Activation of parabrachial nucleus - ventral tegmental area pathway underlies the comorbid depression in chronic neuropathic pain in mice

Abstract

Depression symptoms are often found in patients suffering from chronic pain, a phenomenon that is yet to be understood mechanistically. Here, we systematically investigate the cellular mechanisms and circuits underlying the chronic-pain-induced depression behavior. We show that the development of chronic pain is accompanied by depressive-like behaviors in a mouse model of trigeminal neuralgia. In parallel, we observe increased activity of the dopaminergic (DA) neuron in the midbrain ventral tegmental area (VTA), and inhibition of this elevated VTA DA neuron activity reverses the behavioral manifestations of depression. Further studies establish a pathway of glutamatergic projections from the spinal trigeminal subnucleus caudalis (Sp5C) to the lateral parabrachial nucleus (LPBN) and then to the VTA. These glutamatergic projections form a direct circuit that controls the development of the depression-like behavior under the state of the chronic neuropathic pain.

Graphical abstract

Figure 1

Chronic trigeminal neuralgia induces the depression-like behavior and increases the firing activity of the VTA DA neurons (A) Schedule for the pIONT (partial infraorbital nerve transection) procedure and subsequent experiments. (B) Schematic of the trigeminal nerve anatomy, showing infraorbital branch and the pIONT ligation/transection point (red line). (C) Summary for trigeminal neuralgia, quantified as the frequency of face-grooming behaviors after the pIONT (n = 10) or sham (n = 9) surgeries, respectively; ^∗∗∗p < 0.001 (two-way repeated-measures ANOVA; Bonferroni post hoc test). (D) Summary of the immobility times in tail suspension test (TST; i), and the forced swimming test (FST; ii) at the 28^th day after the pIONT operation (n = 10; ^∗∗∗p < 0.001; two-tailed t test). (E) Example traces of in vivo single-unit recordings of tonic (left) and burst firing (right) recorded from the VTA DA neurons of the pIONT and the sham-operated mice. (F) Summarized firing frequency (i), number of bursts per minute (ii), and average number of spikes in a burst (iii); n = 13–15, ^∗∗p < 0.01, ^∗∗∗p < 0.001, two-tailed t test. (G) Firing frequency of the VTA DA neurons recorded in vitro from the brain slices of the lateral (i) and the medial (ii) VTA; n = 5–7, ^∗p < 0.05, two-tailed t test. See also Figure S1.

Figure 2

RTG reduces the firing activity of the VTA DA neurons and alleviates the depression-like behaviors (A) Schedule of the experimental procedures (left) and schematic illustration for drug delivery. (B) Example recordings from the VTA DA neurons before and during the application of vehicle (ACSF, 0.4 μl) or RTG (200 μM, 0.4 μl), applied in sequence to the same neuron. The recordings were made using in vivo single-unit recordings 28 days after the pIONT or sham operations. (C) Histogram showing the time course of RTG-induced inhibition on the VTA DA neuron firing frequency. (D) Summary data for experiments as those shown in (B). The frequencies were quantified at 5 min after the ACSF or RTG application (n = 8, ^∗∗∗p < 0.001, one-way repeated-measures ANOVA with Bonferroni post hoc test). (E) Summary data for the effects of RTG on the firing frequency (i), burst/min (ii), and spike/burst (iii) under in the pIONT-operated and the sham-operated mice (n = 5–10, ^∗p < 0.05, paired t test). (F) Summary data for the effects RTG and ACSF on the immobility time in the TST at 28 days after the pIONT or the sham operations. RTG (200 μM, 0.4 μl) was given through the cannula implanted into the VTA (n = 8, ^∗∗p < 0.01, one-way repeated-measures ANOVA with Bonferroni post hoc test). The TST was performed before and 15 min after the infusion of either RTG or ACSF, with an interval of 24 h. See also Figure S2.

