Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex

Abstract

Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton-spine interface and an increase in glutamate transporter expression in astrocytic processes.

Figure 1. Up-Regulation of GLAST and GLT1 Protein Levels after Whisker Stimulation

(A) A single barrel column (C2) was removed by aspiration through a glass micropipette, under sodium pentobarbital anesthesia. (B) Tangential section of the barrel cortex, Nissl stained, shows the location of the excised barrel column. A clear hole can be seen in the section in the region of barrel C2, with the neighboring barrels intact. (C) Representative immunoblot microassay of C2 columns dissected immediately after 24 h of C2 whisker stimulation (stim), 4 d after stimulation (4 d post stim), and from unstimulated mice (unstim). Blot was probed for GLAST, GLT1, and actin, and indicates an increase in GLAST and GLT1 levels after 24 h of whisker stimulation, but not 4 d later. (D) These changes were quantified using densitometry with the values being normalized against the actin levels. Results were expressed as percentages of levels in unstimulated mice (100%) and statistically analyzed with a Tukey studentized range test, p < 0.01; error bars indicate SD. Scale bar in (B) indicates 0.5 mm. (E) Representative immunoblots from animals treated as in (C), and analyzed for protein levels of EAAC1, tubulin, and actin. (F) Quantification of the immunoblot signals revealed no significant alteration in EAAC1 levels in stimulated animals. EAAC1 and tubulin values were normalized on actin levels, and expressed as % of level in control animals. Note that the relative level of tubulin was unchanged by the stimulation. Error bars indicate SD.

Figure 2. Serial EM Images, and Reconstruction, of a Dendritic Spine from a C2 Barrel Hollow

(A–D) Show four micrographs from a series of 18 that were used to reconstruct the entire dendritic spine (S); making an asymmetric synaptic contact with a bouton (B) (arrow in [A] and [B]). The astrocytic element that surrounds this spine is marked with an asterisk (*) and can be seen to be closely associated with the interface between the spine head and the axonal bouton. Scale bar in (D) indicates 0.5 μm. (E) Shows the corresponding 3D reconstruction of this spine (green), bouton (grey), PSD (red), and astrocyte (blue) in three images below. The left-hand image shows the spine in the same orientation as the above micrographs, with a transparent astrocyte revealing the shape of the spine beneath; the middle image is in the same orientation, but the astrocyte is now opaque, showing the degree to which the spine is covered. The right-hand image shows the spine and covering astrocyte, viewed after a 180° rotation around the y axis.

Figure 3. Sensory Stimulation Increases Percentage of Spines Whose Bouton–Spine Interface Is Surrounded by Astrocyte

The histogram shows the distribution of four classes of spines, sorted according to their degree of contact with the astrocyte (see examples of classes I–IV), expressed as mean ± standard error of the mean (SEM) (unstimulated, n = 6 mice; stimulated. n = 6 mice). The percentage of spines in class IV, whose bouton–spine interface is completely surrounded by an astrocytic element, was increased significantly in stimulated mice (p < 0.03). Dendritic spines were classified into four classes, I–IV, based on the arrangement of the astrocyte at their surface. Electron micrographs of spines of each class are shown, as well as the 3D reconstruction of the whole spine to the right. (spines are indicated with an S and axonal boutons, B). Examples of spines in classes I–IV, and their reconstructions, are shown. Scale bar in lower micrograph represents 200 nm.

Figure 4. Whisker Stimulation Increases the Astrocytic Participation at the Bouton–Spine Interface

(A) Shows a 3D reconstruction of a spine head (green), its PSD (red), and the associated astrocyte (blue). The orientation of the structure shows the region occupied by the axonal bouton (removed). The line drawing below shows the parameters measured: the total perimeter of the interface between the bouton and the spine, and the part of this perimeter that is occupied by the astrocyte, the astrocytic perimeter. (B) Stimulation did not change the degree of contact between bouton and spine, measured by the total perimeter (p > 0.5). However, the amount of the perimeter occupied by the astrocyte was significantly increased (p < 0.0001), using mean values per animal. (C) Correlation between the length of the perimeter that is occupied by astrocytic membrane and the PSD surface area on spines in unstimulated (light grey diamonds, n = 271; p < 0.001, R² = 0.68) and stimulated neuropil (dark grey diamonds, n = 340; p < 0.001, R² = 0.73).