Development and sensory experience dependent regulation of microglia in barrel cortex

Abstract

The barrel cortex is within the primary somatosensory cortex of the rodent, and processes signals from the vibrissae. Much focus has been devoted to the function of neurons, more recently, the role of glial cells in the processing of sensory input has gained increasing interest. Microglia are the principal immune cells of the nervous system that survey and regulate the cellular constituents of the dynamic nervous system. We investigated the normal and disrupted development of microglia in barrel cortex by chronically depriving sensory signals via whisker trimming for the animals' first postnatal month. Using immunohistochemistry to label microglia, we performed morphological reconstructions as well as densitometry analyses as a function of developmental age and sensory experience. Findings suggest that both developmental age and sensory experience has profound impact on microglia morphology. Following chronic sensory deprivation, microglia undergo a morphological transition from a monitoring or resting state to an altered morphological state, by exhibiting expanded cell body size and retracted processes. Sensory restoration via whisker regrowth returns these morphological alterations back to age-matched control values. Our results indicate that microglia may be recruited to participate in the modulation of neuronal structural remodeling during developmental critical periods and in response to alteration in sensory input.

Keywords: RRID: AB_2224402; RRID: AB_2313661; RRID: AB_2336126; RRID: AB_2339392; RRID: AB_2339427; RRID: AB_2340111; barrel cortex; microglial morphology; sensory deprivation; somatosensory cortex; vibrissae.

Figure 1.

Microglial morphology. Coronal sections through barrel cortex of P2(A),P14 (B), P30 (C), P45 (E), P60 (F) under high magnification labeled with the antibody to Iba-1. Our staining produced high resolution of morphological features of the somata and processes. Scale bar = 10μm for all panels.

Figure 2.

Microglial reconstructions. Representative reconstructed microglial cells within layer IV of the barrel cortex. Reconstructed microglial cells from the control group at different developmental ages; P2 (A), P14 (B), P30 (C), P45 (D), and P60 (E). All scale bars 10 μm.

Figure 3.

Iba-1 expression varies across laminae and development. A: Coronal view of Iba-1 stained barrel cortex with contours indicating layers 2/3, 4, 5, 6 and the cortical white matter. The brightness of the individual layers was divided by the area of the contour map generated and were normalized to the brightness and area of the white matter of that specific animal (Optical density = [brightness of cortical layer]/[brightness of white matter]). Scale bar = 250 μm. B: Optical density measurements on overall expression patterns of Iba-1 as a function of age. Data are from control animals, means and one standard error of the mean are represented. Laminae were determined by cellular density and size determined by Hoechst staining (not shown).

Figure 4.

Quantification of morphological features. Morphological measurements of reconstructed microglial cells in layer IV of barrel cortex as a function of age. Morphological measurements include, A: cell body area, B: cell body perimeter C: somatic aspect ratio, D: number of processes, E: processes ends, F: process length. Data represent population means, error bars indicate one standard error of the mean (SEM). Asterisks indicates statistical significance for all pair-wise comparisons following rank-based ANOVA (p<0.05).

Figure 5.

Sholl analyses. The starting radius was at 10 μm from the cell perimeter with a radius increment of 5 μm. Using concentric sphere analysis, we focused on the process length and number of intersections. A: Process Length as a function of distance away from the soma. B. Number of intersections as a function of distance away from the soma. Data represent population means, Error bars indicate one standard error of the mean (SEM).

Figure 6.

Sensory deprivation does not impact overall expression of Iba-1. (A) Optical density measurements of Iba-1 expression as a function of cortical lamina in control (open bars) and trimmed (solid bars) conditions. (B) Stereological quantification method of Iba-1 expression as a function of cortical lamina in control (open bars) and trimmed (solid bars) conditions, the stereological analysis results in an unbiased estimate of microglia population (see methods). The difference between control and trimmed condition for both experiments was not statistically significant between any pair. Bars represent population means and error bars represent SEM.

Figure 7

Sensory deprivation’s impact on microglia morphology. A Iba-1+ microglia from a P30 animal (A) has a small cell body with spindly processes. In contrast, following 30 days of whisker trimming the cell body enlarges and the processes retract (B). Following 30 days of whisker regrowth P60 microglia are indistinguishable (C, D). See Figure 8 for quantification.

Figure 8.

Sensory deprivation impacts microglial morphology. Comparison between reconstructed barrel cortex layer IV microglial cells in the P30 nondeprived cortex (P30 control, A) and sensory deprived barrel cortex animals (P30 trim, B) showed increased cell body size and retraction of processes following trimming. Following sensory restoration the control (C) and regrow group (D) did not differ from each other. All scale bars show 10 μm. Sensory deprivation did not alter the number of processes across all groups. (E), but a statistical decrease in the process length (F) was observed as well as an increase in the number of processes ends between treatment groups and respective controls (G). Concomitantly with these changes, a statistical increase was observed in the size of the cell body (μm) in the sensory deprived microglia (P30 trim) compared with control P30 mice (H). For the box and whisker plots the solid black lines represent population medians, whereas the dotted white lines represent population means. Error bars indicate standard error of the mean (SEM). Asterisks indicates statistical significance for all pair-wise comparisons following rank-based ANOVA (p<0.05).

Figure 9.

Microglia surface receptor expression following sensory deprivation: Sample confocal images from a sensory deprived (Row A) and a control (Row B) animal. Number of total cells in a z-stack was determined from Hoechst staining (blue, A1, B1). All microglia were labeled with Iba-1 (magenta, A2, B2), and a smaller proportion were also immunoreactive for MHC-II (green, A3, B3). The images show ramified microglia with small somas and many branched processes. The overlay of all the channels shows colocalization between Iba-1+ and MHC-II+ cells (indicated with asterisks) (A4, B4) and Iba-1+ microglia that do not express MHC-II are indicated with arrows. Scale bar (25 μm) is the same for all panels. Neither the overall microglia density (as measured by Iba-1+ cells) nor activated microglia density (as measured by MHC-II+ cells) change significantly following one month of sensory deprivation (C). The relative proportion of MHC-II+ microglia did not change as a result of sensory deprivation (D). Bar charts plot population means and one SEM.