The LHb conveys inhibitory reward signals that inhibit VTA DA neurons and DA release in the NAc19, and neurons in the LHb can be excited by peripheral stimulation20. To determine if ulnar MS might activate the LHb, we measured brain temperature, an indicator of metabolic neural activation21. LHb temperature following ulnar MS increased to 0.073 ± 0.013 °C over baseline and recovered to baseline shortly after stimulation (LHb; Fig. 4A1–3), while no significant changes of temperature were seen during ulnar MS in control sites, 1.3 mm apart from LHb (Con; Fig. 4A1–3). To provide evidence for LHb activation and specific LHb projections to the VTA/RMTg, we examined the expression of c-fos in LHb neurons retrogradely labeled from the VTA/RMTg. The number of c-fos positive cells in the LHb increased approximately 2-fold in rats undergoing ulnar MS compared to naïve rats (p = 0.003; Fig. 4B1–3). Numbers of c-fos positive cells in retrogradely labeled LHb neurons significantly increased in ulnar MS-treated rats compared to naïve rats (Fig. 4C1–4; Con vs. MS. P < 0.05), suggesting that VTA/RMTg-projecting LHb neurons were activated by ulnar MS. To identify the role of the LHb in sensory inhibition of cocaine locomotion, electrolytic lesions of LHb were made bilaterally (Fig. 4D1–2). LHb lesions impaired the inhibition of cocaine locomotion by ulnar MS (LHb X + MS), compared to control group (Sham + MS; Fig. 4D3). To further confirm that the neurons in the LHb were evoked during ulnar MS, extracellular recordings were performed in LHb. The mean firing rates of LHb neurons (n = 21 from 7 rats) during ulnar MS increased from 2.44 ± 0.6 spikes/sec to 28.95 ± 3.8 Hz and returned to baseline (2.43 ± 0.71 Hz) shortly after termination of ulnar MS (p < 0.05, Fig. 5A1–3). To see a putative VPL-LHb link, we explored whether VPL lesion could interrupt the neuronal activation of LHb following peripheral stimulation. The c-fos expression in the LHb increased following ulnar MS (12.29 ± 1.62), compared to controls (Con, 4.04 ± 0.27), which was prevented by VPL lesion (VPL X + MS, 5.42 ± 1.88; Fig. 5B4, p < 0.05).Figure 4The role of VTA/RMTg projections to the lateral habenula (LHb) on ulnar inhibition of cocaine locomotion. (A) Ulnar MS increases LHb temperature (A1–3). (B) Activation of LHb neurons projecting to the VTA/RMTg regions following ulnar stimulation. The number of c-fos positive cells in the LHb increased by approximately 2-fold in ulnar MS rats (MS, B2-B3; n = 5) compared to rats in the control group (Con; B1; n = 5). (C) c-fos LHb positive cells (C2) were labeled retrogradely with fluorogold (FG; yellow; C1) injected into the VTA/RMTg regions (C1–3). The number of c-fos positive cells double-labeled with FG significantly increased in ulnar MS (n = 5), compared to control (Con; n = 5; C4). # p < 0.05 vs. Con. (D) Electrolytic lesions of LHb (LHb X; D1–2) prevented ulnar MS-induced inhibition of cocaine-induced locomotion (LHb X + MS; n = 5), compared to the control groups of LHb lesion only (n = 5) and Sham + MS (n = 5) (D3). # p < 0.05 vs. LHb X. *p < 0.05 vs. Sham + MS. Figure 5The role of the VPL in the neural activation of LHb following ulnar MS. (A) Extracellular single-unit recordings in the LHb during ulnar MS. Representative waveforms (A1) and peri-stimulus time histograms (A2). Single-unit activities of LHb neurons were markedly evoked during ulnar MS (n = 7) (A2–3). # p < 0.05 vs. Before. (B) Ulnar stimulation (MS, n = 5; B2, B4) significantly increased the numbers of c-fos immunopositive cells, compared to control (Con, n = 5; B1, B4), which was prevented in the rats injected with ibotenic acid into VPL (VPL X, n = 5; B3–4). # p < 0.05, Con vs. MS; *p < 0.05, MS vs. VPL X + MS. Bar = 20 µm.