Syntaxin 1B is important for mouse postnatal survival and proper synaptic function at the mouse neuromuscular junctions

Abstract

STX1 is a major neuronal syntaxin protein located at the plasma membrane of the neuronal tissues. Rodent STX1 has two highly similar paralogs, STX1A and STX1B, that are thought to be functionally redundant. Interestingly, some studies have shown that the distribution patterns of STX1A and STX1B at the central and peripheral nervous systems only partially overlapped, implying that there might be differential functions between these paralogs. In the current study, we generated an STX1B knockout (KO) mouse line and studied the impact of STX1B removal in neurons of several brain regions and the neuromuscular junction (NMJ). We found that either complete removal of STX1B or selective removal of it from forebrain excitatory neurons in mice caused premature death. Autaptic hippocampal and striatal cultures derived from STX1B KO mice still maintained efficient neurotransmission compared with neurons from STX1B wild-type and heterozygous mice. Interestingly, examining high-density cerebellar cultures revealed a decrease in the spontaneous GABAergic transmission frequency, which was most likely due to a lower number of neurons in the STX1B KO cultures, suggesting that STX1B is essential for neuronal survival in vitro. Moreover, our study also demonstrated that although STX1B is dispensable for the formation of the mouse NMJ, it is required to maintain the efficiency of neurotransmission at the nerve-muscle synapse.

Keywords: NMJ; SNARE; evoked release; neurotransmitter release; spontaneous release.

Fig. 1.

Deletion of syntaxin 1B (STX1B) in mice influences their postnatal development. A: cloning strategy for the generation of STX1B knockout (KO) mice. The black arrows on the graph represent forward (F) and reverse (R) primers for the genotyping PCR. Neo, neomycin. B: genotyping PCRs from DNA extracted from animals showed wild-type (WT) and KO bands. M, marker. C: growth curve of STX1B WT, heterozygous (Het), and KO mice showing that STX1B KO mice were lighter than the control mice from postnatal day 5 (P5) on and stopped gaining weight after P8. n, Number of animals monitored for the experiment. D: survival curve showing that all monitored STX1B KO mice succumbed to death before P13. Number of animals monitored for each genotype is indicated in the graph. ***P ≤ 0.001.

Fig. 2.

Deletion of STX1B does not affect the brain gross morphology and the mouse neuromuscular junctions (NMJs). A: Nissl staining of brain slices from STX1B WT and KO mice at P10 suggested that STX1B KO mice were able to form proper brain morphology (n = 3 for each genotype). Scale bar, 1 mm. B: immunohistochemistry images of the hippocampus and cerebellum in the brain slices from STX1B WT and KO mice with antibodies recognizing STX1B and axonal marker Tau. Brain slices were prepared from animals at P10. Scale bar, 100 μm. C: micrographs of the NMJs from transversus abdominis (TVA) muscles from STX1B WT and KO mice at P8. Antibody recognizing STX1B identified the presynaptic terminals, whereas α-bungarotoxin conjugated to rhodamine (BTX-Rho) was used to label the postsynaptic ACh receptors. Scale bar, 10 μm.

Fig. 3.

Removal of STX1B in mice results in reduction of Munc18-1. A: Western blot analysis from the whole brain lysates from P10 to P11 animals showed the expression levels of STX1B, STX1A, synaptosomal-associated protein 25 (SNAP-25), synaptobrevin-2 (SYB2), Munc18-1, synaptotagmin-1 (SYT1), synaptophysin-1 (SYN1), Munc13-1, Rab5, and β-tubulin III (b-tub III). β-Tubulin III was used as an internal control. B: quantification of the protein expression levels from the Western blot (n = 3 for all of the proteins analyzed). The value of each protein was normalized (Norm.) to that of β-tubulin III in individual genotype in individual blot and then normalized to the WT ratio. Bar graphs show means ± SE. *P ≤ 0.05; ***P ≤ 0.001.

Fig. 4.

Hippocampal excitatory neurons and striatal inhibitory neurons show normal synaptic transmission in the absence of STX1B. A: sample traces of excitatory postsynaptic current (EPSC) from STX1B WT (black), Het (light gray), and KO (dark gray) neurons after a 2-ms depolarization. B: plot of average EPSC amplitudes (amp.) in STX1B WT, Het, and KO autaptic hippocampal excitatory neurons. C: sample traces of responses from STX1B WT (black), Het (light gray), and KO (dark gray) hippocampal excitatory neurons during 500 mM sucrose application for 5 s. D: plots of average readily releasable pool (RRP) size and vesicular release probability (P_vr) in STX1B WT, Het, and KO autaptic hippocampal excitatory neurons. E: sample traces of mEPSC from STX1B WT (black), Het (light gray), and KO (dark gray) neurons. F: plots of mean mEPSC amplitudes and frequencies are presented. G: sample traces of inhibitory postsynaptic current (IPSC) from STX1B WT (black), Het (light gray), and KO (dark gray) neurons after a 2-ms depolarization. H: average IPSC amplitudes in STX1B WT, Het, and KO autaptic striatal inhibitory neurons are shown. I: sample traces of responses from STX1B WT (black), Het (light gray), and KO (dark gray) striatal inhibitory neurons during 500 mM sucrose application for 5 s. J: plots of average RRP size and P_vr in autaptic striatal inhibitory neurons from STX1B WT, Het, and KO cultures. K: sample traces of mIPSC from STX1B WT (black), Het (light gray), and KO (dark gray) neurons. L: mean mIPSC amplitudes and frequencies are presented. All bar graphs are presented with means ± SE. The total number of neurons examined in each genotype is indicated in the graphs.

Fig. 5.

