Functional glutamate transport in rodent optic nerve axons and glia

Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na+-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake

Primers sequence for PCR amplification.

<p>Primers sequence for PCR amplification.</p

Surface expression of GluA1 is not increased in <i>Tspan6</i> KO hippocampal primary neurons.

<p><b>(A)</b> Representative images from 14 days <i>in vitro</i> WT and <i>Tspan6</i> KO hippocampal primary neurons stained with an extracellular N-terminal domain GluA1 antibody before fixation (surface expression), and with VGlut1 antibody after fixation. <b>(B)</b> Histograms comparing mean (±S.E.M) density <b>(</b>particles/10μm) and area (μm²<b>)</b> from total cell surface GluA1 particles. <b>(C)</b> Histograms comparing mean (±S.E.M) density <b>(</b>particles/10μm) and area (μm²<b>)</b> from synaptic surface GluA1 particles. Scale bar = 10μm. <i>n</i> = 28 (WT) and 31 (<i>Tspan6</i> KO) neurons from 4 different embryos, 2 independent cultures.</p

Generation of Tspan6 KO mice and Tspan6 expression in the brain.

<p><b>(A)</b> The <i>Tspan6</i> KO mouse was generated by insertion of a neomycin cassette in the exon 2 of the <i>Tspan6</i> gene. Right panel show a representative agarose gel electrophoresis with the PCR products amplified with specific primers (a, b and c, shown by arrows in the left panel). <b>(B)</b> RNA was extracted from <i>Tspan6</i> KO and WT animals. Primers were designed between exon 1 and 3 (WT-specific primers), and exon 4 and exon 5 (primers downstream insertion). <b>(C)</b> Real time semi-quantitative PCR shows no RNA amplification between exon 1 and 3 in <i>Tspan6</i> KO mice due to the insertion of the neomycin cassette. <b>(D)</b> RNA amplification downstream the insertion is reduced in <i>Tspan6</i> KO mice (0.35± 0.01 mean fold change compared to WT) suggesting RNA degradation. Histogram shows mean (±S.E.M) fold changes normalized against WT expression, using either WT-specific primers (C) or primers downstream the insertion (D). Two housekeeping genes (Actin and GAPDH) were used for the normalization of the expression. <b>(E)</b> Neuronal lysates from cortical primary cultures from <i>Tspan6</i> WT, heterozygous and KO mice show the absence of Tspan6 protein in the KO condition. <b>(F)</b> RNA scope shows expression of Tspan6 RNA in the pyramidal layer of the hippocampus and granule cells from the dentate gyrus. First panel is a general view of the hippocampus (scale bar = 100μm). Panels 1 to 5 show box section in higher magnification (scale bar = 10μm).White dots are Tspan6 RNA molecules, synaptophysin RNA is stained in red and DAPI in blue.</p

Tspan6 does not affect synapse formation and maturation <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A)</b>Representative images from WT and <i>Tspan6</i> KO hippocampal primary neurons transfected with EGFP and fixed after 14 days <i>in vitro</i> (scale bar = 20 μm). Right panels show box sections in higher magnification (scale bar = 10μm). 15 neurons and more than 90 dendritic sections were examined from 3 independent cultures. Only spines more than 80μm from the soma were analyzed. Histograms compare mean (±S.E.M) filopodia and spine density (number/10μm of dendrite). <b>(B)</b> Golgi staining of 100 μm coronal sections from 10 month-old <i>Tspan6</i> KO and WT mice. Scale bar = 100 μm. <b>(C)</b> Representative images of dendritic spines from basal secondary dendrites from CA1 hippocampal neurons and the IMARIS reconstruction to analyze spine morphology. Scale bar = 5μm. Histograms compare mean (±S.E.M) spine density (number/10μm of dendrite) <b>(D)</b>, length <b>(E)</b> and head width <b>(F)</b> between Tspan6 KO and WT mice. <i>n</i> = 19 to 22 neurons from 3 different mice.</p

<i>Tspan6</i> mice show normal behavior in the Morris water maze test.

<p>Mice were trained in the hidden platform Morris water maze for 10 days (4 trials/day) and tested with a probe trial (100s) on day 6 and 11. <b>(A)</b> Curve shows no differences between <i>Tspan6</i> KO and WT mice in the learning-related decrease in escape latency (time required to find the platform) during the acquisition phase. <b>(B)</b> Probe trial performance illustrate spatial memory for the platform position as both groups show a preference for the quadrant where the platform was located during the training (target quadrant) with no significant differences between the groups. <b>(C)</b> Spatial learning was analyzed in more detail quantifying the time that the mice spend swimming in the specific area of the quadrant were the platform was during the training showing no changes in the KO animals compared to controls. <i>n</i> = 12 WT, 21 KO animals.</p

<i>Tspan6</i> KO mice show an increased basal synaptic transmission and impaired LTP in the CA3-CA1 synapses of the hippocampus.

<p><b>(A)</b> Input-output relations between stimulus intensity applied to the Schaffer collateral fibers and slope of field excitatory postsynaptic potentials (fEPSP) recorded in the <i>stratum radiatum</i> of CA1. Right panel show representative traces of fEPSP at 90μA stimulation from Tspan6 KO and control acute slices. <i>n</i> = 30 WT and 25 <i>Tspan6</i> KO slices from 9 to 12 different mice per group. (F _(11,583) = 9.447, p<0.0001, Repeated measurements ANOVA). <b>(B)</b> LTP was induced in CA1 neurons by theta-burst stimulation (5 trains, each with 10 bursts at 5 Hz, each burst containing 4 pulses at 100 Hz) and fEPSP slope is normalized to 20 minutes baseline. Insets: representative traces averaged from the baseline (thick lines) or from the last 10 minutes of the recordings (thin lines). Histogram compares mean (±S.E.M) normalized fEPSP slope from the last 10 minutes of the recordings from <i>Tspan6</i> KO and control slices (<i>p</i> = 0.026, T-test). Between 10 and 14 slices were analyzed from 9 different mice per group. <b>(C)</b> Paired-pulse facilitation ratios evoked by stimulation of the Schaffer collateral fibers with different interstimulus intervals (50, 100, 200 and 400ms). Representative traces of the paired-pulse facilitation at 50ms interstimulus interval from WT and KO slices are shown in the right. 27 slices from 8 to 10 different mice were analyzed per group.</p

No changes in synaptic markers in the hippocampus from <i>Tspan6</i> KO mice.

<p><b>(A)</b> Levels of synaptic markers are not altered in hippocampal homogenates from <i>Tspan6</i> KO mice compared with controls (n = 7 mice per group). <b>(B)</b> Crude synaptosomes were isolated from 10 month old <i>Tspan6</i> KO (<i>n</i> = 7) and control (<i>n</i> = 6) hippocampus and levels of AMPA and NMDA receptor subunits were analyzed by western blotting. Histograms compare mean (±S.E.M) relative expression normalized to loading control (β-actin).</p

Primers sequence for real time semi-quantitative PCR amplification.

<p>Primers sequence for real time semi-quantitative PCR amplification.</p