A genetic method for selective and quickly reversible silencing of Mammalian neurons

Abstract

Genetic methods for neuronal silencing have great promise for allowing selective inactivation of specific cell types within complex neural systems. Present methods, however, are limited in their reversibility by the slow time scale (days) of transcriptional regulation. We report the rapid and reversible inactivation of mammalian cortical neurons expressing the insect G-protein-coupled receptor AlstR (Drosophila allatostatin receptor) [corrected] after application of its peptide ligand allatostatin (AL). The onset and reversal of inactivation could be achieved rapidly, within minutes. Moreover, the effects of AL were selective for AlstR-transfected neurons. The AlstR/AL system is therefore a promising genetic method for selective and quickly reversible silencing of neuronal activity.

Fig. 1.

Silencing of cortical neurons with the AlstR/AL receptor/ligand system. A1, The spike threshold of a representative AlstR-transfected neuron was determined by a series of depolarizing current pulses (I_stim, 1 sec duration; left panel) before addition of allatostatin to the bath (gradually shaded bar). In this example, the spike threshold was found to be at +14 pA. Before and after the onset of perfusion with 1 nm AL, input resistance was monitored by hyperpolarizing current pulses at 5 sec intervals (middle panel). Input resistance and resting membrane potential decreased within minutes of AL application. The amount of current necessary to elicit an action potential in the presence of AL (+118 pA) was greatly increased with respect to the initial values (right panel). A2, The effects of AL were reversible over the course of several minutes by washing out AL with normal ACSF. Input resistance, membrane potential (left panel), and spike threshold (right panel) returned to approximately initial values after a perfusion time approximately equivalent to the time required for the exchange of one bath volume (see Results). B, Input resistance, membrane potential, and spike threshold of control neurons were unaffected by application of AL.

Fig. 2.

Selectivity and reversibility of AL-induced effects. A1, Effect of AL on membrane potential in AlstR-transfected (n = 15; black bar) and control neurons (n = 9;light bar). AL produced a significant decrease in membrane potential in AlstR-transfected neurons (−6.7 ± 0.7 mV; ***p < 0.0001). Control neurons showed no change (0.1 ± 0.9 mV). A2, Ratio of input resistance before and after application of 1 nm AL in AlstR-transfected and control neurons. Input resistance decreased to 48 ± 7% of the original value (***p < 0.0001) for AlstR-transfected neurons (p < 0.001), but control neurons were unaffected (116 ± 17%).A3, The ratio of spike threshold (current amplitude necessary to elicit an action potential) before and after application of 1 nm AL in AlstR-transfected and control neurons. Spike threshold increased 13-fold for AlstR-transfected neurons (1300 ± 410%; p < 0.05), but control neurons were unaffected (110 ± 10%). B1, After removal of AL from the bath, the decrease in membrane potential induced by 1 nm AL for a subset of eight AlstR-transfected neurons tested (−8.1 ± 0.7 mV; p < 0.001) recovered to its original value (−0.4 ± 1.1 mV). B2, Input resistance also returned from reduced levels (33 ± 43%;p < 0.001) to approximately original values (122 ± 39%). B3, The reversal of AL-induced effects on membrane potential and input resistance was accompanied by a recovery of excitability, as measured by the current amplitude for eliciting an action potential, from 2058 ± 654% (p < 0.05) to approximately original values (186 ± 75%). ***p < 0.0001; **p < 0.001; *p < 0.05. n.s., Not significant. Dotted lines indicate 100% (no change).