Deletion of STX1B from mice causes a reduction of GABAergic transmission in high-density dissociated cerebellar cultures. A: sample traces of mEPSC input on STX1B WT (black), Het (light gray), and KO (dark gray) neurons. B: plots showing mean mEPSC amplitudes and frequencies in high-density cerebellar cultures. C: sample traces of mIPSC input on STX1B WT (black), Het (light gray), and KO (dark gray) neurons. D: plots showing average mIPSC amplitudes and frequencies in high-density cerebellar cultures. Bar graphs in B and D show means ± SE. *P ≤ 0.05; ***P ≤ 0.001. The number of neurons examined in each condition is indicated in the graphs. E: summary plot of the fraction of RRP and its corresponding peak release rate. Data were collected from the 3 genotypes (STX1B WT: black, n = 22; STX1B Het: light gray, n = 17; STX1B KO: dark gray, n = 22) at 500 mM (circle), 250 mM (square), and 150 mM (triangle) sucrose applications. All sucrose responses were normalized to the RRP size (500 mM sucrose response) to determine the fraction of the pool released. F: example images of cerebellar cultures from STX1B WT, Het, and KO cultures. Antibodies recognizing NeuN (blue), vesicular glutamate transporter 1 (VGlut1; green), and vesicular GABA transporter (VGAT; red) were used to quantify the neuronal number and the density of excitatory and inhibitory synapses, respectively. Scale bar, 20 μm. G: bar graphs with means ± SE illustrating neuronal number and the mean density of VGlut1 and VGAT puncta per 0.15 mm². Neuronal number obtained from each genotype was normalized to that from the STX1B WT cultures (STX1B WT: n = 34 images; STX1B Het: n = 35 images; STX1B KO: n = 36 images; with 4 cultures). Densities of VGlut1 puncta and VGAT were obtained from 47 images for STX1B WT, 47 images for STX1B Het, and 48 images for STX1B KO (with 5 cultures). **P ≤ 0.01.

Fig. 6.

STX1B is important to support a normal neurotransmission at the mouse NMJs. A: frequencies of mean miniature end-plate potentials (mEPPs freq.) from STX1B WT (white), Het (light gray), and KO (dark gray). B: cumulative fraction of mEPP amplitudes in STX1B WT (black), Het (light gray), and KO (dark gray). C: bar graph showing amplitudes of mean mEPPs in STX1B WT, Het, and KO NMJs. D: representative EPP traces from STX1B WT (black), Het (light gray), and KO (dark gray) NMJs. E and F: mean EPP amplitudes (E) and quantal content (F) among the 3 genotypes. G: bar graph showing mean input resistance (R_in) of the muscle fibers from STX1B WT (white), Het (light gray), and KO (dark gray) mice. H: amplitude-normalized EPP traces in STX1B WT (black), Het (light gray), and KO (dark gray) fibers. I and J: decay time constant (τ) of EPPs and mEPPs, respectively, in STX1B WT (white), Het (light gray), and KO (dark gray) muscle fibers. All bar graphs show means ± SE. The number of muscle fibers examined in each genotype is indicated in the graph. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 7.

Motor nerve terminals in STX1B KO mice present more short-term depression than those in the control mice. A: sample traces showing EPP responses at the 1st 7 stimuli and at the last 5 stimuli of a 20-Hz train (5 s) in STX1B WT (black) and KO (dark gray) NMJs. B and C: mean and normalized, respectively, quantal content during the train of stimulation in STX1B WT (black), Het (light gray), and KO (dark gray). D: bar graphs showing the release ratio (determined by the average responses of the last 50 stimuli divided by the 1st response) from the 3 genotypes. E: z-stack projections of motor nerve terminals from TVA muscle from STX1B KO mice stained with BTX-Rho (red) and anti-STX1A antibody (white). Scale bar, 10 μm. *P ≤ 0.05; ***P ≤ 0.001.

Fig. 8.

Deletion of STX1B from the mouse forebrain excitatory neurons also results in premature death and hypersynchronous brain activity. A: cloning strategy for the generation of mice carrying floxed (FL) STX1B alleles (STX1B^FL/FL). B: Western blot analysis from the whole brain lysates from mice at P15. The protein expression level of STX1B was reduced in the STX1B^FL/FL;CamKIIα^Cre/+ brain lysate compared with the STX1B^FL/FL;CamKIIα^+/+ brain lysate. β-Tubulin III was used as an internal control. C: growth curve of control and STX1B^FL/FL;CamKIIα^Cre/+ mice showing that STX1B^FL/FL;CamKIIα^Cre/+ mice stopped gaining weight from P10 on. n, Number of animals monitored. D: survival curve showing that all monitored STX1B^FL/FL;CamKIIα^Cre/+ mice succumbed to death before P20. During the observation period, 1 control pup (STX1B^+/+;CamKIIα^Cre/+) also died at P1. Number of animals monitored for each genotype was indicated in the graph. ***P ≤ 0.001. E–G: examples of cortical EEG recorded in freely moving conditional KO and control mice. Control mice (n = 6) exhibited normal EEG (E), whereas STX1B^FL/FL;CamKIIα^Cre/+ mice (n = 6) displayed a hypersynchronous EEG with interictal (F) and ictal (G) activity. Representative traces are continuous recordings from an EEG channel taken from prolonged monitoring of each mouse. The ictal pattern was characterized by bursts of spike-wave complexes. During 2-h recording, this mouse exhibited 8 episodes of such bursts, each of which lasted between 28 and 45 s, and multiple interictal spikes. The boxed regions in traces on the left are amplified on the right (E and F) or top (G). G and H: power spectrum of the recording epochs shown in E control mouse, black line (H), and conditional KO mouse, dark gray line (G). The control spectrum is scaled by the ratio of conditional KO-to-control maximal power. Note the power peak in the conditional KO at ∼15 Hz.