Chorghay Z et al. (2023), The effects of the NMDAR co-agonist D-serine on...

XB-ART-60200

Sci Rep 2023 Aug 17;131:13383. doi: 10.1038/s41598-023-39951-4.

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The effects of the NMDAR co-agonist D-serine on the structure and function of optic tectal neurons in the developing visual system.

Chorghay Z , Li VJ , Schohl A , Ghosh A , Ruthazer ES .

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The N-methyl-D-aspartate type glutamate receptor (NMDAR) is a molecular coincidence detector which converts correlated patterns of neuronal activity into cues for the structural and functional refinement of developing circuits in the brain. D-serine is an endogenous co-agonist of the NMDAR. We investigated the effects of potent enhancement of NMDAR-mediated currents by chronic administration of saturating levels of D-serine on the developing Xenopus retinotectal circuit. Chronic exposure to the NMDAR co-agonist D-serine resulted in structural and functional changes in the optic tectum. In immature tectal neurons, D-serine administration led to more compact and less dynamic tectal dendritic arbors, and increased synapse density. Calcium imaging to examine retinotopy of tectal neurons revealed that animals raised in D-serine had more compact visual receptive fields. These findings provide insight into how the availability of endogenous NMDAR co-agonists like D-serine at glutamatergic synapses can regulate the refinement of circuits in the developing brain.

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Species referenced: Xenopus laevis
Genes referenced: dlg4 psd
GO keywords: brain development [+]
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	Figure 1 Growth of dendritic arbors of tectal cells in animals reared in D-serine over 4 days. (A) Experimental design. Tadpoles at stage 42–44 were electroporated, screened for single-cell GFP expression 48 h later at stage 46–47, and the GFP + cell was imaged daily. After a baseline image on day 0, the animals were reared in control or D-serine medium, and images collected every day for 3 more days. (B) Z-projections of representative tectal neurons. Scale bar: 20 µm. (C, D) Quantification of the (C) length and (D) number of tips of the dendritic arbors from tadpoles reared in D-serine (red squares) compared to control (black circles), subdivided into immature (total dendritic arbor length < 500 µm on day 0) or mature (≥ 500 µm on day 0). Immature cells were labeled by CREMSCLE and mature cells by single-cell electroporation. For immature cells, total dendritic arbor length and branchtip number were reduced in D-serine compared to control. [RM ANOVA interaction p < 0.05, Šídák's multiple comparisons post-hoc test Day 2 p < 0.005, Day 3 p < 0.05. 1 cell per animal, immature cells: n = 6 cells for D-serine, n = 9 for control, mature cells: n = 4 cells for both groups] (E) Sholl analysis of CREMSCLE cells shows tectal dendritic arborization occurs closer to the soma in animals reared in D-serine. [multiplicity-corrected RM ANOVA interaction, Day 3 p < 0.0005, Šídák's post-hoc test for multiple comparisons ****p < 0.0001. 1 cell per animal analyzed with n = 9 cells for D-serine, n = 6 for control].
	Figure 2 Filopodia and branch dynamics of tectal cell dendritic arbors in animals reared in D-serine for 48 h. (A) Experimental design. Tadpoles at stage 42–44 were electroporated, screened for GFP expression 48 h later at stage 46–47, and reared in control or D-serine medium for 48 h to stage 48. After the initial image, cells were imaged every 10 min for 1 h as tadpoles were visually stimulated with 1 Hz strobe flashes. (B) Representative images of dendritic arbors during imaging and a final merged image overlaying timepoints 0 (magenta) and 60 (yellow) min. Scale bar: 10 µm. (C) Density of processes counted per timepoint for filopodia (dendritic processes < 10 µm length; shaded) and branches (≥ 