Abstract
α- and β-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available and targeting all α- and β-neurexins produced by a particular gene is challenging. Using newly generated constitutive and conditional knockout mice that target all neurexin-3α and neurexin-3β isoforms, we found that neurexin-3 was differentially required for distinct synaptic functions in different brain regions. Specifically, we found that, in cultured neurons and acute slices of the hippocampus, extracellular sequences of presynaptic neurexin-3 mediated trans-synaptic regulation of postsynaptic AMPA receptors. In cultured neurons and acute slices of the olfactory bulb, however, intracellular sequences of presynaptic neurexin-3 were selectively required for GABA release. Thus, our data indicate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms.
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Acknowledgements
We thank D. Martinelli, P. Rothwell, L. Chen and R.C. Malenka for advice and technical help. This study was supported by grants from the National Institute of Mental Health (R37MH52804 to T.C.S. and 1K99MH103531to J.A.), the National Institute on Drug Abuse (K99DA034029 to C.F.) and the American Heart Association, Western States (11POST7360078 to J.A.).
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J.A., K.T. and T.C.S. conceived the project. K.T. generated the Nrxn3α/β cKO mice and performed biochemical analyses of protein expression. J.A. designed and performed immunocytochemistry, culture and acute hippocampal slice electrophysiology, and animal behavior experiments. S.M.C.I. performed olfactory bulb immunocytochemistry. J.A. and C.F. performed olfactory bulb acute slice analyses. J.A. and T.C.S. wrote and edited the manuscript.
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Supplementary Figure 1 Protein expression levels are unchanged in constitutive Nrxn3α/β KO brains.
a. Representative Western blots - cropped images for 25 neuronal proteins in brain homogenates harvested from three sets of adult littermate wild-type and Nrxn3a/b KO mice.
b. Quantification of protein concentrations in adult wild-type and Nrxn3α/β KO brains (n=3 mice each).
c. qRT-PCR from mRNA isolated from wild-type, Nrxn3α+/– and Nrxn3α–/– brains and probed for transcripts representing Nrxn3α (left) and Nrxn3β (right). Note that based on the KO strategy (Fig. 1), the 5’ end of the Nrxn3α mRNA is still synthesized, but the resulting RNA is likely to nonsense-mediated decay resulting in a reduced level of mRNA for Nrxn3α. If the low levels of truncated mRNA produced a stable protein despite the presence of a partial C-terminal LNS-domain that is unable to fold due to the truncation, the produced protein would be secreted because it has no membrane anchor, and thus would be non-functional in all assays we have performed (see Fig. 7 this paper, and Aoto et al., 2013; Nrxn3α expression: Nrxn3α/β+/– vs. Nrxn3α/β –/–, p < 0.0001; Nrxn3β expression: Nrxn3α/β+/– vs. Nrxn3α/β –/–, p < 0.0001).
All data are shown as percent of control. Protein concentration levels were determined by phosphorimager analysis using I125 conjugated secondary antibodies. Data are means ± SEM. *, p<0.05 by Student’s t test comparing genotype protein levels and single factor ANOVA compared qRT-PCR.
Supplementary Figure 2 Comparison of Nrxn3α/β cKO and Nrxn3α KO on synaptic properties of cultured hippocampal neurons.
a. Conditional Nrxn3α/β KO does not alter passive membrane properties (left, input resistance; right, membrane capacitance). Hippocampal neurons were cultured from newborn homozygous Nrxn3α/β cKO mice, infected with inactive (control) or active cre-recombinase (cre) at DIV2-4, and analyzed on DIV14-16 (Rm: p = 0.612; Cm: p = 0.646).
b & c. mEPSC and mIPSC kinetics are not changed by the conditional Nrxn3α/β KO. Representative traces (top) and summary graphs (bottom) of mEPSC (b; rise: p = 0.995; decay: p = 0.861) and mIPSC (c; mIPSCs: rise: p = 0.876; decay: p = 0.800) rise and decay kinetics, measured in neurons obtained as in (a).
