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. 2023 Feb 8;12:e78689. doi: 10.7554/eLife.78689

Figure 6. Chemogenetic activation of medial serotonergic hindbrain neurons.

(A). Injection scheme for chemogenetic activation of medial serotonergic hindbrain neurons. Stereotaxic coordinates used for activating midline serotonergic neurons of Tph2-Cre animals with hM3D(q)-containing AAVs (–6, 0, 5.9). (B). Example of an injection site from an experiment to activate midline serotonergic neurons with hM3D(q) (scale bar = 500 μm). (C). Lumbar spinal cord from the injection site shown in B. showing mCherry-expressing terminals located ventral to the dense 5-HT innervation of the superficial dorsal horn (scale bar = 200 μm). (D). Sensory and sensorimotor coordination assays of animals following CNO or vehicle injections. Repeated one-way ANOVA, followed by Bonferroni’s post-hoc tests for von Frey (F(3, 6)=13.84, p<0.0001) show significant decreases between post-vehicle and post-CNO injections (P=0.0005) as well as pre-CNO and post-CNO (p<0.0001). Significance: *p<0.05, **p<0.01, **p<0.001.

Figure 6.

Figure 6—figure supplement 1. Injection sites for chemogenetic activation of medial serotonergic neurons of the hindbrain.

Figure 6—figure supplement 1.

In all cases, spread is restricted to the medial neurons with limited spread to the LPGi serotonergic neurons. The targeted region of the hindbrain was the NRM at (–6, 0, 5.9) relative to Bregma according to the mouse brain atlas. In many cases, hM3Dq-mCherry-expressing neurons are also seen in the Raphe Obscuris (ROb), and the Raphe Pallidus (RPa) (scale bar = 500 μm).
Figure 6—figure supplement 2. Both male and female animals show increase in mechanical hypersensivity during chemogenetic activation of medial serotonergic hindbrain neurons.

Figure 6—figure supplement 2.

Sensory and motor tests of male and female animals 1–3 hr after CNO or vehicle injection. A decrease in withdrawal threshold is seen in both male and female animals for the von Frey test (paired t-tests post vehicle vs post CNO with Holm-Sidak correction). N=5 male and 6 female animals for all tests. Significance: *p<0.05, **p<0.01, **p<0.001.
Figure 6—figure supplement 3. Proportion of neurons labeled in chemogenetic experiments that express TPH2.

Figure 6—figure supplement 3.

Summary of the proportion of hM3Dq-mCherry neurons labeled in the medial hindbrain that also express detectable TPH2 (n=3 animals).