Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains

Abstract

Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominantly on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only approximately 15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.

Morphological plasticity of astroglia: Understanding synaptic microenvironment

“…3B). The GFAP‐positive main branches of an astrocyte make up only ∼15% of its total volume (Bushong et al, 2002), thus leaving the bulk of astroglial morphology largely beyond the diffraction limit of conventional optical microscopy. Structural changes in astroglial filopodia‐like protrusions can be routinely observed using time‐lapse light microscopy in cultured hippocampal or cortical astrocytes (Cornell‐Bell et al, 1990a; Lavialle et al, 2011; Molotkov et al, 2013).…”

“…In accord with these observations, a global increase in GFAP expression in the aged brain is detectable, suggesting an increased number of astrocytes in older brains (Cotrina and Nedergaard, 2002). Alternatively, because only ∼15% of astrocytes stain for GFAP in the mouse brain (Bushong et al, 2002), global increases in GFAP labeling could also suggest that various astrocyte subtypes start expressing GFAP during aging. It also appears that the interaction of astrocytes with nearby neurons changes with age.…”

“…Depending on the brain region, the tissue volume occupied by one (nonoverlapping) astrocyte in rodents varies between 20,000 and 80,000 µm 3 (Bushong et al, 2002; Halassa and Haydon, 2010; Oberheim et al, 2012), and 300 to 600 dendrites of principal neurons normally approach a single astrocyte (Halassa and Haydon, 2010). These relationships indicate that a single astrocyte approaches 20 to 120 thousand synapses in the rodent brain, and ∼270 thousand to 2 million synapses in the human brain (Bushong et al, 2002; Oberheim et al, 2009, 2006). …”

Supportive or information-processing functions of the mature protoplasmic astrocyte in the mammalian CNS? A critical appraisal

“…Bushong et al (2002) emphasized in their combined immunohistochemical and dye-filling studies in the CA1 region of 1-monthold Sprague-Dawley rats, that the dye-filled astrocytes were morphologically homogenous and their highly bushy processes (1000-10 000 per cell body) occupied separate territories of ~70 000 μm 3 . This is an important concept.…”

“…The large number of processes of mature astrocytes, that the processes of separate astrocytes overlap by only ~10%, and that gap junctional, electrophysiological, synaptic and domain maturity all occur at around the same time in the hippocampus (Bushong et al, 2002;Bushong et al, 2004;Zhou et al, 2006) can be incorporated into a general hypothesis of astrocyte function in the mature brain, or at least in the hippocampus (Fig. 6).…”