Loss of laminin‐a4 results in pre‐ and postsynaptic modifications at the neuromuscular junction

Abstract

Synaptic basal lamina such as laminin-421 (α4β2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (4 mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in 4 and wild-type mice. 4 NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. 4 NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of 4 NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP‐43 Q331K transgenic mouse model of amyotrophic lateral sclerosis

“…The peroneal nerve of extensor digitorum longus muscle was then sucked carefully into a glass stimulating pipette that was filled with Tyrode's solution. The nerve was stimulated with a 0.1‐ms, 10‐ to 20‐V square wave pulse at a frequency of 0.5 Hz using a Grass Instruments stimulator (SD48; Natus Neurology, Warwick, RI, USA) (35, 37).…”

“…We investigated the facilitation of NMJs by applying twin pulses at a 10‐ms delay in 0.3 mM [Ca 2+ ]/1.0 mM [Mg 2+ ] and 2.0 mM [Ca 2+ ]/1.0 mM [Mg 2+ ] Tyrode's solutions. The degree of facilitation was evaluated by using the facilitation index ( fi ): fi = A 2 /Α 1 , where A 2 is the average of the EPP amplitudes elicited by the second stimulus, and A 1 is the average of the EPP amplitudes elicited by the first stimulus (37). A minimum of 3 mice—14–18 NMJs in total per genotype—were studied at all age groups under low extracellular [Ca 2+ ] conditions.…”

“…Vesicles and motor axons that were targeted with mouse anti‐SV2 and rabbit anti‐neurofilament, respectively, were detected with Alexa Fluor 488–conjugated secondary Abs, whereas postsynaptic AChRs were labeled with Alexa Fluor 555 α‐bungarotoxin. These stained NMJs were reconstructed into three dimensions (3D) utilizing a surface‐rendering algorithm that was based on local contrast in fluorescent z‐stacks as previously described (37, 40, 41). Using this approach, we were able to investigate changes in the expansion of SV2 volume—the size of the total vesicle pool—in relation to the postsynaptic AChRs, rather than the standard 2‐dimensional colocalization approach (37, 42).…”

“…These stained NMJs were reconstructed into three dimensions (3D) utilizing a surface‐rendering algorithm that was based on local contrast in fluorescent z‐stacks as previously described (37, 40, 41). Using this approach, we were able to investigate changes in the expansion of SV2 volume—the size of the total vesicle pool—in relation to the postsynaptic AChRs, rather than the standard 2‐dimensional colocalization approach (37, 42). To solely quantify the volume of SV2 staining at these 3D‐rendered NMJs, neurofilament‐positive motor axons were carefully and accurately cut off from the reconstructed SV2‐labeled vesicles.…”

“…We subsequently undertook an examination of SV2 volume relative to the postsynaptic end plate (SV2/AChR volume ratio) by dividing the volume of 3D‐reconstructed, SV2‐stained synaptic vesicles over the volume of 3D‐reconstructed postsynaptic end plates. The postsynaptic NMJ (end plate) volume was defined as the entire 3D‐reconstructed endplate, including interstitial spaces, as we have previously reported (37, 42).…”