d. The Nrxn3α/β cKO does not decrease the release probability at excitatory synapses, measured using the stimulus-dependent NMDAR blockade by MK-801 during low-frequency stimulation. Neurons were obtained as described in a (p = 0.710).
e. Constitutive Nrxn3α KO does not alter passive membrane properties (left, input resistance; right, membrane capacitance). (Rm: p = 0.569; Cm: p = 0.670).
f. Constitutive Nrxn3α KO does not alter spontaneous mEPSCs (left, representative traces; right, summary graphs of the mEPSC frequency (p = 0.0676) and amplitude (p = 0.613)..
g-i. Constitutive Nrxn3α KO does not alter evoked AMPAR-mediated (g; p = 0.842) or NMDAR-mediated EPSCs (h; p = 0.767) or the PPR of NMDAR-mediated EPSCs (i; 100ms ISI: p = 0.553). For each panel, representative traces are shown on the left and summary graphs on the right..
Data shown represent means ± SEM of individual experiments; number of cells patched/experiment analyzed are shown in the bars or plots. For (i), mean ± SEM was calculated from number of cells. Statistical significance was assessed by single-factor ANOVA.
Supplementary Figure 3 Morphological quantification of synaptic parameters and GluA1 endocytosis in cultured hippocampal neurons.
a-c, Summary graphs representing the absolute puncta quantification of GluA1, GluA2 VGluT1 and PSD95 density (a; GluA1: p = 0.694; GluA2: p = 0.714; PSD95: p = 0.819; vGluT1: p = 0.758), size (b; GluA1: p = 0.0213; GluA2: p = 0.0424; PSD95: p = 0.188; vGluT1: p = 0.421) and intensity (c; GluA1: p = 0.579; GluA2: p = 0.267; vGluT1: p = 0.796) in control and cre infected Nrxn3α/βcKO neurons.
d, Summary graph representing levels of GluA1 internalization normalized to the control condition (p = 0.0110).
Data shown represent means ± SEM. Data were collected from 5 independent experiments. Statistical significance was assessed by single-factor ANOVA.
Supplementary Figure 4 Representative traces of neurexin rescue experiments.
a & b. Nrxn3α/β cKO neurons were co-infected with inactive (control) or active (cre) cre-recombinase at DIV 4 and with a virus that expressed (a) an endogenously expressed soluble isoform of Nrxn3SS4– (solNrxn3βSS–-) that results from alternative splicing in splice-site 5 and (b) the extracellular domain of neurexin-1βSS4– was fused to the transmembrane and intracellular domains of PDGFR (pDisNrxn1βSS4-). This construct permits trans-synaptic signaling while inhibiting intracellular signaling.
Supplementary Figure 5 Characterization of burst and regular firing subicular neurons.
a & b, Sample traces of intrinsic spiking properties used to identify burst (a) and regular (b) firing subicular projection neurons. Postsynaptic subicular neurons were recorded in whole cell current clamp mode and currents steps were injected.
c & d, Summary graphs representing the passive membrane properties of burst (c; input resistance: p = 0.719; capacitance: p = 0.796) and regular (d; input resistance: p = 0.696; capacitance: p = 0.929) firing cells. Measurements were recorded from uninfected postsynaptic subicular neurons in Nrxn3α/β cKO slices that had been stereotactically injected with AAVs expressing GFP fused to inactive (control) or active (cre) cre-recombinase in the CA1 region of the intermediate hippocampus.
Data shown represent means ± SEM calculated from number of cells. Numbers in bar graphs represent number of cells/independent experiments. Statistical significance was assessed by single-factor ANOVA.
