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. 2022 Aug 25;13:5014. doi: 10.1038/s41467-022-32558-9

Fig. 5. Strong attenuation of inflammatory mechanical and thermal hypersensitivity by optogenetic stimulation of NBM cholinergic-GABAergic projections to the prelimbic cortex (PL) and the role of layer 5 PL neurons.

Fig. 5

a–c Scheme for optogenetically stimulating NBM-PL cholinergic-GABAergic projections, and its impact on inflammatory mechanical (day 2; b) and heat hypersensitivity (c); P values in inset (b) represent ANOVA-based comparison of the two entire stimulus–response curves. n = 6 ChAT-Cre- and 11 ChAT-Cre+ mice; P < 0.05 (*0.0043, right panel in b; *,# < 0.0001 for CFA days 3 and 8 in (c)), two-way ANOVA with Sidak’s multiple comparisons test. d Scheme of connectivity between NBM cholinergic-GABAergic projections, excitatory afferents and diverse PL neurons impacting on the firing of PL pyramidal neurons via nicotinic and muscarinic cholinergic signaling. IN GABAergic interneuron, PN pyramidal neuron; types of GABAergic interneurons: PV parvalbumin-type, SOM somatostatin-type, VIP vasoactive intestinal peptide-type. e Example images of anti-YFP immunohistochemistry showing NBM projections to the PL (magnification to the right) and adjoining cortices (left: different high-resolution confocal fields stitched together). Scale bars = 250 µm (left) and 100 µm (right). f–h Fos quantification of all PL layers or layer 5 (f) and in excitatory projection neurons (SATB2- or Ctip2; g) and inhibitory neurons (PV or SOM; h) in response to optogenetic stimulation of NBM-PL cholinergic-GABAergic projections (represented by Cre+ mice) or control (represented by Cre- mice) (all mice are “Laser ON”). N = 5 mice/group for panels f–h; *P < 0.05 (all layers: 0.0091, Sham, <0.0001, CFA; layer 5: 0.0036, Sham, <0.0001, CFA), two-way ANOVA followed by post hoc Sidak’s test for f, and unpaired two-tailed t test for g (P = 0.0166, for SATB2 & 0.0066 for Ctip2) and h. Only CFA-injected mice are represented in g, h. i Inflammatory mechanical hypersensitivity in Rbp4-Cre mice expressing the excitatory DREADD, hm3D(Gq) in a Cre-dependent manner. n = 8 mice/group; *P < 0.05 (0.0019, 0.0019, 0.0002 for 0.07, 0.16, & 0.4 g filaments, respectively), two-way ANOVA with Sidak’s multiple comparisons test. Data are presented as mean +/− SEM.