ASC transporters mediate D-serine transport into astrocytes adjacent to synapses in mouse brain
Background: D-serine is an important signaling molecule, which activates N-methyl D-aspartate receptors (NMDARs) in conjunction with its fellow co-agonist, the neurotransmitter glutamate. Question: Despite its involvement in plasticity and memory related to excitatory synapses, its cellular source and sink remain a question. Importance: This removal of synaptic D-serine and its subsequent metabolic degradation is expected to reduce its extracellular availability, influencing NMDAR activation and NMDAR-dependent synaptic plasticity. Hypothesis: We hypothesise that astrocytes, a type of glial cell that surround synapses are likely candidates to control the extracellular concentration of D-Serine by removing it from the synaptic space. Notable finding: We observed D-serine induced transport-associated currents upon puff-application of 10 mM D-serine on astrocytes. Further, O-benzyl-L-serine and trans-4-hydroxy-proline, known substrate inhibitors of the alanine serine cysteine transporters (ASCT) reduced D-serine uptake. These results indicate that ASCT is a central mediator of astrocytic D-serine transport and plays a role in regulating its synaptic concentration by sequestration into astrocytes. Similar results were observed in astrocytes of the somatosensory cortex and Bergmann glia in the cerebellum, indicative of a general mechanism expressed across a range of brain areas. How was it gathered: We used patch clamping technique, to study the transmembrane electrical signals from individual identified astrocytes in the CA1 region of hippocampus, primary somatosensory cortex and Bergmann glia. It involved placing a tiny glass patch pipette, onto the surface of the astrocytic membrane, to record D-serine transporter currents. Methods: Using in situ patch clamp recordings and pharmacological manipulation of astrocytes in CA1 region of the mouse hippocampal brain slices, we investigated transport of D-serine across the plasma membrane. How to interpret data: The spread sheet contains all the mean astrocytic D-serine currents that were recorded from individual cells. How to use it: The raw data traces are most compatible with Clampfit 10.7.0.3 (P Clamp - Molecular devices 2016).
Steps to reproduce
Drugs used: The recordings were performed in a cocktail of pharmacological agents that inhibited any possible current evoked due to activation of voltage gated sodium channels, voltage gated potassium channels, NMDA receptors (both APV and mk801), AMPA receptors, GABA A receptors and glycine receptors. Solutions used: Puff application was done in a HEPES based ACSF solution. Bicarbonate based ASCF was avoided due to puff generated bicarbonate transporter currents. How we arrived at our data: Import raw data traces (.abf files) into the clampfit software. Channel In1 (pA) with the current trace was selected. Out of the four cursors, two baseline cursors (3&4) were placed before the substrate application. Substrate application starts at 5 seconds, as indicated by channels IN0(pA) and IN4(V). Two recording or search cursors (1&2) were placed between 14 and 15 seconds. After placing cursors, click on 'Analyze' tab - and then select 'Statistics'. In peak polarity, select channel IN1 - negative going. In measurements, select mean (pA) and click OK. The raw data values for a particular trace can be found by selecting 'Window' tab - swap to results window. Details regarding instruments, software version, protocols can be found in the methods section of the paper.