Astrocyte Specificity and Coverage of hGFAP-CreERT2 Tg(GFAP-Cre/ERT2)13Kdmc Mouse Line in Various Brain Regions

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.

Astrocytic proBDNF and Tonic GABA Distinguish Active versus Reactive Astrocytes in Hippocampus

Astrocytes are the most abundant cell type in the brain and they make close contacts with neurons and blood vessels. They respond dynamically to various environmental stimuli and change their morphological and functional properties. Both physiological and pathological stimuli can induce versatile changes in astrocytes, as this phenomenon is referred to as ‘astrocytic plasticity’. However, the molecular and cellular mechanisms of astrocytic plasticity in response to various stimuli remain elusive, except for the presence of hypertrophy, a conspicuous structural change which is frequently observed in activated or reactive astrocytes. Here, we investigated differential characteristics of astrocytic plasticity in a stimulus-dependent manner. Strikingly, a stab wound brain injury lead to hypertrophy of astrocytes accompanied by increased GABA expression and tonic GABA release in mouse CA1 hippocampus. In contrast, the mice experiencing enriched environment exhibited astrocytic hypertrophy with enhanced proBDNF immunoreactivity but without GABA signal. Based on the results, we define proBDNF-positive/GABA-negative hypertrophic astrocytes as ‘active’ astrocytes and GABA-positive hypertrophic astrocytes as ‘reactive’ astrocytes, respectively. We propose for the first time that astrocytic proBDNF can be a bona fide molecular marker of the active astrocytes, which are distinct from the reactive astrocytes which show hypertrophy but with aberrant GABA.

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes.