Synapse elimination and learning rules co-regulated by MHC class I H2-Db


The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-Db is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-Kband H2-Db (KbDb−/−), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-Db in KbDb−/− mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in KbDb−/− mice. This change is due to an increase in Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-Db to KbDb−/− neurons renders AMPA receptors Ca2+ impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-Db in functional and structural synapse pruning in CNS neurons.

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Figure 1: Failure of retinogeniculate synapse elimination despite intact retinal waves in KbDb−/−.
Figure 2: H2-Db expression in neurons rescues synapse elimination and eye-specific segregation in KbDb−/− LGN.
Figure 3: Impaired LTD but intact LTP at retinogeniculate synapses in KbDb−/− induced with natural activity patterns.
Figure 4: Increased Ca2+-permeable AMPA receptors at retinogeniculate synapses in KbDb−/− LGN.
Figure 5: Neuronal expression of H2-Db restores Ca2+-impermeable AMPA receptors and rescues LTD.


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We thank members of the Shatz laboratory for helpful comments. For technical assistance, we thank N. Sotelo-Kury, C. Chechelski and P. Kemper. For training in retinogeniculate slice methods, we thank C. Chen, P. Kanold and D. Butts. This work was supported by NIH grants R01 MH071666 and EY02858, and the G. Harold and Leila Y. Mathers Charitable Foundation (C.J.S.); NIH grant RO1 EY13528 (M.B.F.); NDSEG and NSF Graduate Research Fellowships (J.D.A.); and an NSF Graduate Research Fellowship (L.A.K.).

Author information




H.L. and C.J.S. designed all experiments, analysed and reviewed all results and wrote manuscript. Data contributions are as follows: electrophysiology experiment by H.L.; multi-electrode array experiments by L.A.K. and M.B.F. B.K.B. designed H2-Db monoclonal antibody and performed western blots. H.L. designed and performed RT–PCR experiments. A.D. performed RGC neuronal tract tracing experiments and analysis. J.D.A. and S.C. performed Taqman qPCR.

Corresponding author

Correspondence to Carla J. Shatz.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Comparison of retinogeniculate synaptic responses in wild type versus KbDb−/−.

a, b, Examples of minimal stimulation for wild type (open circles) and KbDb−/− (filled circles). Plot of EPSC peak versus number of stimulations (grey box represents failures, >50%). c, No difference in onset latency of SF-AMPA between all genotypes. Onset latency of SF-AMPA was estimated using minimal stimulation as time (ms) to reach 10% of peak IAMPA from stimulation artefact (wild type: 3.0 ± 0.3 (n = 12 cells/N = 6 animals); KbDb−/−: 2.7 ± 0.1 (n = 23/N = 8); KbDb−/−;NSEDb+ : 3.0 ± 0.2 (n = 17/N = 7); KbDb−/−;NSEDb: 2.6 ± 0.2 (n = 19/N = 5); P > 0.5, t-test). d, Cumulative probability histogram shows no difference in Max-AMPA between wild type and KbDb−/−. Inset: mean ± s.e.m. Wild type: 2.6 ± 0.4 nA (n = 14/N = 6); KbDb−/−: 2.9 ± 0.4 nA (n = 22/N = 8); P > 0.1, Mann–Whitney U-test. eh, Presynaptic release probability at KbDb−/− retinogeniculate synapses is similar to wild type at P20–24. e, f, Examples of EPSCs evoked by paired-pulse stimulation of optic tract (20 Hz) recorded in whole-cell mode in individual LGN neurons from wild type (e) versus KbDb−/− (f). g, h, Paired-pulse depression (PPD) (%) (EPSC 2/EPSC 1) over varying intervals. g, Wild type (open circle) versus KbDb−/− (filled circle) without cyclothiazide (CTZ), a blocker of AMPA receptor desensitization. Wild type versus KbDb−/−: 1 Hz: 82.3 ± 2.6 (n = 10) versus 77.4 ± 3.4 (n = 8); 10 Hz: 44.9 ± 2.3 (n = 9) versus 42.8 ± 3.9 (n = 9); 20 Hz: 37. 1 ± 2.1 (n = 10) versus 37.3 ± 2.8 (n = 9) (P > 0.1 for each). h, Wild type (open circle) versus KbDb−/− (filled circle) with CTZ (20 μM). Wild type versus KbDb−/−: 1 Hz: 79.0 ± 2.4 (n = 9) versus 81.8 ± 1.1 (n = 7); 10 Hz: 59.2 ± 3.0 (n = 8) versus 57.4 ± 3.7 (n = 7); 20 Hz: 58.5 ± 2.3 (n = 8) versus 56.6 ± 4.1 (n = 7) (P > 0.1 for each). N = 4 for wild type; N = 3 for KbDb−/− for g, h. There was no significant difference in PPD between wild type and KbDb−/−, but note significant decrease of PPD +20 μM CTZ versus 0 μM CTZ application for both wild type and KbDb−/− at 10 Hz and 20 Hz (P < 0.05). t-test. mean ± s.e.m. n = cells/N = animals.

Extended Data Figure 2 Intact spatio-temporal pattern of retinal waves in KbDb−/− mice at P5–12.

a, Correlation indices as a function of inter-electrode distance for all cell pairs for wild type (grey) and KbDb−/− (black) at P5–P8. Data points correspond to mean values of medians from individual data sets and error bars represent s.e.m. be, Summary of temporal firing patterns for retinas isolated from wild-type (open squares) and KbDb−/− (filled squares) mice at P5–P8 (stage II, cholinergic waves) and P10–P12 (stage III, glutamatergic waves). Open circles correspond to the mean values of individual retinas. b, Interburst interval for stage II (in min): wild type: 1.9 ± 0.2; KbDb−/−: 1.6 ± 0.2; stage III: wild type: 1.0 ± 0.3; KbDb−/−: 0.9 ± 0.1. c, Firing rate during burst for stage II (in Hz): wild type: 8.6 ± 1.9; KbDb−/−: 8.4 ± 1.7; stage III: wild type: 19.7 ± 2.1; KbDb−/−: 18.6 ± 2.2. d, Burst duration for stage II (in seconds): wild type: 4.2 ± 0.2; KbDb−/−: 3.8 ± 0.1; stage III: wild type: 2.6 ± 0.6; KbDb−/−: 2.1 ± 0.2. e, Mean firing rate for stage II (in Hz): wild type: 0.5 ± 0.1; KbDb−/−: 0.5 ± 0.2; stage III: wild type: 1.0 ± 0.1; KbDb−/−: 0.8 ± 0.1, mean ± s.e.m. (P > 0.05 for each, t-test, N = 6 animals for each group, except when N = 5 for stage III wild type; non-blind experiments).

Extended Data Figure 3 Rescue of H2-Db expression in brain of KbDb−/−;NSEDb+ mice.

a, Diagram of breeding strategy to generate KbDb−/−;NSEDb+ mice. KbDb−/− (white indicates absence of H2-Db) were crossed to NSEDb transgenic mice (black indicates presence of H2-Db in both body and brain). From F1 offspring, H2-Kb+/−H2-Db+/−;NSEDb+ mice (grey body, black brain) were selected and crossed to KbDb−/− mice further, generating KbDb−/−;NSEDb+ (black brain with white body indicates rescue of H2-Db expression in brain alone) and KbDb−/−;NSEDb littermate controls (white body, white brain). b, H2-Db-specific primers. Solid arrows: forward (exon 2) and reverse (exon 3) for 200 bp spliced mRNA as well as unspliced proRNA (500 bp) (e; exon, i; intron). Dotted arrows: NSEDb -A (forward, NSE promoter region) and NSEDb -B1 and/or B2 (reverse, exon 2) for genotyping and mRNA detection20. c, RT–PCR showing rescue at P10 in thalamus of KbDb−/−;NSEDb+ mice cDNAs. Wild-type thalamus (WT(T)) shown as positive control; KbDb−/− thalamus (KbDb−/− (T)) and spleen (KbDb−/− (S)) as negative controls, and various organs from KbDb−/−;NSEDb+ mice (spleen (S), liver (L), gut (G), thalamus (T), hippocampus (H), cortex (C), retina (R)) were used as templates. d, Quantitative PCR comparing relative H2-Kb/H2-Db gene expression in wild type, KbDb−/− and KbDb−/−;NSEDb+ thalami. Left: results show small but highly significant rescue of H2-Db mRNA expression in KbDb−/−;NSEDb+ (KbDb−/−: 0.00003 ± 0.00002; KbDb−/−;NSEDb+ : 0.0035 ± 0.00044 relative to wild type: 1.0056 ± 0.032, ***P = 0.0001). Each point represents average relative gene expression for one mouse. Right: raw H2-Kb/H2-Db Ct values for each genotype (wild type: 27.4 ± 0.1; KbDb−/−: 44.3 ± 0.4; KbDb−/−;NSEDb+ : 35.9 ± 0.3; ***P < 0.001), one-way ANOVA, mean ± s.e.m. N = 5 animals for wild type, 7 for KbDb−/−, 10 for KbDb−/−;NSEDb+ . e, Rescue of H2-Db protein in KbDb−/−;NSEDb+brains at P60. Western blot of immunoprecipitation from whole brain (three pooled brains) lysate from KbDb−/− and KbDb−/−;NSEDb+ . H2-Db-specific signal from KbDb−/−;NSEDb+ appears below IgG band.

