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Mechanotransduction is a process fundamental to life. It under-pins a variety of sensory modalities from hearing to blood pressure regulation. However, the molecular components of the mechanosensory mechanisms in primary sensory endings are poorly understood. Experimental approaches to solving this problem are long and laborious. Therefore, a theoretical approach was proposed as an efficient means to circumventing this process. A mathematical, biophysical model of mechanosensory endings was implemented, which reproduced existing experimental data of the receptor potential of the mammalian muscle spindle primary ending. This probabilistic model combines mathematical representations of different ion channel types to produce an output which is the predicted receptor potential of the sensory ending, given the presence of specific ion channels. The model outputs the tension-dependent electrical response of the receptor, given a stretch stimulus. The parameters required for this model identify the necessary molecular entities required for this behaviour to occur. The dbd (dorsal bipolar dendritic) neuron in D. melanogaster larvae fulfils a similar role to the muscle spindle in mammals. Electrophysiological data was obtained from these neurons via whole-cell patching. It was shown that the dbd neuron can respond to both electrical and mechanical stimuli, but that these responses are noticeably distinct. Furthermore the stretch-evoked data obtained from these receptors was equivalent to that predicted by the model, demonstrating a cross-taxa correlation between the behaviour of neurons in this class. This finding enables simple genetic assays to be carried out in D. melanogaster to ascertain the identity of molecules which are involved in primary mechanotransduction at the sensory terminal. A simple bioinformatics search has yielded a short-list of candidates which fulfil the criteria of the model predictions. These can now be experimentally tested in a simple and direct approach. [ABSTRACT FROM AUTHOR]

Mechanotransduction by proprioceptive sensory organs, such as muscle spindles, is poorly understood. We recently reported that stretch releases glutamate from synaptic-like vesicles within spindle terminals, and activates a non-canonical mGluR (Bewick et al, 2005) coupled to phospholipase D (PLD), modulating afferent firing. In further investigations into this receptor's pharmacology, ligands selective for classical mGluRs were screened for their ability to alter stretch-evoked spindle firing. We now report on the development of a fluorescent and a biotinylated agonist. These agonists will be in the key in the next stages of the investigation, isolating the receptor using a pull-down/precipitation assays. Nerve-muscle preparations were excised from humanely killed (Schedule 1, ASPA, 1986) adult male Sprague-Dawley rats. Spindle discharges during 1mm stretch-and-hold cycles were compared with and without drug. Differences in mean firing frequencies were evaluated by two-way ANOVA with Bonferroni post test (sig. thresh- old P=0.05). Four compounds were found to increase afferent firing; kainate (1 µM, n = 7, P = 0.01) quisqualate (1 µM, n = 8, P = 0.05), t-ADA (1 µM, n = 9, P = 0.05) and L-CSA (0.1 µM, n = 7, P = 0.05). NBQX, a kainate and AMPA receptor antagonist, had no effect on afferent firing when applied alone (n=4) or in combination with kainate (n = 7), adding further evidence that classical kainate or AMPA ionotropic receptors are not responsible for the increase in stretch-induced afferent firing with application of kainate. In addition PCCG-13 was able to attenuate the effect of kainate (n = 4) suggesting kainate is acting on the PLD-coupled mGluR. Kainate was used as the parent compound in the synthesis of click-chemistry amenable (Kolb et al, 2001) analogues; ZCZ49, ZCZ50 and ZCZ90. Only ZCZ90 retained activity in the system, increasing afferent firing at 1 µM (n = 7, P = 0.01). From this fluorescent (ZCZ172) and biotinylated (ZCZ180) versions were synthesised. Both retained activity, increasing afferent firing at 10 µM (n = 8, P = 0.001) and 1 µM (n = 8, P = 0.01) respectively. These data show kainate is a useful base ligand for the development of tools to isolate and further characterise this intriguing receptor. [ABSTRACT FROM AUTHOR]