A closer look at astrocyte morphology: Development, heterogeneity, and plasticity at astrocyte leaflets

Abstract

Astrocytes represent an abundant type of glial cell involved in nearly every aspect of central nervous system (CNS) function, including synapse formation and maturation, ion and neurotransmitter homeostasis, blood-brain barrier maintenance, as well as neuronal metabolic support. These various functions are enabled by the morphological complexity that astrocytes adopt. Recent experimental advances in genetic and viral labeling, lineage tracing, and live- and ultrastructural imaging of miniscule astrocytic sub-compartments reveal a complex morphological heterogeneity that is based on the origin, local function, and environmental context in which astrocytes reside. In this minireview, we highlight recent findings that reveal the plastic nature of astrocytes in the healthy brain, particularly at the synapse, and emerging technologies that have advanced our understanding of these morphologically complex cells.

FIGURE 1. Astrocytes are morphologically complex cells.

Ai) Hand drawn illustrations by Santiago Ramón y Cajal depicting astrocytes in the brain contacting neurons and Aii) the vasculature. Ramon y Cajal’s illustrations revealed the stellate-nature of astrocyte morphology^[1]. Nearly a century after Ramón y Cajal’s illustrations, Bi-Biv) dye-filling of single astrocytes in rat hippocampus revealed that astrocytes are densely ramified cells composed of numerous leaflet-like processes. It was observed that astrocytes undergo a dynamic phase of morphological maturation between the first 4 postnatal weeks ^[2]. These processes have little overlap with other astrocytes in such a way that astrocytes tile the brain parenchyma. This tiling may not have been fully appreciated until C) the first glial “Brainbow” mice were developed and enabled for mosaic expression of numerous combinations of fluorescent proteins to depict the complexity of astrocytes and their ability to tile and infiltrate the CNS^[53].

FIGURE 2. Astrocyte Morphological Development.

A) Clavreul et al.’s recent work examined astrocyte cortical developmental by tracing numerous astrocyte progenitors marked prior to gliogenesis. This revealed that both pre- and postnatal astrocyte progenitors undergo a period of sporadic proliferation and randomized spreading across the cortex during early development, before finally adopting unique morphological profiles during the period of maturation^[13]. The underlying mechanisms of this morphological maturation are beginning to be resolved and they include B) signaling mechanisms such as Fibroblast Growth Factor (FGFs)/astrocytic Heartless signaling^[17], glutamate/astrocytic mGluR5 signaling^[18], BDNF/astrocytic TrkB.T1 signaling^[19]. C) Cell adhesion mechanisms that involve astrocytic neuroligin contact with neuronal neurexins have also been demonstrated to underlie astrocyte morphology^[20]. D) Interestingly, the ability for astrocytes to form their non-overlapping territories and gap-junctional networking has been shown to rely on astrocyte-to-astrocyte contacts via astrocyte HepaCAM interactions^[21]. Altogether, both neuronal-derived cues and astrocytic collaborations sculpt the morphology of individual astrocytes and give rise to their heterogeneity.

FIGURE 3. Astrocyte cytoskeletal dynamics give rise to plasticity at the synapse.

A) The astrocyte cytoskeleton is composed of microtubules and intermediate filaments such as GFAP that reside in the major primary and secondary branches. Actin dynamics such as Ezrin and its kinases (e.g. PKCɛ) have been identified to reside in astrocyte leaflets that associate with synapses. In this way, glutamatergic signaling can trigger astrocyte plasticity. One possible mechanism this occurs through is by direct glutamate signaling onto the astrocytic mGluR5 receptor. Glutamate binding activates and releases receptor-associated PKCɛ which can in turn phosphorylate ezrin. Phosphorylation of ezrin triggers a conformational change to its open state. Activated and open ezrin can then tether actin filaments to the membrane by linking with membrane-bound proteins such as CD44. This enables membrane motility and overall plasticity. B) This plasticity may vary depending on the synapse and the degree of leaflet coverage at a synapse. Smaller spines have been observed to have larger degrees of leaflet coverage and GLT1/GLAST-mediated glutamate clearance, perhaps enabling immature spines to enlarge and mature. Activity such as that from LTP has been observed to trigger leaflet rearrangement at spines. C) In vivo imaging has revealed that leaflet plasticity can occur in the span of minutes. Following LTP, leaflet plasticity at potentiated synapses begins with the initial withdrawal of leaflets. This allows for inter-synaptic glutamate communication. The increase in glutamate at potentiated synapses and consequent release of K⁺ leads to NKCC1 transporter-mediated actin engagement and leaflet motility at potentiated synapses.

FIGURE 4. Astrocyte sub-compartments revealed with high resolution microscopy.

Ai) 3D reconstruction of neighboring astrocytes (purple, pink, white) associated with dendrites (orange). A closer look at astrocytic processes reveals that many leaflets create reflexive processes. Bi) 2D EM traces of reflexive process pseudo-colored as pink and purple with Bii) their corresponding 3D reconstructions reveal these reflexive processes are loops-like structures. In the same study, evaluation of each astrocyte sub-compartment revealed that synapses associate with astrocytic B1-B2) somas, C1-C2) root processes and D1-D2) intermediate processes, E1-E2) terminal leaflets, F1-F2) reflexive loops, and G1-G2) endfeet. Interestingly, similar loop-like structures are also found in situ and in vivo. H) Left: 2Photon-STED image of the spongiform domain of an astrocyte from an acute slice of dentate gyrus. Right: Increased magnification reveals that astrocyte processes form loop-like structures termed Nodes. The same structures are observed using 2Photon microscopy in vivo in the whisker barrel cortex. I) Left: spongiform domain of an astrocyte in barrel cortex imaged with 2 Photon microscopy. Right: Increased magnifications reveal hot spot nodes and loop like structures. J) Ca²⁺ imaging reveals that astrocyte nodes/loop-like structures are compartments of high Ca²⁺ dynamics.