Extended Data Figure 4 Cumulative probability distribution for SF-AMPA and Max-AMPA recorded at retinogeniculate synapses according to H2-Db genotype.

a, b, Max-AMPA (a) and SF-AMPA (b) showed similar cumulative probability histograms between wild type (black line), KbDb−/−;NSEDb+ (dashed grey line), KbDb−/− (dashed black line) and KbDb−/−;NSEDb (grey line). Number of experiments are the same as in the main text, except for Max-AMPA: for KbDb−/−;NSEDb: n = 18 cells/N = 5 animals; for KbDb−/−;NSEDb+ : n = 16/N = 7 (P > 0.05, Mann–Whitney U-test). Fibre fraction calculated from Max-AMPA and SF-AMPA measurements is similar between wild-type and KbDb−/−;NSEDb+ mice (Fig. 2d).

Extended Data Figure 5 Neuronal H2-Db expression in KbDb−/− mice rescues impaired eye-specific axonal segregation at P34.

a, Coronal sections of dLGN of KbDb−/−;NSEDb (top row) and KbDb−/−;NSEDb+ (rescue; bottom row) showing pattern of RGC axonal projections from the two eyes after intraocular tracer injections of CTB AF594 (red channel; contralateral eye injected) or AF488 (green channel; ipsilateral eye injected). Left: thresholded fluorescent images of dLGN at 60% maximum signal intensity (see Fig. 2e). Right: overlap of RGC projections (white pixels) from ipsilateral and contralateral eyes displayed for 20%, 40% and 60% maximal threshold for KbDb−/−;NSEDb (top) and KbDb−/−;NSEDb+ (bottom). Overlap = pixels labelled in both red and green channels. b, Mean percentage dLGN area ± s.e.m. pixel overlap for KbDb−/−;NSEDb (filled squares; N = 3) versus KbDb−/−;NSEDb+ (open triangles; N = 4): 0% threshold: 8.3 ± 1.1 versus 1.2 ± 0.3; 20% threshold: 12.2 ± 1.5 versus 2.9 ± 0.7; 40% threshold: 17.9 ± 1.8 versus 5.3 ± 0.9; 60% threshold: 21.1 ± 1.8 versus 7.2 ± 0.9; 80% threshold: 23.2 ± 1.3 versus 11.1 ± 1.2; 100%: 25.3 ± 1.9 versus 14.0 ± 1.5 (P < 0.05, two-way ANOVA). See Methods and ref. 47.

Extended Data Figure 6 Intact release probability at KbDb−/− retinogeniculate synapses before eye opening.

Paired-pulse stimulation was delivered to the optic tract at 10 Hz, similar to the natural firing frequency of RGCs (Extended Data Fig. 2c), and whole-cell recordings were made from LGN neurons in slices aged between P8–13. Paired-pulse stimulation resulted in synaptic depression, represented as EPSC 2 divided by EPSC 1 (%). In 0 μM CTZ (left panel): wild type: 67.0 ± 2.9 (n = 11/N = 4); KbDb−/−: 64.2 ± 2.8 (n = 7/N = 2). In 20 μM CTZ (right panel): wild type: 48.6 ± 4.9 (n = 8/N = 3); KbDb−/−: 46.6 ± 2.5 (n = 7/N = 2) (P > 0.1 for each, t-test), mean ± s.e.m. 20 mM BAPTA containing Cs+-internal solution was used for this experiment due to prolonged kinetics of EPSCs in KbDb−/−. The identical paired-pulse ratios between wild type and KbDb−/− are consistent with the conclusion that presynaptic release probability is intact at P8–13 retinogeniculate synapses in KbDb−/− mice. (See also Extended Data Fig. 1e–h for similar conclusion at P20–24, after synapse elimination is largely complete.)

