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. 2019 Jul 3;103(1):102–117.e5. doi: 10.1016/j.neuron.2019.04.022

Figure 7.

Figure 7

In Vivo Optogenetic Stimulation of GRP Neurons Requires Burst Stimulation Paradigms to Elicit Aversive Behavior

(A) Experimental setup of in vivo optogenetic activation of spinal GRP neurons with single 4 ms pulses (at 0.5 Hz; blue) or burst stimulation (5 pulses at 25 Hz intra-burst frequency and repeated at 0.5 Hz; magenta).

(B) Waterfall plot showing aversive behavior during unilateral optogenetic single pulse (4 ms) or burst-like (bursts of 5 pulses at 25 Hz with 0.5 Hz repetition rate; magenta) stimulation of GRP neurons in Grp-ChR2 mice (ChR2+) and Grp::cre–;ChR2 mice (ChR2–, n, number of trials; N, number of mice). Shaded lines represent mean ± SEM.

(C) Categorical dot plots showing population data of aversive behavior during stimulation. Circles are individual trials. Error bars indicate mean ± SEM. Two-way ANOVA, followed by Bonferroni post hoc tests, F(1,56) = 39.31, p < 0.0001.

(D) Time course of onset and cessation of light-evoked aversive behavior (black lines; n = 15 individual trials) in the five Grp-ChR2 mice. In two trials, blue light stimulation did not trigger aversive behaviors.

(E) Strategic location of the GRP-GRPR neuron synapse in the spinal itch pathway. Synaptically released GRP is essential for the suprathreshold activation of GRPR neurons by glutamatergic input and induces spontaneous activity. GRP acts not only on synaptically connected neurons but also depolarizes GRPR neurons not directly connected via so-called volume transmission. SPB, spinoparabrachial projection neurons; BNP, B-type natriuretic peptide B; NMB, neuromedin B.