Fig 2: Transfection of OXTR plasmid in HEK-293-TrkB cells produces increased phospho-TrkB. Immunocytochemistry of cells with pTrkB in red transfected with myc-tagged OXTR in green. Co-expression of OXTR led to activation of TrkB. Next day, cells were serum starved for 2 h and then treated with 1 µM oxytocin. Images were taken at 120 min of oxytocin treatment. (A) Left panel, staining with phosphor-TrkB Y816 Cell Signaling 4168 antibody (red). Middle panel, staining with myc-tagged OXTR (green; 18). Right panel, Merge of pTrkB and OXTR. (B) Images from time points of 0 (no oxytocin), 30, 60 and 120 min were collected with matching laser intensities and detector responses. The results were quantitated by Image J analysis as a ratio of Red (pTrkB) to Green (OXTR) staining. The results were plotted in GraphPad Prism 8.2.0. Zeiss CZI files were imported into Image J using Bio-formats 6.5.0. (C) Upper panel, untransfected HEK293/TrkB cells. Staining with antibodies against p-TrkB (Cell Signaling 4168); OXTR-myc, DAPI and total TrkB (R & D System AF1494) is shown.Lower panel, HEK293/TrkB cells are transfected with 0.5 µg/well myc-taggedOXT receptor plasmid for 16 h. The effect of OXTR expression on TrkB activation in cells was evaluated by detecting the levels of p-TrkB (Y816), leftlower panel and Merge.(D) Negligible oxytocin ligand RNA is detected in HEK293-TrkB cells.Expression of oxytocin ligand RNA is measured by RT-qPCR from total RNAisolated from cortex, hypothalamus, and HEK293-TrkB cells. Relative expressionlevels of OXT mRNAs was normalized to actin, statistical calculations were madeusing one-way ANOVA with multiple comparisons (Prism 9).

Fig 3: PRDM12 Controls Development of TRKA+ Nociceptors(A) Immunohistochemistry analysis on E18.5 DRG sections reveals complete absence of nociceptive (TRKA+) neurons in Prdm12-/- embryos, leading to reduced size of the DRG. Scale bar, 20 µm.(B–E) Immunohistochemistry analysis on E12.5 DRG sections confirms the complete absence of nociceptive (TRKA+) neurons in Prdm12-/- embryos (B), while A-LTMR (C, TRKC+; D, TRKB+; and E, RET+) populations of neurons are generated in the mutant. Scale bar, 50 µm.(F) Quantification of neuronal populations on DRG section labeled with ISL1 and TRKC or TRKB or RET. Results are represented in a bar graph and show a 70% decrease in number of neurons in Prdm12-/- embryo, while the overall number of A-LTMRs (TRKC+, TRKB+, and RET+) is unchanged. Note that RET number decreases while TRKC increases in Prdm12-/- DRG at E12.5. n = 2. *p = 0.05, **p = 0.01; ns, not significant; Student’s t test. Data are presented as mean ± SEM.(G) Combined immunostainings with mixed antibodies for TRKB, TRKC, and RET (red) and NF200 (green), which allows visualization of A-LTMR population co-labeled with ISL1 (blue), show that all neurons observed at E18.5 in the Prdm12-/- are A-LTMR mechanoreceptors. n = 4.(H–J) Specific markers of nociceptive neurons (H, substance P; I, CGRP; and J, TH) are not expressed in Prdm12-/- DRG neurons. n = 4.(K) In the epidermis, sensory nerve endings stained by PGP9.5 are detectable in Prdm12+/+ embryos (white arrows) and are absent in Prdm12-/- embryos at E18.5. Arrowhead in Prdm12-/- section indicates Meissner corpuscle (innervated by A-LTMR subtype). Dotted lines indicate boundary between epidermis and dermis. n = 3. Scale bar, 20 µm.(L) Whole-mount of forelimbs using peripherin (PERIPH) immunolabeling at E14.5 reveals a general decrease in limb innervation (arrowheads point to the same nerve in Prdm12+/+ and Prdm12-/- embryos). Insets show higher magnification of digit innervation; arrowheads indicate reduced nerve branching in Prdm12-/- embryos. n = 3. Scale bar, 100 µm.(M) While TRKA+ neurons in DRG and their projections in spinal cord (SC) are absent in Prdm12-/- embryos at E18.5, TRKA expression in sympathetic ganglia (SG) is not affected. n = 3. Scale bar, 200 µm.

Fig 4: Interaction between CB1, TRPV1 and TRKB regulating burying behavior.The activation of CB1 by endocannabinoids (eCB) leads to TRKB coupling, activation and decrease in burying, putatively in GABAergic cells (dashed blue). On the other hand, the activation of TRPV1 receptors by eCB triggers the release of BDNF and activation of TRKB, possibly in pyramidal (dashed red) and/or neighbor cells (dashed green) leading to increase in burying.

Fig 5: Blockade of 5-HT2A with ketanserin reversed TrkB autophosphorylation in vitro and in vivo. (A) Neuroblastoma N1E-115 cells expressing HA-tagged 5-HT2A receptor, GFP-tagged TrkB, or co-expressing both receptors were treated with the 5-HT2A receptor antagonist ketanserin (1 µM) for 10 min, followed by treatment with either 7,8-DHF (500 nM) or vehicle for 30 min. Cell lysates were then subjected to SDS-PAGE and Western blot (WB) analysis with antibodies against phosphorylated or total TrkB. ß-tubulin was used as a loading control. Representative WBs are shown. Bars show means ± SEM (n = 8). * p < 0.05; *** p < 0.001 (two-way ANOVA). (B) Mice were intraperitoneally injected with ketanserin (1 mg/kg) or 25CN-NBOH (1 mg/kg), resulting in increased TrkB phosphorylation in the hippocampus and frontal cortex. At 10 min (for 25CN-NBOH) or 30 min (for ketanserin) after injection, the animals were euthanized, and brain lysates were prepared from the frontal cortex, hippocampus, and striatum. This was followed by SDS-PAGE and WB to detect phosphorylated and total TrkB. GAPDH was used as a loading control. Representative WBs are shown. Bars represent means ± SEM (5 = n = 8) * p < 0.05; ** p < 0.01 (two-way ANOVA).