Spatiotemporal limits of optogenetic manipulations in cortical circuits

Abstract

Neuronal inactivation is commonly used to assess the involvement of groups of neurons in specific brain functions. Optogenetic tools allow manipulations of genetically and spatially defined neuronal populations with excellent temporal resolution. However, the targeted neurons are coupled with other neural populations over multiple length scales. As a result, the effects of localized optogenetic manipulations are not limited to the targeted neurons, but produces spatially extended excitation and inhibition with rich dynamics. Here we benchmarked several optogenetic silencers in transgenic mice and with viral gene transduction, with the goal to inactivate excitatory neurons in small regions of neocortex. We analyzed the effects of the perturbations in vivo using electrophysiology. Channelrhodopsin activation of GABAergic neurons produced more effective photoinhibition of pyramidal neurons than direct photoinhibition using lightgated ion pumps. We made transgenic mice expressing the light-dependent chloride channel GtACR under the control of Cre-recombinase. Activation of GtACR produced the most potent photoinhibition. For all methods, localized photostimuli produced photoinhibition that extended substantially beyond the spread of light in tissue, although different methods had slightly different resolution limits (radius of inactivation, 0.5 mm to 1 mm). The spatial profile of photoinhibition was likely shaped by strong coupling between cortical neurons. Over some range of photostimulation, circuits produced the "paradoxical effect", where excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks. The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, which can be mitigated by slowly varying photostimuli, but at the expense of time resolution. Our data offer guidance for the design of in vivo optogenetics experiments and suggest how these experiments can reveal operating principles of neural circuits.

Recently formed context fear memories can be retrieved without the hippocampus

“…Consistent with a recent studies, we found that direct hyperpolarization of pyramidal cells produces less inhibition than stimulation of interneurons [27]. We hypothesize that restricted hippocampal silencing is the reason that some of our acute manipulations did not affect the retrieval of context fear.…”

“…Stimulation of inhibitory neurons also produced more extensive inhibition than direct hyperpolarization of pyramidal cells, as recently reported [27]. Unexpectedly, some inhibitory manipulations (e.g.…”

“…Similar to published work, this increase extended at least .8 mm AP from the fiber tip (Fisher's LSD post-hoc tests, Anterior (p = .042), Intermediate (p = .0048)). Elevated c-Fos expression in inhibitory neurons was not observed in the ventral hippocampus (Fisher's LSD post-hoc test, Posterior (p = .3294)) [27,30]. A similar spatial profile was found when GABAergic neurons were activated with hM3Dq ( Figure 4C) (Group x AP coordinate interaction, F (2,12) = 42.2, p = <.0001; Fisher's LSD post-hoc tests, Anterior (p <.0001), Intermediate (p=.0003), Posterior (p=.1060)).…”

“…It was recently shown that activation of GABAergic neurons produces more efficient and widespread inhibition of pyramidal cells than direct photoinhibition using light-gated ion pumps [27,30]. Given that the dorsal hippocampus is a large structure, we first silenced it by stimulating inhibitory neurons with ChR2 or hM3Dq.…”

“…Stimulation of GABAergic neurons with ChR2 can produce inhibition that extends 1-2mm from the center of the optic fiber [27,30]. To determine the extent of GABAergic stimulation in our experiments, we quantified c-Fos expression in inhibitory neurons at multiple anteroposterior (AP) coordinates in the dorsal, intermediate and ventral hippocampus in a subset of animals from each group (ChR2 n=5, control n=5; hM3Dq CNO n=4, vehicle n=4).…”