The Emerging Role of the Interplay Among Astrocytes, Microglia, and Neurons in the Hippocampus in Health and Disease

Abstract

For over a century, neurons have been considered the basic functional units of the brain while glia only elements of support. Activation of glia has been long regarded detrimental for survival of neurons but more it appears that this is not the case in all circumstances. In this review, we report and discuss the recent literature on the alterations of astrocytes and microglia during inflammaging, the low-grade, slow, chronic inflammatory response that characterizes normal brain aging, and in acute inflammation. Becoming reactive, astrocytes and microglia undergo transcriptional, functional, and morphological changes that transform them into cells with different properties and functions, such as A1 and A2 astrocytes, and M1 and M2 microglia. This classification of microglia and astrocytes in two different, all-or-none states seems too simplistic, and does not correspond to the diverse variety of phenotypes so far found in the brain. Different interactions occur among the many cell populations of the central nervous system in health and disease conditions. Such interactions give rise to networks of morphological and functional reciprocal reliance and dependency. Alterations affecting one cell population reverberate to the others, favoring or dysregulating their activities. In the last part of this review, we present the modifications of the interplay between neurons and glia in rat models of brain aging and acute inflammation, focusing on the differences between CA1 and CA3 areas of the hippocampus, one of the brain regions most susceptible to different insults. With triple labeling fluorescent immunohistochemistry and confocal microscopy (TIC), it is possible to evaluate and compare quantitatively the morphological and functional alterations of the components of the neuron-astrocyte-microglia triad. In the contiguous and interconnected regions of rat hippocampus, CA1 and CA3 Stratum Radiatum, astrocytes and microglia show a different, finely regulated, and region-specific reactivity, demonstrating that glia responses vary in a significant manner from area to area. It will be of great interest to verify whether these differential reactivities of glia explain the diverse vulnerability of the hippocampal areas to aging or to different damaging insults, and particularly the higher sensitivity of CA1 pyramidal neurons to inflammatory stimuli.

Keywords: CA1; CA3; acute inflammation; confocal microscopy; inflammaging; neurodegeneration; triads.

Figure 1

Representation of the different stages of microglia activation, marked with the anti IBA1 antibody [from Cerbai et al. (2012)].

Figure 2

(A) Z-projections of confocal stacks acquired in CA1 hippocampus showing GFAP (green) immunostaining in control (A1) and aged (A2) rats. (B–D) Confocal acquisitions of IBA1 (green), GFAP (blue), and integrin-β1 (red) immunostaining in CA1 region of control (B), LPS (C), and aged (D) rats. (B1–D2b) Immunostaining of Integrin-β1 in microglia of control, LPS-treated and aged rats [magnifications of framed regions in (B–D)]; in (B1,C1,D1b,D2b) integrin-β1 colocalization on microglial cells is highlighted omitting GFAP immunostaining. Scale bars: (A1,A2): 7.5 μm; (B–D): 20 μm; (B1–D2b): 7.5 μm [Modified from Lana et al., 2019)].

Figure 3

Characterization of neurons-astrocytes-microglia interplay and apoptosis. (A) Confocal microscopy acquisition of GFAP (green) and NeuN immunostaining (red). Scale bar: 5 μm (A1–A3). Digital subslicing of the neuron in (A). Images are the merge of two contiguous confocal acquisitions. Serial confocal images that digitally sub-slice the neuron cell body (A1–A3) clearly demonstrate that astrocyte branches are present inside and infiltrate the neuron body [open arrows in (A2,A3)]. Scale bar: 5 μm. (B) The arrow shows a neuronal debris closely apposed to an astrocyte branch. The presence in the neuron cytoplasm of AIF (C1,C2) or of diffuse CytC immunostaining (D2), are clear signs of apoptosis (Suen et al., 2008). Scale bar: 15 μm. (C1,C2) Immunostaining for AIF (red) and GFAP (green). Scale bar: 5 μm. (D1–D4) Confocal acquisitions of NeuN (green), CytC (red), and IBA1 (blue) immunostaining acquired in CA3 region of aged rats. Digital subslice, total thickness 0.3 μm, and the merge of the three other images (D4). Scale bar: 15 μm. (E) Digital subslicing of a neuron-astrocyte-microglia triad, obtained stacking seven contiguous confocal acquisitions. Open arrows point to astrocytes branches intermingling the body of a neuron. It is possible to appreciate the presence of an engulfed neuronal fragment within the body of the microglial cell (arrowhead). Scale bar: 10 μm. (F1–F3) Digital subslicing of the Str. Radiatum of an aged rat of a IBA1-positive microglia (green). It is possible to highlight the presence of a NeuN-positive neuronal fragment [red, (F2)] inside the microglial cell body [yellow-orange, (F3), open arrow]. Scale bar: 7 μm [Modified from Cerbai et al. (2012), Lana et al. (2016)].

Figure 4

Schematic representation of neuron-astrocytes-microglia interplay during neurodegeneration. (A) Apoptotic neurons of pyramidal layer show diffuse cytoplasmic staining of CytC or AIF. Gap junctions (Connexine 43) mediate the recruitment of astrocytes on neurons. (B) The apoptotic neuron is detached from the Pyramidal layer to becoming an ectopic pyramidal neuron. CX3CL1 (fraktalkine) is released by the ectopic neuron and the astrocyte branches take close contact with the neuron. (C) Microglia are recruited and astrocytes branches infiltrate the body of the damaged neuron. (D) The damaged neuron is bisected and disgregated in cell debris. Phagocytic microglia mediate the clearance of cell debris.