10 µm length; unshaded) from control (black) and D-serine (red) arbors. Density was calculated as the number of dendritic processes divided by arbor length. (D) Number of filopodia added to the dendritic arbor over 1 h of imaging. (E) Number of filopodia lost showed a trend towards a decrease in D-serine arbors compared to control. [t-test #p = 0.06] (F) Length of filopodial elongations and (G) retractions over 1 h of imaging. (H) Filopodial motility (sum of elongations and retractions) per timepoint. (I–M) Quantification for branches, including (I) number added and (J) lost over 1 h. (K) Branches showed a trend towards elongating less over 1 h in D-serine arbors compared to control. [t-test #p = 0.06] (L) Length of branch retractions over 1 h was significantly less in the D-serine group [t-test p < 0.05], as was the (M) branch motility per timepoint [RM ANOVA main effect of treatment p < 0.05]. 1 cell per animal analyzed with n = 6 for D-serine, n = 7 for control.
	Figure 3 Synaptic density in the tectum of animals reared in D-serine for 48 h. (A) Experimental design: Tadpoles at stage 46–47 were reared in control or D-serine medium for 48 h to stage 48, then fixed, sectioned and immunostained for SV2 and GluA1 for confocal imaging. (B) Colocalized puncta of SV2 (presynaptic) and GluA1 (postsynaptic) immunofluorescence on brain sections. For a sample field, (i) the tectal neuropil is shown, (ii) zoomed into a dashed 20 µm × 20 µm region-of-interest (top) with automated processing to identify “anatomical synapses” (bottom, white overlay, see “Materials and methods”). (C) Anatomical synapse density in the optic tectum was significantly elevated for animals reared in D-serine. [t-test p < 0.05. 2–3 fields per tectal hemisphere were analysed i.e. at least 5 fields per animal from n = 5 D-serine, 3 control animals]. Only colocalized puncta that fit the synapse size criterion of 0.1–5.0 µm2 were included. (D) Experimental design: Tadpoles at stage 42–44 were electroporated, screened for dsRed and PSD95-GFP expression 48 h later at stage 46–47, reared in control, D-serine, or D-serine + MK-801 medium for 48 h and imaged at stage 48. (E) Magnified dendritic arbors of tectal neurons expressing PSD95-GFP + puncta (yellow) in dsRed + cells (magenta). Scale bar: 10 µm. Arrows: examples of synaptic puncta. (F) Quantification of the densities of PSD-95 puncta per volume of dendritic arbor for single tectal neurons reveals that rearing in D-serine increases synapse density, which is prevented when NMDARs are blocked by MK-801. [one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons p < 0.01, p < 0.05. n = 6 control, 10 D-serine, 8 D-serine + MK-801 cells].
	Figure 4 Postsynaptic retinotopic maps in stage 48 animals reared in D-serine from stage 37. (A) Experimental design. mRNA for GCaMP6s was injected into one blastomere at the two-cell stage to generate tadpoles with mosaic GCaMP6s expression restricted to half the animal. Tadpoles were screened at stage 37 for GCaMP6s expression, then reared in control or D-serine for two-photon imaging of calcium fluorescence in the optic tectum at stage 48. Scale bar: 50 µm. (B, C) Representative images showing receptive field center locations (left) and receptive field sharpness (right) of individual tectal cells in response to a bar flashed at each of 5 locations across the (B) azimuth and (C) elevation axes. The maps are color-coded by the optimal stimulus position that evokes a response in each cell. Scale bar: 50 µm. (D, E) Cumulative probability distributions of receptive field sharpness of tectal cell bodies in individual animals (lighter lines) and grouped by treatment (darker lines) for the (D) azimuth [n = 365 cells from 6 animals for D-serine, n = 181 cells from 5 animals for control] and (E) elevation axes [n = 326 cells from 6 animals for D-serine, n = 172 cells from 5 animals for control. Kolmogorov–Smirnov test for pooled values *p < 0.05].

