Laser-scanning astrocyte mapping reveals increased glutamate-responsive domain size and disrupted maturation of glutamate uptake following neonatal cortical freeze-lesion

Abstract

Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL) model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM), which combines glutamate transport current (TC) recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes.

Astrocytic glutamate uptake is slow and does not limit neuronal NMDA receptor activation in the neonatal neocortex

“…For analysis involving TC amplitude or charge transfer ( Fig. 1F,G), membrane and input resistance were corrected for on a cell-by-cell basis (Armbruster et al, 2014). Dihydrokainate (DHK; 300 mM), a GLT-1-specific antagonist (Arriza et al, 1994) was applied in a separate subset of cells to isolate the GLT-1-mediated current.…”

α2δ-1 Signaling Drives Cell Death, Synaptogenesis, Circuit Reorganization, and Gabapentin-Mediated Neuroprotection in a Model of Insult-Induced Cortical Malformation

“…In parallel to measuring lesion volume, we also examined immunoreactivity for GFAP, a protein that is strongly upregulated by reactive astrocytes in the cerebral cortex. Our previous work, and the work of others, shows that FL causes significant reactive astrocytosis ( Bordey et al, 2001 ; Dulla et al, 2013 ; Armbruster et al, 2014 ; Campbell et al, 2014 ; Hanson et al, 2016 ). To quantify GFAP labeling in the cortex, we examined immunolabeled serial sections through the entire FL volume at P28.…”

“…In addition to driving cell death, FL also induces robust astrocytosis ( Bordey et al, 2001 ; Campbell and Hablitz, 2008 ; Dulla et al, 2013 ; Armbruster et al, 2014 ). Here, we show that α2δ-1 signaling plays an important role in driving reactive astrocytosis.…”

Short and long TNF‐alpha exposure recapitulates canonical astrogliosis events in human‐induced pluripotent stem cells‐derived astrocytes

“…Although conflicting results have been reported regarding gene expression and function of glutamate/aspartate transporters in different pathological conditions (Armbruster, Hampton, Yang, & Dulla, ; Pregnolato, Chakkarapani, Isles, & Luyt, ; van Landeghem, Weiss, Oehmichen, & von Deimling, ), the dysfunction of glutamate/aspartate transporters has been shown in situations where reactive astrocytes are present (Maragakis & Rothstein, ). In order to accurately measure uptake activity of these transporters in human iPSC‐derived astrocytes, the nonmetabolized analog D‐[ 3 H] aspartate was used.…”