The precise development and organisation of the neuromuscular junction (NMJ) is critical for the production of efficient neurotransmission signals. Disruption of these specialisations may result in severely altered transmission properties at the NMJ. Signalling and adhesion molecules have long been established to be key mediators in the distribution of synaptic specialisations. The basal lamina acts to maintain stability of the associated tissue and contains a number of these signalling and adhesion molecules. The laminins, a family of large multimeric proteins, are one of the key components of the basal lamina that act as specific cell regulators and provide mechanical stability. Laminins have been shown to form an interconnecting network that assists in stabilisation of the basal lamina and provides attachment sites for other basal lamina components. The synapse specific laminin chains, -α4, -α5, and -β2, play an essential role in differentiation and organisation of the NMJ. These chains form the laminin isoforms, laminin-221 (α2β2γ1), laminin-421 (α4β2γ1), and laminin-521 (α5β2γ1).
Laminin-β2, found in each of the three synapse specific isoforms, has been shown to organise presynaptic specialisations at the developing NMJ. In vitro laminin-β2 is able to interact directly with N-type and P/Q-type voltage-gated calcium channels (VGCCs), suggesting a role in organising VGCCs in close proximity to active zones. During NMJ development and maturation different VGCCs mediate neurotransmission, with N-type VGCCs being dominant during early development but later switching to P/Q-type VGCC mediation as the NMJ matures. Mice deficient in laminin-β2 (lamb2-/-), present severely disrupted neurotransmission properties, and morphological abnormalities including invasion of the synaptic cleft by perisynaptic Schwann cell (PSCs) processes. The poor transmission properties observed at lamb2-/- NMJs may be attributed to the entry of the processes into the cleft, with laminin-521, containing the β2 chain, shown to directly prevent invasion of the synaptic cleft by PSCs. The laminin-α4 chain forms the unique short arm laminin-421 heterotrimer. Targeted mutation of the lama4 gene does not alter formation of active zones and junctional folds, though disruptions in the precise alignment of active zones and postsynaptic folds are seen at NMJs of laminin-α4 deficient mice (lama4-/-). The functional consequences of loss in laminin-α4 had not been previously reported.
This study examined the role of laminin-β2 in the organisation of N- and P/Q-type VGCCs at active zones of developing postnatal day 8 (P8) and matured, postnatal day 18 (P18) NMJs. The contribution of each channel to the release of transmitter was assessed using intracellular electrode recordings of end-plate potentials (EPPs). The VGCC toxins, ω-conotoxin-GVIA and ω-agatoxin-IVA, were used to specifically target N- and P/Q-type VGCCs respectively in order to assess the relative contribution of each subtype to neurotransmission in wild type (WT) and lamb2-/- mice. Utilising live Ca2+ imaging techniques we also assessed the capabilities of PSCs at postnatal day 14 (P14) lamb2-/- NMJs. The present study also investigated the functional consequences of loss in laminin-α4 at both P8 and P18. Utilising electrophysiological recordings we characterised the neurotransmission properties of mutant mice compared to age-matched wild-type littermates. Immunohistochemical analyses were undertaken to support our functional studies. This study found that loss of either laminins-α4 or -β2 resulted in markedly different deficits in neurotransmission. Lamb2-/- NMJs demonstrated decreased calcium sensitivity with no change in calcium dependence compared to wild-type littermates. Mutants did not display the characteristic change in VGCC subtype involved with mediating neurotransmitter release. These mutants maintained dependence on N-type VGCCs with P/Q-type channels playing a minor role, in contrast to age-matched wild-type NMJs which predominantly relied on P/Q-type VGCCs at P18. Immunohistochemical analyses confirmed this finding with lamb2-/- NMJs displaying a significantly higher expression of N-type channels at the presynaptic membrane than wild-type littermates. Laminin-β2 deficient junctions also demonstrate poor maturation of perisynaptic Schwann cells. At lamb2-/- NMJs, PSCs fail to mature and remain dependent on the developmental purinergic signalling receptor (P2YR) rather than switch to the muscarinic signalling receptor (MR) family associated with maturation. The failure to switch signalling receptors resulted in poor decoding of neurotransmission signals, as shown by our functional work on the lamb2-/- soleus preparation at P14. At lama4-/- NMJs, we observed early perturbations in neurotransmission from P8, which became more evident by P18. Lama4-/- NMJs displayed significantly higher levels of synaptic depression under high frequency stimuli and altered paired pulse facilitation compared to wild-type littermates. Analysis of the binomial parameters of neurotransmitter release demonstrated a decrease in quantal release as a result of a decrease in the number of active release sites, but not in the average probability of transmitter release from these sites. Our functional findings suggest alterations in the short-term plasticity of the NMJ and possibly defective recycling of synaptic vesicles and/or the calcium handling at lama4-/- NMJs. We propose that alterations to synapsin I and its associated molecules may be partly responsible for the changes in neurotransmission observed at lama4-/- NMJs. Our findings demonstrate altered distribution and expression of presynaptic components associated with active zones, specifically an increase in the number of synaptic vesicles and a decrease in the density of the fluorescently labelled Bassoon at the active zone region. Our results suggest that the fewer active release sites may compensate for the deficits of the lama4-/- NMJs by alterations to pre- and postsynaptic specialisations.
In conclusion, this thesis has identified that laminins-α4 and -β2 play fundamental roles in establishing efficient neurotransmission properties at the NMJ. Results suggest that laminin-β2 is a key regulator in development of the neuromuscular junction, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure in the developmental switch from N- to P/Q-type VGCCs in mediation of transmitter release. Associated with this failed switch in VGCCs, we note a failure in the developmental expression and clustering of presynaptic N- and P/Q type VGCCs and associated presynaptic molecules at lamb2-/- NMJs. We are also the first to characterise the functional consequences of the loss of laminin-α4, finding subtle changes in neurotransmission properties at mutant NMJs. We suggest laminin-α4 plays a role in organisation of the NMJ during development, with its loss resulting in altered neurotransmission properties that do not significantly compromise the survival of the animal.