Neuroscience

Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem

He-Hai Jiang
Ruoxuan Xu
Xiupeng Nie
Zhenghui Su
Xiaoshan Xu
Ruiqi Pang
Yi Zhou
Fujun Luo author has email address

Guangzhou National Laboratory, 510005, Guangzhou, Guangdong, China
Bioland Laboratory, 510005, Guangzhou, Guangdong, China
School of Basic Medical Sciences, Guangzhou Medical University, 510005, Guangzhou, Guangdong, China
Department of Neurobiology, School of Basic Medicine, Army Medical University, 400038, Chongqing, China
Advanced Institute for Brain and Intelligence, School of Medicine, Guangxi University, 530004, Nanning, China

https://doi.org/10.7554/eLife.94315.1

Open access
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Figures and data

Neurexins are highly expressed in MNTB neurons.
(a) Immunohistochemistry and RNAscope FISH showing expression of various isoforms of neurexins in the MNTB, which make glycinergic synapses with LSO neurons (b) Summary of expression of various isoforms of neurexins in the MNTB of WT mice. (c) Diagram of auditory brainstem and virus injection in Nrxn123 cTKO mice. (d) Representative images of Nrxn3 FISH in the MNTB of mice injected with AAV-EGFP or AAV-Cre-EGFP. Scale bar: 50 μm. (e) Selected ROI in high magnification showing Nrxn3 FISH in MNTB neurons expressing EGFP or Cre-EGFP. Scale bar: 10 μm. (f) Summary of expression of various neurexins in the MNTB of Nrxn123 cTKO mice injected with AAV-EGFP and AAV-Cre-EGFP. Data are means ± SEM. Number of sections analyzed are indicated in the bars (b, f); Statistical differences were assessed by Student’s t test. (**P < 0.01, ***P < 0.001). Source data are provided as a Source Data file.

Deletion of neurexins reduces the strength and kinetics of glycinergic synapse. (a) Representative IPSC traces of LSO neurons evoked by optogenetic stimulation of ChR2 expressed in MNTB neurons. (b-d) Summary of IPSC amplitude, rise time, and decay time constant. P = 0.0119, P = 0.0004, P = 0.002. unpaired two-sided t-test. (e-h) Same as a-d, except that IPSCs were evoked by afferent fiber stimulation. (i) Representative IPSCs traces of LSO neurons evoked by a pair of fiber stimulation at 50 ms interval. (j) Summary of paired-pulse ratio (PPR) of IPSCs in relationship to different paired pulse intervals. P < 0.0001, two-way ANOVA. Data are means ± SEM. Number of neuron are indicated in the bars (b-d, f-h) or graph (j). Statistical differences were assessed by Student’s t test or two-way ANOVA test. (*P < 0.05, **P < 0.01, ***P < 0.001).

Deletion of neurexins increases sIPSC frequency and glycinergic synapse density of LSO neurons. (a) Representative sIPSC traces of LSO neurons. (b) Summary of sIPSC amplitude and frequency. P = 0.057, P = 0.015, unpaired two-sided t-test. (c) Representative sIPSC waveforms of LSO neurons. (d) Summary of sIPSC rise time and decay time constant P = 0.376, P = 0.82, unpaired two-sided t-test. (e) Representative confocal microscopy images of MNTB-LSO-containing brainstem slice with specific labeling of GlyT2 (red) and EGFP (green) from both control and Nrxn123 TKO mice at P14. Scale bar, 10 µm. (f) Summary of GlyT2 immunostaining intensity (normalized to control). P = 2.49E-07, unpaired two-sided t-test. (g-h), Same as (e-f) except for specific labeling of VGAT (red) and EGFP (green). P = 0.002, unpaired two-sided t-test. Data are means ± SEM. Number of neuron or sections/animals for immunostaining are indicated in the bars (b, d, f and h). Statistical differences were assessed by Student’s t test. (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are provided as a Source Data file.

Deletion of neurexins increases the Ca²⁺ sensitivity of release at the glycinergic synapse. (a) Representative time courses of IPSC amplitudes for individual LSO neurons of control mice during the perfusion of low Ca²⁺ ACSF. (b) Representative IPSC traces of control mice before and during low Ca²⁺ perfusion at the indicated points in a. (c & d) Same as a & b, except of TKO mice. (e) Summary of normalized IPSC amplitudes plotted against the calculated extracellular Ca²⁺ concentration for control and TKO mice. P = 0.0311 for 0.92 mM Ca²⁺, P = 0.0104 for 1.10 mM Ca²⁺, P = 0.0208 for 1.28 mM Ca²⁺, P = 0.0067 for 1.46 mM Ca²⁺, P = 0.0007 for 1.64 mm Ca²⁺, unpaired two-sided t-test. (f) Summary of IC₅₀ of Ca²⁺ for half-blocking of IPSCs and the blocking effect of IPSCs by 0.2 mM Ca²⁺. P = 0.0139, P = 0.3985, unpaired two-sided t-test. Data are means ± SEM. Number of neuron are indicated in the graph (e) or bars (f). Statistical differences were assessed by Student’s t test. (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are provided as a Source Data file.

Deletion of neurexins increases the blocking effect of EGTA on glycinergic IPSCs. (a) Representative time courses of IPSC amplitudes for individual LSO neurons of control mice during the treatment of high concentration of EGTA-AM. (b) Representative IPSC traces of control mice before and after EGTA. (c, d) Same as a, b, except of TKO mice. (e) Summary of IPSC amplitude before and after EGTA treatment of control mice. P = 0.0201, paired two-sided t-test. (f) Summary of IPSC amplitude before and after EGTA treatment of TKO mice. P = 0.0123, paired two-sided t-test. (g) Summary of the blocking percentage of IPSCs by EGTA. P = 0.0001, unpaired two-sided t-test. Data are means ± SEM. Number of neuron are indicated in the bars (e to g). Statistical differences were assessed by Student’s t test. (*P < 0.05, ***P < 0.001). Source data are provided as a Source Data file.

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