Key pointsr Neuromuscular junctions from β2-laminin-deficient mice exhibit lower levels of calcium sensitivity.r Loss of β2-laminin leads to a failure in switching from N-to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation.r The motor nerve terminals from β2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature.r There is decreased co-localisation of presynaptic specialisations in β2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression.r These findings support the idea that β2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse.Abstract β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N-to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N-to P/Q-type VGCC-mediated synaptic transmission. N. A. Lavidis and P. G. Noakes contributed equally to this work.

Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis.

Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.

Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca 2+ (Ca v 2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Ca v 2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca 2+ influx in synaptosomes with an EC 50 of 50 pM. Both these effects were prevented using selective Ca v 2.2 channel blockers suggesting the functional involvement of Ca v 2.2 channels in mediating glutamate release in this system. We further show that both Ca v 2.1 (P/Q-type) and Ca v 2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Ca v 2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Ca v 2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Ca v 2.2-expressing mammalian neurons.