Research
Genetic control of neural circuits
Contemporary genetic methods allow cell-selective targeting within complex neural circuits. We have recently developed a novel genetic method, called “tethered toxins”, to manipulate ionic currents in vitro and in vivo by recombinant expression of membrane-tethered neurotoxins. The ongoing projects in our group are aimed at silencing or manipulating specific ion channels in defined neuronal circuits. The questions we are addressing are: how a particular class of ion channels in one cell population contributes to the function of a given neuronal circuit, and whether silencing one cell population has an impact in only that circuit and/or also affects the next circuit. Another question of interest is whether these functions can be restored upon reversibly inhibiting the expression of the cell-surface toxin or peptide. To approach these questions, we are focusing on specific neuronal circuits in which manipulation of certain ion channels could help dissecting the cascade of events that leads to chronic pain, hearing impairment, and nicotine mediated effects. To target these circuits, we are using genetic approaches such as BAC and knock-in transgenesis and lentiviral vectors to achieve cell-specific and stable expression in specific neuronal populations in vivo.
Tethered toxins as modulators of ion channels and receptors
The cholinergic modulators lynx1 and lynx2 are a unique class of cell-surface regulatory molecules. These molecules are members of the Ly6 superfamily that also include the three-finger fold snake venom toxins, α- and κ-bungarotoxin. Both lynx1 and lynx2 form stable associations with nicotinic acetylcholine receptors (nAChRs) and alter their function in vivo. Lynx1-like molecules are well conserved across species, both in structure and function, suggesting the importance of cell-surface modulators of nicotinic receptors in nature.
Peptide toxins from predatory animals that have been routinely employed for neuroscience research do not normally exist as cell-surface anchored molecules. Using the scaffold of the lynx1-like gene family, i.e. secretory signal and consensus sequences for GPI processing and recognition, it is possible to produce a series of tethered toxins (t-toxins) that are highly effective modifiers of neuronal activity. Approximately 40 different chimeric t-toxins derived from the venom of several predatory animals have been cloned and their activity characterized on voltage and ligand-gated ion channels. The t-peptide strategy has also been successfully extended to other bioactive peptides, such as ligand peptides for constitutive activation of GPCRs, illustrating the general applicability of this approach for cell-surface modulation of receptors. Tethered toxins and peptides are being used for very diverse applications pertaining to experimental animal physiology. Because of their mode of action at the cell-surface, membrane-anchored peptide molecules act only on ion channels and receptors present in the cell that is expressing the t-toxin or t-peptide, and not on identical receptors present on neighboring cells that do not express the tethered construct. We are applying the t-toxin strategy combined with transgenesis and viral-mediated genetic approaches for investigations regarding the physiology of neuronal circuits in the mouse nervous system.
Silencing neurotransmission with tethered toxins
Recently, we were able to demonstrate the ability of tethered toxins directed against Cav2.1 and Cav2.2 calcium channels to achieve silencing of neurotransmission in vitro. Furthermore, we could show specific inhibition of striatal dopamine release in vivo in mice injected with lentivirus in the substantia nigra pars compacta and suppression of chronic pain in t-toxin transgenic mice, by expression of t-toxins in nociceptive peripheral neurons of the doral root ganglia. For reference, see official press release and Auer, S. et al. Silencing neurotransmission with membrane-tethered toxins. Nat Meth (2010).
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Silencing of neurotransmission with membrane-tethered toxins.
T-toxins directed against Cav2.1 and Cav2.2 voltage-gated calcium channels are able to inhibit calcium influx upon membrane depolarization, and thus block neurotransmitter release and neurotransmission. |
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