Morphological analysis of activity-reduced adult-born neurons in the mouse olfactory bulb

Abstract

Adult-born neurons (ABNs) are added to the olfactory bulb (OB) throughout life in rodents. While many factors have been identified as regulating the survival and integration of ABNs into existing circuitry, the understanding of how these factors affect ABN morphology and connectivity is limited. Here we compare how cell intrinsic [small interfering RNA (siRNA) knock-down of voltage gated sodium channels Na(V)1.1-1.3] and circuit level (naris occlusion) reductions in activity affect ABN morphology during integration into the OB. We found that both manipulations reduce the number of dendritic spines (and thus likely the number of reciprocal synaptic connections) formed with the surrounding circuitry and inhibited dendritic ramification of ABNs. Further, we identified regions of ABN apical dendrites where the largest and most significant decreases occur following siRNA knock-down or naris occlusion. In siRNA knock-down cells, reduction of spines is observed in proximal regions of the apical dendrite. This suggests that distal regions of the dendrite may remain active independent of Na(V)1.1-1.3 channel expression, perhaps facilitated by activation of T-type calcium channels and NMDA receptors. By contrast, circuit level reduction of activity by naris occlusion resulted in a global depression of spine number. Together, these results indicate that ABNs retain the ability to develop their typical overall morphological features regardless of experienced activity, and activity modulates the number and location of formed connections.

Keywords: activity; morphology; naris occlusion; neurogenesis; olfaction; sodium channels.

Figure 1

Summary of experimental conditions and injection paradigm. (A) Schematic of EGFP coding lentivirus. EGFP was expressed downstream of the ubiquitin promoter. (B) Schematic of siRNA + EGFP coding lentivirus. Two U6 promoters, each expressing one shRNA against voltage gated sodium channel 1.1–1.3 (Na_V1.1–1.3) expression were followed by the ubiquitin promoter expressing EGFP. Each group received the following manipulations: (C) – Control EGFP lentivirus [Control, see (A)] in one hemisphere, siRNA sodium channel knock-down lentivirus [siRNA, see (B)] opposite hemisphere. (D) – Control EGFP lentivirus in both hemispheres, naris occlusion (NO) in one hemisphere. (E) Experimental timeframe. Mice were allowed to survive for 35 days post-injection (DPI)/naris occlusion before sacrifice, allowing for expression of Na_V in progenitor cells born the day of injection (DPI-0). (F) Example neuron from individual slices of an image stack. Scale bar, 100 μm. Inset shows resolution of apical dendrite spines. Scale bar, 25 μm. (G) Example ABN reconstruction from images in (F). Scale bar, 100 μm. Inset shows reconstruction of inset in (F). Scale bar, 25 μm.

Figure 2

Complete cellular reconstructions of randomly selected EGFP⁺ ABNs. (A,C) Group 1 cells; four animals, five cells per hemisphere, n = 20 per condition. Scale bar, 100 μm. (B,D) Raw image of dendrite and spines taken from image stacks of control and siRNA cells respectively. Scale bar, 5 μm. (E,G) Group 2 cells; three animals, seven cells per hemisphere, n = 21 per condition. Scale bar, 100 μm. (F,H) Raw image of dendrite and spines taken from image stacks of control and naris-occluded cells respectively. Scale bar, 5 μm.

Figure 3

Reducing activity reduces the number of functional synapses formed in ABNs. (A) Total spines per cell (gray dots) and group means (bars with SE) for reconstructed ABNs. Both siRNA knock-down and naris occlusion showed significant reductions in total numbers of spines compared to controls (control/siRNA: p = 0.031, n = 20; control/NO: p = 0.003, Wilcoxon Rank-Sum test, n = 21) in the number of formed synapses. (B,C) Distribution of spines plotted as total number of spines per 40 μm bin vs. distance along apical dendrite path with SE. Significant differences occurred in early segments (40–80 μm) of apical dendrites in siRNA knock-down cells (B); (p < 0.05 and p < 0.05 respectively, n = 20).

Figure 4

Reducing activity decreases spine densities in specific regions of ABNs. (A) Spine density per cell (gray dots) and group means (bars with SE). Spine density calculated as total spines per cell divided by total apical dendrite length. Both siRNA knock-down and naris-occluded cells show significant overall reductions in spine (control/siRNA: p < 0.001, n = 20; control/NO: p = 0.039, n = 21), without significant difference in total apical dendritic length (see Tables 1 and 2). (B,C) Spine density plotted as total number of spines divided by total apical dendrite length in 40 μm bins vs. distance along apical dendrite path with SE. siRNA knock-down cells (B) show significant decreases in spine density in proximal and intermediate regions of the dendrite (at 40 μm: p < 0.01; at 80 μm: p < 0.05; at 160 μm: p < 0.05; n = 20). No significant differences were observed in naris-occluded cells (C).

Figure 5

Reductions in activity reduce the overall complexity of newborn ABNs. (A1) Classical Sholl analysis with radii increasing in 20 μm increments. Sholl analysis measures apical dendrite length within each sphere plotted against radius from soma. (A2) siRNA knock-down cells show a significant reduction in length in proximal-intermediate distances from soma (at 120 μm: p < 0.01, n = 20). (A3) NO cells show no significant differences in Sholl analysis. (B1) Modified Sholl analysis of branch intersections. Modified Sholl analysis represents the number of branches at a fixed distance along the apical dendrite (not counting terminated branches). (B2) siRNA knock-down cells show a significant reduction in the number of branches at proximal-intermediate distances from soma (at 120 μm: p < 0.001, n = 20). (B3) NO cells show no significant differences in modified Sholl analysis. (C1) Cumulative branch order along length of apical dendrite. Plotted as the cumulative branch order (where branch order increments by one following bifurcation) vs. distance from soma along apical dendrite length. (C2) siRNA knock-down cells show a significant reduction in cumulative branch order at proximal-intermediate distance from soma (at 100 μm: p < 0.05, n = 20). (C3) NO cells show no differences in cumulative branch order.