References [+] :

Andrade-Talavera, Presynaptic Spike Timing-Dependent Long-Term Depression in the Mouse Hippocampus. 2016, Pubmed

	Figure 1 Growth of dendritic arbors of tectal cells in animals reared in D-serine over 4 days. (A) Experimental design. Tadpoles at stage 42–44 were electroporated, screened for single-cell GFP expression 48 h later at stage 46–47, and the GFP + cell was imaged daily. After a baseline image on day 0, the animals were reared in control or D-serine medium, and images collected every day for 3 more days. (B) Z-projections of representative tectal neurons. Scale bar: 20 µm. (C, D) Quantification of the (C) length and (D) number of tips of the dendritic arbors from tadpoles reared in D-serine (red squares) compared to control (black circles), subdivided into immature (total dendritic arbor length < 500 µm on day 0) or mature (≥ 500 µm on day 0). Immature cells were labeled by CREMSCLE and mature cells by single-cell electroporation. For immature cells, total dendritic arbor length and branchtip number were reduced in D-serine compared to control. [RM ANOVA interaction p < 0.05, Šídák's multiple comparisons post-hoc test Day 2 p < 0.005, Day 3 p < 0.05. 1 cell per animal, immature cells: n = 6 cells for D-serine, n = 9 for control, mature cells: n = 4 cells for both groups] (E) Sholl analysis of CREMSCLE cells shows tectal dendritic arborization occurs closer to the soma in animals reared in D-serine. [multiplicity-corrected RM ANOVA interaction, Day 3 p < 0.0005, Šídák's post-hoc test for multiple comparisons ****p < 0.0001. 1 cell per animal analyzed with n = 9 cells for D-serine, n = 6 for control].
	Figure 2 Filopodia and branch dynamics of tectal cell dendritic arbors in animals reared in D-serine for 48 h. (A) Experimental design. Tadpoles at stage 42–44 were electroporated, screened for GFP expression 48 h later at stage 46–47, and reared in control or D-serine medium for 48 h to stage 48. After the initial image, cells were imaged every 10 min for 1 h as tadpoles were visually stimulated with 1 Hz strobe flashes. (B) Representative images of dendritic arbors during imaging and a final merged image overlaying timepoints 0 (magenta) and 60 (yellow) min. Scale bar: 10 µm. (C) Density of processes counted per timepoint for filopodia (dendritic processes < 10 µm length; shaded) and branches (≥ 10 µm length; unshaded) from control (black) and D-serine (red) arbors. Density was calculated as the number of dendritic processes divided by arbor length. (D) Number of filopodia added to the dendritic arbor over 1 h of imaging. (E) Number of filopodia lost showed a trend towards a decrease in D-serine arbors compared to control. [t-test #p = 0.06] (F) Length of filopodial elongations and (G) retractions over 1 h of imaging. (H) Filopodial motility (sum of elongations and retractions) per timepoint. (I–M) Quantification for branches, including (I) number added and (J) lost over 1 h. (K) Branches showed a trend towards elongating less over 1 h in D-serine arbors compared to control. [t-test #p = 0.06] (L) Length of branch retractions over 1 h was significantly less in the D-serine group [t-test p < 0.05], as was the (M) branch motility per timepoint [RM ANOVA main effect of treatment p < 0.05]. 1 cell per animal analyzed with n = 6 for D-serine, n = 7 for control.
	Figure 3 Synaptic density in the tectum of animals reared in D-serine for 48 h. (A) Experimental design: Tadpoles at stage 46–47 were reared in control or D-serine medium for 48 h to stage 48, then fixed, sectioned and immunostained for SV2 and GluA1 for confocal imaging. (B) Colocalized puncta of SV2 (presynaptic) and GluA1 (postsynaptic) immunofluorescence on brain sections. For a sample field, (i) the tectal neuropil is shown, (ii) zoomed into a dashed 20 µm × 20 µm region-of-interest (top) with automated processing to identify “anatomical synapses” (bottom, white overlay, see “Materials and methods”). (C) Anatomical synapse density in the optic tectum was significantly elevated for animals reared in D-serine. [t-test p < 0.05. 2–3 fields per tectal hemisphere were analysed i.e. at least 5 fields per animal from n = 5 D-serine, 3 control animals]. Only colocalized puncta that fit the synapse size criterion of 0.1–5.0 µm2 were included. (D) Experimental design: Tadpoles at stage 42–44 were electroporated, screened for dsRed and PSD95-GFP expression 48 h later at stage 46–47, reared in control, D-serine, or D-serine + MK-801 medium for 48 h and imaged at stage 48. (E) Magnified dendritic arbors of tectal neurons expressing PSD95-GFP + puncta (yellow) in dsRed + cells (magenta). Scale bar: 10 µm. Arrows: examples of synaptic puncta. (F) Quantification of the densities of PSD-95 puncta per volume of dendritic arbor for single tectal neurons reveals that rearing in D-serine increases synapse density, which is prevented when NMDARs are blocked by MK-801. [one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons p < 0.01, p < 0.05. n = 6 control, 10 D-serine, 8 D-serine + MK-801 cells].
	Figure 4 Postsynaptic retinotopic maps in stage 48 animals reared in D-serine from stage 37. (A) Experimental design. mRNA for GCaMP6s was injected into one blastomere at the two-cell stage to generate tadpoles with mosaic GCaMP6s expression restricted to half the animal. Tadpoles were screened at stage 37 for GCaMP6s expression, then reared in control or D-serine for two-photon imaging of calcium fluorescence in the optic tectum at stage 48. Scale bar: 50 µm. (B, C) Representative images showing receptive field center locations (left) and receptive field sharpness (right) of individual tectal cells in response to a bar flashed at each of 5 locations across the (B) azimuth and (C) elevation axes. The maps are color-coded by the optimal stimulus position that evokes a response in each cell. Scale bar: 50 µm. (D, E) Cumulative probability distributions of receptive field sharpness of tectal cell bodies in individual animals (lighter lines) and grouped by treatment (darker lines) for the (D) azimuth [n = 365 cells from 6 animals for D-serine, n = 181 cells from 5 animals for control] and (E) elevation axes [n = 326 cells from 6 animals for D-serine, n = 172 cells from 5 animals for control. Kolmogorov–Smirnov test for pooled values *p < 0.05].