Transplantation of Quail Collagen-tailed Acetylcholinesterase Molecules Onto the Frog Neuromuscular Synapse

The highly organized pattern of acetylcholinesterase (AChE) molecules attached to the basal lamina of the neuromuscular junction (NMJ) suggests the existence of specific binding sites for their precise localization. To test this hypothesis we immunoaffinity purified quail globular and collagen-tailed AChE forms and determined their ability to attach to frog NMJs which had been pretreated with high-salt detergent buffers. The NMJs were visualized by labeling acetylcholine receptors (AChRs) with TRITC-α-bungarotoxin and AChE by indirect immunofluorescence; there was excellent correspondence (>97%) between the distribution of frog AChRs and AChE. Binding of the exogenous quail AChE was determined using a speciesspecific monoclonal antibody. When frog neuromuscular junctions were incubated with the globular G4/G2 quail AChE forms, there was no detectable binding above background levels, whereas when similar preparations were incubated with the collagen-tailed A12 AChE form >80% of the frog synaptic sites were also immunolabeled for quail AChE attached. Binding of the A12 quail AChE was blocked by heparin, yet could not be removed with high salt buffer containing detergent once attached. Similar results were obtained using empty myofiber basal lamina sheaths produced by mechanical or freeze-thaw damage. These experiments show that specific binding sites exist for collagen-tailed AChE molecules on the synaptic basal lamina of the vertebrate NMJ and suggest that these binding sites comprise a “molecular parking lot” in which the AChE molecules can be released, retained, and turned over

Novel Mouse Model Reveals Distinct Activity-Dependent and –Independent Contributions to Synapse Development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ

Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells

Kuhnian revolutions in neuroscience: the role of tool development.

The terms "paradigm" and "paradigm shift" originated in "The Structure of Scientific Revolutions" by Thomas Kuhn. A paradigm can be defined as the generally accepted concepts and practices of a field, and a paradigm shift its replacement in a scientific revolution. A paradigm shift results from a crisis caused by anomalies in a paradigm that reduce its usefulness to a field. Claims of paradigm shifts and revolutions are made frequently in the neurosciences. In this article I will consider neuroscience paradigms, and the claim that new tools and techniques rather than crises have driven paradigm shifts. I will argue that tool development has played a minor role in neuroscience revolutions.The work received no fundin

Degradation of two AChR population at rat neuromuscular junctions: regulation in vivo by electrical stimulation.

The effect of electrical stimulation on the stability of junctional ACh receptors (AChR) on soleus muscles of Wistar rats was compared to that of denervation and reinnervation. Denervation causes the degradation rate of the slowly degrading AChRs (Rs) at the neuromuscular junction to accelerate and be replaced by rapidly degrading AChRs (Rr), while reinnervation restabilizes the accelerated Rs. Electrical stimulation initiated at the time of denervation prevented the acceleration of the Rs. It could not, however, reverse the effect of denervation if initiated after the AChRs became destabilized, nor could it slow the degradation rate of the Rr. We conclude that electrical stimulation of denervated muscle downregulates the expression of the Rr and prevents the destabilization of Rs

Fast to slow transformation of denervated and electrically stimulated rat muscle

Denervated fast extensor digitorum longus (EDL) muscles of adult rats were stimulated electrically for up to 4 months with a ‘slow’ pattern resembling the activity in soleus (Sol) motor units and examined with antibodies against myosin heavy chains (MHCs).The normal EDL contained, on average, 45 % type IIB, 29 % type IIX, 23 % type IIA and 3 % type I fibres. All type IIB and almost all type IIX fibres disappeared during the first 3 weeks of stimulation. They were replaced by type IIA and type I fibres, whose percentages increased to about 75 and 15, respectively. Type IIA fibres remained at 75 % for nearly 2 months and were then gradually replaced by type I fibres during the next 2 months. The transformation occurred sequentially in the order IIB/IIX → IIA → I, the first step (IIB/IIX → IIA) occurring after a short delay (2 weeks) and the last step (IIA → I in originally IIB or IIX fibres) after a long delay (> 2 months). During the transformation coexpression of MHCs occurred.It appears that the transformation to type I fibres occurred in pre-existing type II fibres since no signs of fibre damage or regeneration were observed.Normal EDL was also stimulated through an intact nerve with the same pattern for up to 37 days. The effects on fibre type distributions were identical to those observed in the denervated EDL. The result indicated that the Sol-like pattern of evoked muscle activity, rather than nerve-derived trophic influences or denervation per se, was primarily responsible for the fast to slow transformation