In vivo intracellular recording suggests that gray matter astrocytes in mature cerebral cortex and hippocampus are electrophysiologically homogeneous

Abstract

Previous anatomical and in vitro electrophysiology studies suggest that astrocytes are heterogeneous in physiology, morphology, and biochemical content. However, the extent to which this diversity applies to in vivo conditions is largely unknown. To characterize and classify the physiological and morphological properties of cerebral cortical and hippocampal astrocytes in the intact brain, we performed in vivo intracellular recordings from single astrocytes using anesthetized mature rats. Astrocytes were classified based on their glial fibrillary acidic protein (GFAP) immunoreactivity and cell body locations. We analyzed morphometric measures such as the occupied volume and polarity, as well as physiological characteristics such as the mean membrane potential. These measurements did not show obvious segregation into subpopulations, suggesting that gray matter astrocytes in the cortex and hippocampus are composed of a homogeneous population in mature animals. The membrane potential of astrocytes in both cortex and hippocampus fluctuated within a few millivolts in the presence of spontaneous network activity. These membrane potential fluctuations of an astrocyte showed a significant variability that depended on the local field potential state and cell body location. We attribute the variability of the membrane potential fluctuations to local potassium concentration changes due to neuronal activity.

Figure 1.

Morphological reconstruction of in vivo labeled cortical astrocytes. Confocal images of two astrocytes labeled with BDA-10000 by in vivo intracellular injection (A, D; green) and immunohistochemistry against GFAP (B, E; red) are displayed. Overlay of the images (C, F) distinguishes the GFAP(+) astrocyte from the GFAP(−) astrocyte. G, Bar graph shows the total numbers of intracellularly recorded GFAP(+) (gray) and GFAP(−) (white) astrocytes and their locations used in this study. Astrocytes are clearly distinguished from other glial cell types by their morphology, as exemplified by the morphology of an intracellularly filled presumed oligodendrocyte (H). Scale bars are 20 μm. CTX, Cortex; HPC, hippocampus; ori, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; DG, dentate gyrus.

Figure 2.

Occupied area and morphological polarity of astrocytes. A, The occupied volume of GFAP(+) and GFAP(−) astrocytes were similar. B, The occupied volume did not depend on the cell body location. C, Astrocyte morphology was more polarized in hippocampus than cortex (*p < 0.05, t test).

Figure 3.

Membrane potential (MP) recording from single astrocytes. Stable membrane potential recording is achieved from a single astrocyte. A–C, Representative trace of single astrocyte membrane potential fluctuations recorded shortly after intracellular penetration (A), after 5 min (B), and after 10 min (C). D, Distribution of the mean membrane potential of all recorded astrocytes shows a mode at around −86 mV. E, Distributions of the mean membrane potential of GFAP(+) and GFAP(−) astrocytes are represented in box plots. GFAP(+) and GFAP(−) cells did not differ significantly in the mean membrane potential. F, Mean membrane potential of astrocytes in different brain regions are represented in box plots (*p < 0.05, one-way ANOVA).

Figure 4.

Membrane potential (MP) fluctuations of cortical astrocytes in different LFP states. A, Extracellular LFP (upper trace) and membrane potential fluctuations of a single cortical astrocyte (bottom trace) are plotted during the synchronized state (U/D). B, Similar plots taken from the same cell as in A during the desynchronized state (DS). C, D, Fluctuation amplitude is significantly larger during the synchronized state (***p < 0.001, t test) and the upper cortical layer (I–IV) astrocytes elicit larger membrane potential fluctuations during synchronized states (*p < 0.05), but they are indistinguishable during the desynchronized state. E, Mean membrane potential was similar in synchronized and desynchronized states.

Figure 5.

Astrocytic membrane potential (MP) change triggered by UP and DOWN states. Example of triggered averaging of LFP (upper panel) by onset and offset UP states. The traces are from a multichannel silicon probe with linearly arranged recording sites (100 μm apart). Superficial layer traces (layer II–IV) are in gray and deep layer traces (layer V–VI) are in black. The channel indicated by the arrowhead was used to calculate onsets and offsets of UP states for triggered averaging. Population data of the triggered averaging of membrane potential transition of astrocytes with UP and DOWN states (lower panel). Gray lines are triggered averaging of membrane potential transitions of individual astrocytes. The means of the traces are in bold lines.

Figure 6.

Membrane potential fluctuations of hippocampal astrocytes in different LFP states. A, Extracellular LFPs from the CA1 stratum pyramidale (upper trace), stratum radiatum (middle trace), and the membrane potential of a single hippocampal astrocyte (bottom trace) are plotted during the nontheta state. Arrowheads indicate the occurrences of sharp-wave-associated ripple oscillations. B, Similar plots taken from the same cell (bottom trace) as in A during the theta state. Pyr, Stratum pyramidale; Rad, stratum radiatum. C, Mean membrane potential was similar in nontheta and theta states (p = 0.2, t test). D, Membrane potential fluctuation amplitude is significantly larger during nontheta states than theta states (***p < 0.001, t test). E, Similar decreases of fluctuations at the single cell level are observed in those experiments that allow pairwise comparisons of membrane potential fluctuations from the same cells (***p < 0.001, paired t test). F, Sharp-wave-triggered averaging of astrocyte membrane potential in different layers of hippocampal CA1 before and after trigger (solid line). Gray lines show ±SEM.