initial segment (AIS) in vivo Our data reveal remarkable AIS structural remodeling throughout the maturation of these cells under physiological conditions. Moreover, AIS development can be modulated extrinsically by both neuroprotective (environmental enrichment) and detrimental (lipopolysaccharide from Escherichia coli) stimuli.

Keywords: adult neurogenesis; axon initial segment; environmental enrichment; hippocampus; neuroinflammation; structural plasticity.

Figure 1.

Experimental design. A, Seven-week-old mice received a stereotaxic injection of a retrovirus that encodes the fluorescent protein Venus. After different periods of time (i.e., 10 d, and 2, 3, 4, or 8 weeks after injection), mice were sacrificed, and newborn DGCs were examined (B). C–F, Representative images showing the morphology (C), dendritic spines (D), potential location of the AIS (E), and axonal terminals (named MFTs) (F) of 8-week-old newborn DGCs. Yellow scale bar, 100 μm. Pink scale bar, 10 μm. E, Yellow triangles represent the potential location of the AIS.

Figure 2.

Maturation of newborn DGCs. A–E, Representative images of 10-day-old (A) and 2- (B), 3- (C), 4- (D), or 8- (E) week-old newborn DGCs and their respective high-power magnification images showing the presence of dendritic spines (red triangles). F, Total dendritic length in cells of different ages. G, Sholl's analysis of dendritic branching. H, Density of dendritic spines. I–M, Representative high-power magnification images of MFTs of 10-day-old (I) and 2- (J), 3- (K), 4- (L), or 8- (M) week-old newborn DGCs. N, Area of individual MFTs. Yellow scale bar, 50 μm. Pink scale bar, 10 μm. Red triangles represent dendritic spines. Pink triangles represent MFTs. *0.05 > p ≥ 0.01. ***0.001 ≥ p. Asterisks indicate statistically significant differences with respect to 10-day-old newborn DGCs. Error bars represent SEM.

Figure 3.

AIS formation during adult hippocampal neurogenesis. A–C, Representative images of the AIS of 10-day-old (A) and 3- (B) or 8- (C) week-old newborn DGCs. To visualize the starting and ending point of the AIS, orthogonal cross-sections were examined throughout the axon to detect the colocalization between Venus and the AIS marker Ankyrin G. As an example, orthogonal YZ (right) and XZ (bottom) cross-sectional views of the axon obtained at axonal points located closer to the soma (a), within (b), and beyond (c) the AIS location are shown. D, Percentage of cells with a clearly identifiable axonal projection. E, Percentage of cells with a clearly identifiable AIS. F, Percentage of axons with a clearly identifiable AIS. G, AIS length. H, AIS starting point. I, Thickness at the proximal, medial, and distal points of the AIS. J, Average AIS thickness. Yellow scale bar, 10 μm. White scale bar, 5 μm. Pink scale bar, 1 μm. *0.05 > p ≥ 0.01. **0.01 > p ≥ 0.001. ***0.001 ≥ p. Asterisks indicate statistically significant differences with respect to 10-day-old newborn DGCs. Error bars represent SEM.

Figure 4.

EE accelerates the maturation of newborn DGCs. A, Schematic experimental design. Briefly, animals received a stereotaxic injection of a retrovirus that encodes the fluorescent protein Venus. One week later, half the animals were exposed to a week of EE, whereas the other half were housed under standard conditions. B, C, Representative images of newborn DGCs belonging to CH (A) or EE (B) mice, together with their respective high-power magnification images showing the presence of dendritic spines. D, Total dendritic length. E, Sholl's analysis of dendritic branching. F, Length of the primary apical dendrite. G, Percentage of cells with basal dendrites. H, Migration into the GCL. I, Distance between the axonal hillock and the first change of axonal trajectory. J, Density of dendritic spines. K, L, Representative high-power magnification images of MFTs of newborn DGCs belonging to CH (K) and EE (L) animals. M, Area of individual MFTs. Yellow scale bar, 50 μm. Pink scale bar, 10 μm. Red triangles represent dendritic spines. Pink triangles represent MFTs. *0.05 > p ≥ 0.01. Error bars represent SEM.

Figure 5.

EE effects on the development of the AIS of newborn DGCs. A, B, Representative images of newborn DGCs belonging to animals exposed to CH (A) and EE (B). To visualize the starting and ending point of the AIS, XZ and YZ orthogonal cross-sectional images obtained at different points of the axon located closer to the soma (a), and within (b) or beyond (c) the AIS location are shown. It should be noted that colocalization between Venus and Ankyrin G occurred only within the AIS region. C, Percentage of cells with a clearly identifiable axonal projection. D, Percentage of cells with a clearly identifiable AIS. E, Percentage of axons with a clearly identifiable AIS. F, AIS length. G, AIS starting point. H, Average AIS thickness. Yellow scale bar, 50 μm. White scale bar, 10 μm. Blue scale bar, 5 μm. Pink scale bar, 1 μm. *0.05 > p ≥ 0.01. ⁺ 0.1 > p ≥ 0.05. Error bars represent SEM.

Figure 6.

EE effects on the activation of newborn DGCs. A–D, Representative images of cfos⁻ (A,C) and cfos⁺ (B,D) cells in CH (A,B) and EE (C,D) animals. To visualize the starting and ending point of the AIS, XZ and YZ orthogonal cross-sectional images obtained at different points of the axon located closer to the soma (Aa,Ba,Ca,Da), and within (Ab,Bb,Cb,Db) or beyond (Ac,Bc,Cc,Dc) the AIS location are shown. E, Density of cfos⁺ nuclei in the GCL. F, Percentage of Venus⁺ cells that show cfos nuclear staining. G, Percentage of cfos⁺/Venus⁺ and cfos⁻/Venus⁺ cells with a clearly identifiable axonal projection. H, Percentage of cfos⁺/Venus⁺ and cfos⁻/Venus⁺ cells with a clearly identifiable AIS. I, AIS starting point in cfos⁺/Venus⁺ and cfos⁻/Venus⁺ cells. J, AIS length in cfos⁺/Venus⁺ and cfos⁻/Venus⁺ cells. Yellow squares represent Venus⁺/cfos⁻ cells. Red squares represent Venus⁺/cfos⁺ cells. Red triangles represent cfos⁺ cells. Yellow scale bar, 50 μm. Blue scale bar, 5 μm. Pink scale bar, 1 μm. E, F, Asterisks indicate differences with CH animals. G, J, Asterisks indicate statistical significance in Tukey post hoc analyses. *0.05 > p ≥ 0.01. **0.01 > p ≥ 0.001. ***0.001 ≥ p. Error bars represent SEM.

Figure 7.

LPS from E. coli impairs the maturation of newborn DGCs. A, Schematic experimental design. Briefly, animals received a stereotaxic injection of a retrovirus that encodes the fluorescent protein Venus and were implanted with osmotic pumps filled with PBS or LPS. Two weeks later, animals were sacrificed. B, C, Representative images of newborn DGCs belonging to PBS-treated (B) or LPS-treated (C) animals, together with their respective high-power magnification images showing the presence of dendritic spines. D, Total dendritic length. E, Sholl's analysis of dendritic branching. F, Length of the primary apical dendrite. G, Percentage of cells with basal dendrites. H, Migration into the GCL. I, Distance between the axonal hillock and the first change of axonal trajectory. J, Density of dendritic spines. K, L, Representative high-power magnification images of MFTs of newborn DGCs belonging to PBS-treated (K) or LPS-treated (L) animals. M, Quantification of the area of individual MFTs. Yellow scale bar, 50 μm. Pink scale bar, 10 μm. Red triangles represent dendritic spines. White triangles represent MFTs. *0.05 > p ≥ 0.01. Error bars represent SEM.

Figure 8.

LPS from E. coli alters the development of the AIS of newborn DGCs. A, B, Representative images of newborn DGCs belonging to PBS-treated (A) or LPS-treated (EE) (B) mice. To visualize the starting and ending point of the AIS, XZ, and YZ orthogonal cross-sectional images obtained at different points of the axon closer to the soma (a), and within (b) or beyond (c) the AIS location are shown. It should be noted that colocalization between Venus and Ankyrin G occurred only within the AIS region. C, Percentage of cells with a clearly identifiable axonal projection. D, Percentage of cells with a clearly identifiable AIS. E, Percentage of axons with a clearly identifiable AIS. F, AIS length. G, AIS starting point. H, Average AIS thickness. Yellow scale bar, 50 μm. White scale bar, 10 μm. Blue scale bar, 5 μm. Pink scale bar, 1 μm. ⁺0.1 > p ≥ 0.05. **0.01 > p ≥ 0.001. Asterisks indicate statistically significant differences with respect to PBS-treated mice. Error bars represent SEM.

Figure 9.

Effects of LPS from E. coli on newborn DGC activation and neuroinflammation in the DG. A, B, Representative images of the DG belonging to PBS-treated (A) or LPS-treated (EE) (B) mice, showing cfos staining (red) and retrovirally labeled newborn DGCs (green). C, Density of cfos⁺ nuclei in the GCL. D, Percentage of Venus⁺ cells that show cfos nuclear staining. E–I, Microglial activation in the DG. E, Density of Iba1⁺ microglial dells in the ML, GCL, and H. F, Intensity of CD68 fluorescence signal in the ML, GCL, and H. G, M1 Mander's coefficient showing colocalization between Iba1 and CD68. H, I, Representative images showing staining with Iba1 and CD68 in the DG of PBS-treated (H) or LPS-treated (I) animals. Yellow scale bar, 50 μm. Yellow triangles represent Venus⁻/cfos⁺ cells. Red triangles represent Venus⁺/cfos⁺ cells. White triangles represent Venus⁺/cfos⁻ cells. Orange triangles represent microglial cells. **0.01 > p ≥ 0.001. ***0.001 ≥ p. Error bars represent SEM.