Extended Data Figure 7 Normal NMDA/AMPA ratio but increased Ca2+-permeable AMPA receptors at retinogeniculate synapses in KbDb−/− mice.

a, b, NMDA/AMPA ratio is unchanged in KbDb−/− mice. a, NMDA/AMPA ratio (%): peak IAMPA measured at −70 mV (+20 μM SR95531) versus peak INMDA at +40 mV (+20 μM SR95531 + 20 μM DNQX): wild type: 61 ± 6.8 (n = 10/N = 4); KbDb−/−: 70.6 ± 12.1 (n = 7/N = 3) (P > 0.1, t-test) mean ± s.e.m. b, Example recordings from individual neurons for wild type (left) and KbDb−/− (right). APV (100 μM) was added at the end of each experiment to confirm NMDA-mediated synaptic currents. D600 in pipette. c, Example showing effect of NASPM (100 μM bath) on IAMPA: note significant blockade of IAMPA in KbDb−/−. Grey line, before NASPM; black line, after NASPM (5 traces averaged for single cell). SR95531 (20 μM) added to bath for ac. d, Examples for IAMPA normalized to EPSC amplitude at −40 mV. Note reduction in EPSC amplitude at +40 mV in KbDb−/− but not wild type. 100 μM APV + 20 μM SR95531 in bath. Spermine (100 μM) and D600 (100 μM) in pipette. Ages: P8–13. Experimenter was aware of genotype due to obvious differences in time course of EPSCs and effects of NASPM. e, Example western blot (left) and GluR1/GluR2 ratio (right) of P22 thalamus; wild type: 1.0 ± 0.1 (N = 12); KbDb−/−: 1.3 ± 0.1 (N = 13) (P = 0.07). f, Example western blot (left) and GluR1/GluR2 ratio (right) of cultured cortical neurons; wild type: 1.0 ± 0.1 (N = 4); KbDb−/−: 2.3 ± 0.7 (N = 4) (*P = 0.03). Mann–Whitney U-test for e, f, n = cells/N = animals.

Extended Data Figure 8 NASPM-dependent rescue of LTD in KbDb−/− LGN at P8–13.

a, Summary of all 1,100 ms latency experiments: EPSC peak amplitude (% change from baseline) versus time (n = 7/N = 7 for 0 μM NASPM; n = 5/N = 5 for 20 μM NASPM). Grey bar = induction period (Methods). 1 min data binning. b, Average of per cent change (mean ± s.e.m.); KbDb−/− 0 μM NASPM: 105 ± 8.2 (N = 7); KbDb−/− +20 μM NASPM: 72.5 ± 2.2 (N = 5). **P < 0.01, t-test, n = cells/N = animals.

Extended Data Figure 9 Neuronal H2-Db expression decreases Ca2+ permeability of AMPA receptors at retinogeniculate synapses in KbDb−/−;NSEDb+ mice.

a, NASPM blockade of IAMPA is significantly reduced in KbDb−/−;NSEDb+ mice. Example trace of NASPM effect on IAMPA recorded from KbDb−/−;NSEDb (left) or KbDb−/−;NSEDb+ (right) individual LGN neuron. Grey line, before NASPM; black line, after NASPM application (5 traces averaged for single cell); 20 µM SR95531 in bath. b, Internal spermine-dependent block of IAMPA at positive membrane potentials is rescued in KbDb−/−;NSEDb+ LGN neurons. Example recordings for IAMPA normalized to EPSC amplitude at −40 mV from individual neurons. Note reduction in EPSC amplitude at +40 mV in KbDb−/−; NSEDb, but restored to wild-type level in KbDb−/−;NSEDb+ . 100 μM APV + 20 μM SR95531 in bath. Spermine (100 μM) and D600 (100 μM) in internal solution.

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Lee, H., Brott, B., Kirkby, L. et al. Synapse elimination and learning rules co-regulated by MHC class I H2-Db. Nature 509, 195–200 (2014).

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