Calcium, protease activation, and cytoskeleton remodeling underlie growth cone formation and neuronal regeneration.

The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca2+]i) to 200-300 microM induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca2+]i and of intraaxonal proteolytic activity, we found that the elevated [Ca2+]i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca2+]i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.

Depolarization affects the binding properties of muscarinic acetylcholine receptors and their interaction with proteins of the exocytic apparatus.

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca(2+) channels open, level of intracellular Ca(2+) increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K(d) of approximately 20 nM) and they shift to a low affinity state (K(d) of tens of microM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca(2+) influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca(2+) influx, enables release of ACh to commence.

Voltage-dependent interaction between the muscarinic ACh receptor and proteins of the exocytic machinery.

1. Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage dependent. 2. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the soluble NSF-attachment protein receptor (SNARE) complex. 3. Using rat synaptosomes we show that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP) and synaptotagmin as shown using both cross-linking and immunoprecipitation. 4. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. 5. This voltage-dependent interaction of mAChRs with the secretory core complex appears suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals.

alpha-latrotoxin is a potent inducer of neurotransmitter release in Torpedo electric organ--functional and morphological characterization.

In this report we show that alpha-latrotoxin from black widow spider venom is a potent activator of neurotransmitter release in synaptosomes from the Torpedo electric organ. Binding of the purified toxin (5 nM) to the synaptosomal fraction occurs already at 4 degrees C and is dependent on the presence of divalent ions. However, neurotransmitter release commences only after temperature elevation (22 degrees C) and is completed within 2 min. The effect of alpha-latrotoxin on release is achieved at 1 nM and is already saturated at 5 nM. The release is stimulated by the presence of Ca2+ ions. Activation of release by alpha-latrotoxin is accompanied by morphological changes in electric organ synaptosomes. The synaptosomes swell, resulting in a 55% increase in section area. Moreover, the number of synaptic vesicles per unit area decreases about three-fold, and rows of docked synaptic vesicles are rarely detected as opposed to control synaptosomes. These morphological changes indicate that the massive release is mainly due to synaptic vesicle fusion. alpha-Latrotoxin binding sites are highly concentrated in the innervated face of the electrocytes. Immunoelectron microscopy on electric organ sections reveals alpha-latrotoxin binding sites over the entire plasma membrane at release sites and facing Schwann cells surrounding Torpedo nerve terminals. Surprisingly, a high concentration of binding sites is also found at structures surrounding branching unmyelinated axons. This staining is in close proximity to Schwann cell envelopes and to the basal lamina around axonal tips. The mode of action of alpha-latrotoxin in view of the localization of its binding sites is discussed.

The effect of calcium levels on synaptic proteins. A study on VAT-1 from Torpedo.

In this study we compare major synaptic proteins from Torpedo electric organ to their homologues from mammalian brain. Most of these proteins are members of small gene families. We demonstrate a high degree of evolutionary conservation of most synaptic proteins. However, in the electric organ each gene family is represented only by a single member. We focus on VAT-1, a major protein of the vesicle membrane in Torpedo. VAT-1 is located on the synaptic vesicle membrane and is highly concentrated on the plasma membrane following the application of alpha-latrotoxin. Taking advantage of the relative simplicity of Torpedo synapses, we performed an in vitro study on the properties of VAT-1 affected by changes in Ca2+ levels. VAT-1 is a low affinity Ca2+ binding protein whose ability to bind Ca2+ resides mainly, but not entirely, on the carboxy-terminal domain of the protein. In the presence of Ca2+, the protein is organized in a high molecular mass complex, which is destabilized by depleting Ca2+. This effect occurs only by chelating Ca2+ ions, but not with other divalent ions. VAT-1 is not complexed to any of the proteins which were implicated in the docking/fusion complex such as VAMP, synaptophysin or syntaxin, regardless of Ca2+ levels. Dependence of the stability of protein complexes on Ca2+ levels is also demonstrated on Torpedo n-Sec1. The possible physiological implications of such Ca2+ dependence are discussed.