Pain inhibition by optogenetic activation of specific anterior cingulate cortical neurons

Abstract

Cumulative evidence from both humans and animals suggests that the anterior cingulate cortex (ACC) is important for pain-related perception, and thus a likely target for pain relief therapy. However, use of existing electrode based ACC stimulation has not significantly reduced pain, at least in part due to the lack of specificity and likely co-activation of both excitatory and inhibitory neurons. Herein, we report a dramatic reduction of pain behavior in transgenic mice by optogenetic stimulation of the inhibitory neural circuitry of the ACC expressing channelrhodopsin-2. Electrophysiological measurements confirmed that stimulation of ACC inhibitory neurons is associated with decreased neural activity in the ACC. Further, a distinct optogenetic stimulation intensity and frequency-dependent inhibition of spiking activity in the ACC was observed. Moreover, we confirmed specific electrophysiological responses from different neuronal units in the thalamus, in response to particular types of painful stimuli (i,e., formalin injection, pinch), which we found to be modulated by optogenetic control of the ACC inhibitory neurons. These results underscore the inhibition of the ACC as a clinical alternative in inhibiting chronic pain, and leads to a better understanding of the pain processing circuitry of the cingulate cortex.

Fig 1. GAD1 and GFP positive labeled cells in the anterior cingulate cortex.

A: GFP, B: GAD1, C: DAPI, D: GFP+ labeled cells overlaid with GAD1 and DAPI. High magnification image at the anterior cingulate cortex (E: DAPI, F: GAD1, G: GFP, H: co-expression). (I) Number of cells expressing GFP+, GAD1+, or GFP & GAD1 in the ACC. Confocal images of immunostaining of the ACC neurons with GAD1-FITC (J), and YFP-Alexa 555 (K). Scale bar: 50 μm.

Fig 2. In-vivo Electrophysiology of inhibition of ACC neurons by optogenetics.

(a) Schematic showing the mechanism of post-synaptic silencing of excitatory neurons due to optogenetic-stimulation of inhibitory neurons in ACC. (b) Representative neuronal firing in ChR2 transgenic mouse without (left) and with (right) light stimulation for 250 ms. (c) Rate histogram of neuronal spikes with varying intensity of light stimulation in the ACC. (d) Rate histogram of neuronal spikes with varying frequencies of light stimulation in the ACC. (e) Intensity-dependent firing rate of neurons in the ACC. (f) Frequency-dependent firing rate of ACC neurons. Rebound activity of excitatory neurons after switching off the laser is encircled. *P < 0.05 vs. control (laser off).

Fig 3. Behavioral assay of pain inhibition by optogenetic modulation of ACC.

(a) Experimental scheme for Formalin test. (b) Optical fiber mounted via cannula in mouse brain (upper panel). Lower panel shows saline or Formalin injection in hind paw. (c) Pain scoring. (d) Pre-formalin pain score of different mice groups, (e) pain score of same groups of mice in post-formalin condition. *P < 0.05 vs. others (transgenic laser off, wild type laser on and wild type laser off). (f) Histogram for comparison of pre and post formalin pain scores for various mice groups at two different time points. *P < 0.05 vs. control (laser off), n = 10 for each group.

Fig 4. Modulation of neuronal activities in the thalamus in response to brush and pinch.

(a) Representative raw data of spiking activity (left) and waveforms (right). The waveforms of different firing units are grouped as blue and green colors. (b) Raster scan. (c) Response of different neuronal groups in the thalamus due to pinch and brush. C: Laser off (control), P: Pinch, B: Brush, L: Laser on, P+L: Pinch + Laser on, B+L: Brush + Laser on.