Supplementary Figure 6 Characterization of excitatory synaptic transmission in Nrxn3 mutant olfactory bulb neurons.
a-c, Quantification of immunocytochemistry of Nrxn3α/β cKO olfactory bulb cultures infected with lentiviruses expressing inactive (control) or active (cre) cre-recombinase fused to EGFP. Puncta density (b; gephyrin: p = 0.884; vGAT: p = 0.176; vGluT1: p = 0.465), size (c; gephyrin: p = 0.262; vGAT: p = 0.170; vGluT1: p = 0.645) and intensity (d; gephyrin: p = 0.737; vGAT: p = 0.851; vGluT1: p = 0.693) representing gephyrin, vGAT and vGluT1 immunoreactivity.
d, Summary graphs of the passive membrane properties measured in mitral/tufted cells in Nrxn3α/β cKO olfactory bulb cultures infected with lentiviruses expressing inactive (control) or active (cre) cre-recombinase fused to EGFP (input resistance: p = 0.728; capacitance: p = 0.583).
e, Sample traces (left) and summary graphs representing mEPSC kinetics measured in Nrxn3α/β cKO olfactory bulb cultures infected with lentiviruses expressing inactive (control) or active (cre) cre-recombinase fused to EGFP (mEPSC rise: p = 0.816; decay: p = 0.286).
f, Same as in (d) but passive membrane properties measured in mitral/tufted cells from Nrxn3SS4+ KI olfactory bulb cultures infected with lentiviruses expressing inactive (Nrxn3SS4+) or active (Nrxn3SS4–) cre-recombinase fused to EGFP (input resistance: p = 0.921; capacitance: 0.894).
g, mEPSC kinetics from Nrxn3SS4+ or Nrxn3SS4– primary olfactory bulb cultures (mEPSC rise: p = 0.626; decay: p = 0.693).
Data shown represent means ± SEM calculated from total number of experiments. Numbers in bar graphs represent number of cells/independent experiments. Statistical significance was assessed by single-factor ANOVA.
Supplementary Figure 7 Properties of mIPSCs in cultured olfactory bulb neurons from the three different types Nrxn3 mutant animals.
a. mIPSC cumulative probability plots for frequency (left) and amplitude (right) from Nrxn3α/β cKO mitral/tufted neurons in (p < 0.0001).
b, Representative traces (left) and summary graphs (right) of mIPSC decay and rise kinetics from Nrxn3α/β cKO mitral/tufted neurons in (mIPSC rise: p = 0.462; decay: p = 0.991).
c, Representative traces (left) and summary graph (right) measuring the sucrose-evoked readily releasable pool (RRP) of GABAergic vesicles in Nrxn3α/β cKO olfactory bulb cultures (p = 0.303).
d & f, mIPSC cumulative probability plots as in (a) but from cultured Nrxn3α KO mitral/tufted neurons (d; p < 0.0001) or from cultured Nrxn3 SS#4 KI neurons infected with lentiviruses expressing inactive Cre (Nrxn3SS4+) or active Cre (Nrxn3SS4-) recombinase (f; p = 0.671).
e & g, mIPSC kinetics as in (b) but in Nrxn3α KO mitral/tufted neurons (e; mIPSC rise: p = 0.939; decay: p = 0.943) and Nrxn3SS#4 KI (g; mIPSC rise: p = 0.520; decay: p = 0.713).
h & i, Cartoon schematic of full-length (h) and GPI-anchored (i) Nrxn3α rescue constructs used in Figs. 7d and 7e. Note: GPI-Nrxn3α is incapable of activating intracellular signaling.
Data represent means ± SEM calculated from independent experiments. Numbers in bar graphs represent number of cells/independent experiments. Statistical significance in b, c, e, g was assessed by single-factor ANOVA. Statistical significance for cumulative probability plots was determined by K-S test.
Supplementary Figure 8 Dendrodendritic synapse diagram and confirmation of proper AAV targeting.
a, Cartoon model of a dendrodendritic granule cell-mitral cell synapse. Antidromc stimulation of mitral cell axons in the lateral olfactory tract evoke the dendritic release of glutamate onto a granule cell spine. Ca2+ influx via NMDARs located in the granule cell spine cause the release of GABA onto the mitral cell dendrite.
b, Representative coronal section of a Nrxn3α/β cKO olfactory bulb stereotactically injected with AAVs expressing GFP fused to inactive or active cre-recombinase. Note the complete infection (GFP+) of the granule cell layer.
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Aoto, J., Földy, C., Ilcus, S. et al. Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses. Nat Neurosci 18, 997–1007 (2015). https://doi.org/10.1038/nn.4037
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DOI: https://doi.org/10.1038/nn.4037
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