Figure 4. Axon projection-selective silencing with hM4D^nrxn.

A) hM4D^nrxn was constructed by addition of an intracellular amino acid sequence from neurexin-1 (a.a. 1425 −1479) to the C-terminus of hM4D that also contained a C-terminal hemagglutinin (HA) tag.

B) Cell surface distribution of N-terminal (extracellular) epitope tagged HA-hM4D (Armbruster et al., 2007) and HA-hM4D^nrxn (see Experimental Procedures) was determined in hippocampal neuronal cultures that were co-transfected with EGFP. For surface labeling, anti-HA immunofluorescence was measured without membrane permeabilization. HA-hM4D^nrxn showed reduced intensity in the somatic compartment but not the axonal compartment, compared to HA-hM4D. Label intensity was quantified using linescans (hashed lines) across the cell body or synaptic boutons. EGFP fluorescence (HA/EGFP overlay) was used to trace axons and identify transfected neurons. Representative soma and axon images of anti-HA immunofluorescence were adjusted identically for brightness and contrast for display purposes. Cells transfected with EGFP alone did not show detectable signal for surface label anti-HA immunofluorescence.

C) The surface expression of HA-hM4D^nrxn is distributed to the axonal compartment, as illustrated by a reduction in soma:axon (S/A) immunofluorescence ratio, relative to HA-hM4D.

D) In cortical brain slices co-expressing hM4D^nrxn and ChR2, synaptic inhibition of L2/3→L5 transmission is as effective with hM4D^nrxn (n = 5) as with hM4D (n = 8).

E) The dose response relationship for synaptic inhibition with CNO is similar for hM4D^nrxn and hM4D.

F) Hyperpolarization in L2/3 cortical neurons expressing hM4D^nrxn was negligible with CNO (1 µM) (n = 6).

G) Conductance change in response to CNO (1 µM) is markedly reduced in L2/3 cortical neurons expressing hM4D^nrxn (n = 6), as compared to hM4D (n = 7). Values are means ± SEM. n.s. p > 0.05, **p < 0.01, ***p < 0.001.