ANALYSIS OF COMPOUNDS FOR PAIN AND SENSORY DISORDERS
The invention generally relates to optical methods for characterizing the effects of compounds on pain and other sensory phenomena. The effect of compounds on pain and other sensory phenomena may be characterized using dorsal root ganglion (DRG) neurons or sensory neurons expressing optogenetic proteins that allow neural activity to be stimulated and detected optically. The invention provides cell-based optical assays for studying the molecular and cellular bases of pain and sensory phenomena and as platforms to screen and validate drugs, e.g., for pre-clinical trials.
1 . A method for screening a compound for pain treatment, the method comprising:
presenting a compound to a sample comprising a dorsal root ganglion (DRG) neuron, wherein the DRG neuron expresses an optical reporter of membrane electrical potential and a light-gated ion channel;
receiving, via a microscopy system, an optical signal generated by the optical reporter in response to optical stimulation of the sample following presentation of said compound; and
identifying the compound as a candidate for pain treatment based on said optical signal.
2 . The method of claim 1 , wherein the DRG neuron comprises a target ion channel with a suspected aberrant characteristic.
3 . The method of claim 2 , wherein the target ion channel is the TRPV1 channel and the suspected aberrant characteristic comprises over expression of TRPV1.
4 . The method of claim 1 , wherein the microscopy system further comprises a charge-coupled device camera configured to capture the optical signal from the DRG neuron.
5 . The method of claim 1 , wherein the microscopy system comprises a digital micromirror device that provides the optical stimulation.
6 . The method of claim 1 , wherein the DRG neuron also expresses a protein that reports a change in an intracellular calcium level.
7 . The method of claim 6 , wherein the DRG neuron is stimulated by a second neuron that expresses the light-gated ion channel.
8 . The method of claim 7 , wherein the second neuron also expresses the optical reporter of change in membrane potential.
9 . The method of claim 6 , wherein:
the light-gated ion channel comprises an algal channelrhodopsin; and
the protein that reports changes in intracellular calcium levels comprises a GCaMP variant.
10 . The method of claim 6 , further comprising detecting a change in AP waveform and a change in the intracellular calcium level upon exposure of the neuron to the compound.
11 . The method of claim 1 , wherein the DRG neuron is an hiPSC-derived DRG neuron.
12 . The method of claim 1 , further comprising spatially patterning a plurality of DRG neurons in a cell culture on a substrate.
13 . The method of claim 1 , wherein the obtaining the optical signal is performed using an optical microscopy system.
14 . The method of claim 13 , wherein the optical microscopy system comprises at least one digital micromirror device.
15 . The method of claim 1 , wherein analyzing the optical signal comprises detecting an effect of the compound on AP waveform.
16 . The method of claim 1 , further comprising exposing a cell culture to an agent known to stimulate a nociceptor as well as the compound and determining an effect of the compound with the agent on the neuron.