The glycine transporter GlyT1 controls N-methyl-D-aspartic acid receptor coagonist occupancy in the mouse retina

Abstract

We examined the role of GlyT1, the high-affinity glycine transporter, in the mouse retina with an emphasis on the role of glycine as a coagonist of N-methyl-D-aspartic acid (NMDA) receptors. We pursued this objective by studying heterozygote mice deficient in the GlyT1 transporter (GlyT1(-/+)) and compared those results with wild-type (WT) littermate controls (GlyT1(+/+)). Capillary electrophoresis was used to separate and quantitatively measure glycine release from isolated retina preparations; pharmacologically blocking GlyT1 with N-[3-([1,1-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine in the WT retina generated a significantly larger accumulation of glycine into the bathing environment when compared with the GlyT1(-/+) retinas. The relative occupancy state of the NMDA receptor coagonist sites was tested using whole-cell recordings from ganglion cells while bath applying D-serine or D-serine + NMDA. The interpretation of these studies was simplified by blocking post-synaptic inhibition with picrotoxinin and strychnine. NMDA receptor coagonist sites were more saturated and less enhanced by D-serine in the GlyT1(-/+) mice compared with the WT controls. Immunoblots of NMDA receptor subunits (NR1, NR2A and NR2B) in WT and GlyT1(-/+) animals showed that the NR1 subunits were identical. These observations are discussed in view of contemporary issues about NMDA receptor coagonist function in the vertebrate retina and the role of glycine vs. D-serine as the endogenous coagonist.

Fig. 1

A single ganglion cell dye-filled with Alexa 488; the axon is marked with an arrow. Using a stack of images obtained by focusing through the plane of the ganglion cell, the soma, axon and dendrites could be followed. This ganglion cell was identified physiologically as a sustained On cell with its dendritic tree confined to the inner part of the inner plexiform layer.

Fig. 2

Glycine release is different in WT vs. GlyT1^−/+ retinas. (A) Capillary electrophoresis electropherogram that shows elution of the glycine peak between that of taurine (Tau) and glutamine (Gln) and the late peak of the internal standard [α-amino adipic acid (Aad)]. (B) Glycine peak identification. An expanded eletropherogram shows the glycine peak region with an elevated glycine level (top trace) reflecting glycine added to the medium, the glycine level from the bathing chamber (middle trace) and the elimination of glycine (bottom trace) by adding glycine oxidase (GO) to the solution. (C) Expanded glycine peak regions of superimposed electropherograms. The two top traces show virtually no change in glycine levels when NFPS was added to the chamber (top trace, dotted line) vs. the release level of the control retina from the same animal (second trace, solid line). In contrast, glycine from a WT retina exposed to NFPS resulted in a substantial level of glycine enhancement (third trace, dashed line) compared with that of the companion control retina (bottom trace, solid line). (D) Comparison of WT and GlyT1^−/+ retinas exposed to NFPS. Differences between WT and WT + NFPS are significant (t₄ = 2.63, P = 0.058), whereas the difference between GlyT1^−/+ and GlyT1^−/+ + NFPS is not significant (t₄ = 1.17, P = 0.31). The right two bar graphs compare the differences between the WT + NFPS and GlyT1^−/+ retinas expressed as a percent increase over the control values (t₈ = 2.37, P = 0.046). Gly, glycine; RFU, relative fluorescence units.

Fig. 3

D-serine potentiates the light-evoked responses of ganglion cells. (A) A current-clamp recording from a WT sustained On ganglion cell showed enhancement of the peak light response in D-serine and a significant reduction in the light response when D,L-AP7 was added to the bathing solution. (B) Voltage-clamp records from a sustained On ganglion cell of a WT retina; D-serine enhanced the light response, whereas the application of D,L-AP7 reduced the peak response and plateau components. (C) Voltage-clamp recording from an On cell of a GlyT1^−/+ retina, which had a prominent peak and small plateau components. When D-serine was added, a very slight increase in the peak response was observed, with little change in the plateau response, whereas adding D,L-AP7 reduced the peak and plateau components. (D) Bar graph summary of D-serine enhancement using both peak amplitude and response integration values for the GlyT1^−/+ and WT retinas (±SE). Both measurement techniques gave similar results, with a clear difference evident when comparing the two animal groups (t₇ = 3.90, P = 0.0059). Each light-evoked response illustrated in A-C was averaged from 20 consecutive light stimuli. All recordings were performed in continuous perfusion with Ames medium to which 1 μM TTX, 50 μM picrotoxinin and 10 μM strychnine were added.

Fig. 4

D-serine (DS) enhances the NMDA-evoked responses of retinal ganglion cells in WT mice more than it does in GlyT1^−/+ mice. (A) Whole-cell recording from a GlyT1^−/+ retina in response to 500 μM NMDA (top trace) in the presence of TTX (peak amplitude −142 pA), followed by an enhanced response (middle trace, −219 pA) when D-serine was added to the NMDA. The bottom trace shows the result of repeating the NMDA application after washing the retina for several minutes (bottom trace, −124 pA). (B) Exposure of a ganglion cell from a WT retina in the presence of TTX, where the peak response went from −27 pA (control, top trace) to −40 pA (NMDA + D-serine, middle trace) followed by a second NMDA application (bottom trace, −31 pA). (C and D) NMDA-evoked responses in TTX (1 μM), picrotoxinin (50 μM) and strychnine (10 μM). (C) Responses from a GlyT1^−/+ mouse retina. The control NMDA response evoked a peak inward current of −18 pA (upper trace), whereas NMDA + D-serine (middle trace) evoked an inward current of −121 pA; the repeat control response was −63 pA. (D) Responses from a WT retinal ganglion cell; NMDA in the control + toxin environment (upper trace) evoked a slow inward current of −81 pA; the NMDA + D-serine response (middle trace) was −244 pA, whereas the repeat control response (bottom trace) was −63 pA. (E) Bar graph summary comparing NMDA-evoked currents in WT and Glyt1^−/+ as a percent increase over the normalized control response in the presence of TTX. In these studies, D-serine potentiated NMDA-evoked currents of ganglion cells in both the GlyT1^−/+ mice (5/5) as well as the WT controls (4/5). The average enhancement of the NMDA-evoked current when D-serine was coapplied was greater for the WT when compared with the GlyT1^−/+ heterozygotes (t₇ = 3.40, P = 0.014). (F) A comparison between the NMDA-evoked responses of ganglion cells in WT and GlyT1^−/+ ganglion cell recordings that illustrates the greater enhancement of the NMDA response when the experiments were carried out in the presence of TTX, strychnine and picrotoxinin. For these studies, D-serine potentiated NMDA-evoked currents of ganglion cells in some of the GlyT1^−/+ (2/6) but it potentiated the NMDA-evoked currents for all of the WT controls (t₈ = 2.31, P = 0.049). For both panels, error bars represent ±SE. Physiological recordings were processed with offline filtering at 2 Hz with an eight-pole Bessel filter, with further noise reduction carried out in Origin using Fourier transform filtering.

Fig. 5

Western blot comparing levels of NR1 (~ 116 kDa), NR2A and NR2B (~160 kDa) subunits in extracts from age-matched WT and GlyT^+/− mouse retinas. GAPDH (38 kDa) was used as a loading control. All lanes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the same gel; after transfer the membrane was cut into strips for primary antibody incubation. The figure was constructed by realigning digitally scanned strips in Adobe Photoshop using molecular weight markers; any subsequent level and contrast adjustments were performed simultaneously on all parts of the blot.