Figure 3

Specific manipulations with the VTA DA neuron activity alter depression-like behaviors (A) Specific inhibition of the VTA DA neuron activity was realized with the use of chemogenetic activation of the hM4D receptor selectively expressed in the VTA DA neurons through the DAT-controlled Cre activity; schematic depicts the schedule of experimental procedures. (B) Top, schematic illustrating the injection of the viral construct carrying the hM4D receptor (AAV-DIO-hM4D-mCherry) or the control virus (AAV-DIO-mCherry) into the VTA of the DAT-Cre mice (21 days after the pIONT operation). Bottom, mCherry fluorescence was clearly visible in the VTA DA neurons; nuclear DAPI (4',6-diamidino-2-phenylindole) labeling is in blue. (C and D) Specific inhibition of the VTA DA neurons in the pIONT mice reduced depression-like behaviors. The immobility time of the AAV-DIO-hM4D-mCherry-injected (C) and the AAV-DIO-mCherry-injected (D) mice in the TST (i) or the FST (ii) were measured before and 15 min after the administration of CNO (0.33 mg/kg, i.p.); there was a 24-hr interval between the tests (n = 7, ^∗∗p < 0.01, ^∗∗∗p < 0.001, one-way repeated-measures ANOVA with Bonferroni post hoc test [C] or paired t test [D]). (E) Specific activation of the VTA DA neurons was achieved with the use of the hM3D receptor selectively expressed in the VTA DA neurons through DAT-controlled Cre activity (DAT-Cre mice); specific expression of hM3D in the VTA DAT neurons was confirmed by the co-expressed mCherry (bottom panel). (F and G) The mice injected with the hM3D-carrying virus (F) and with the control virus (G) were subjected to TST (i) or FST (ii), and the immobility time was quantified (n = 6, ^∗∗∗p < 0.001, paired t test). (H) Optogenetic activation of the VTA DA neurons in the naive mice. AAV-DIO-ChR2-mCherry or AAV-DIO-mCherry (control) viral particles were injected into the VTA of DAT-Cre mice. The ChR2 was activated by a 475-nm laser through an implanted optical fiber. (I and J) The immobility time in the TST of mice with (I) and without (J) ChR2 expression were measured under 475-nm (20 Hz, 5 pulses per 10 s) light off and light on; n = 8, ^∗∗∗p < 0.001, paired t test. See also Figure S3.

Figure 4

Glutamatergic projections from LPBN to VTA control depression-like behaviors (A) Retrobeads (100 nL) and Cre-dependent virus AAV-DIO-GFP were injected to the VTA of Vglut2-Cre mice to visualize a Vglut2-controlled expression of GFP and retrograde tracing in the LPBN. (B) Retrobead fluorescence in an image from a 30-μμ coronal brain section from the ventral side at Bregma −5.34 mm. The brain regions most prominently labeled by the retrobeads are shown with dotted ellipses. (C) Higher-magnification images showing the retrobeads and GFP fluorescence in the LPBN neuron. Co-labeling of GFP and retrobeads is indicated in the top panel (arrowheads). The area indicated by the white square is shown in the bottom panel with higher magnification. (D) Experimental schedule (left) and schematic of a cannula placement (right) for the local application of the NMDA blocker AP-5. The cannula was implanted into the VTA at 21 days after the pIONT/sham operation. (E) Example traces of single-unit recordings showing firing of the VTA DA neurons before and during AP-5 (200 μM, 300 nL) application (left). Shown on the right is the time course for the effect of AP-5 on the firing frequency. (F) Summary data for effects of AP-5 on firing frequency (i), burst number/min (ii), and spike number/burst (iii); n = 10–12, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05, paired t test. (G) Summary data for effects of AP-5 and the vehicle (ACSF) on the immobility time in the TST. The TST was performed 15 min after application, with an interval of 24 h between ACSF and AP-5; n = 14, ^∗∗∗p < 0.001, one-way repeated-measures ANOVA with Bonferroni post hoc test. See also Figures S4 and S5.

Figure 5

Selective manipulations with the glutamatergic LPBN-VTA projections alter the depression-like behaviors (A) Experimental schedule. (B) Schematic of viral labeling. AAV-DIO-NpHR-eYFP/AAV-DIO-eYFP (200 nL) virions were injected into the LPBN of the Vglut2-Cre mice bilaterally at 21 days after the pIONT operation, and an optical fiber was implanted in the VTA. The 590-nm laser was used to activate NpHR. (C) eYFP fluorescence confirmed the expression of NpHR at the projection terminals of the glutamatergic neurons from the LPBN (top panel) around the neuronal cell bodies in VTA (bottom panel). (D) Summary data for effects of the 590-nm laser illumination (constant) on the immobility time in the TST. The TST was performed before and during the laser illumination on the mice at 28 days after the pIONT surgery; n = 8, ^∗∗p < 0.001, paired t test. (E–G) Selective activation of the glutamatergic neurons in the LPBN projecting to the VTA DA neurons increased the immobility time of the TST in the naive mice. AAV-DIO- ChR2-mCherry/AAV-DIO-mCherry (200 nL) virions were injected bilaterally to the LPBN of the naive Vglut2-Cre mice (E), and an optical fiber was implanted into the VTA. A 475-nm laser was used to activate ChR2 (F). (G) Expression of the ChR2 (visualized by mCherry) at the projection terminals of the glutamatergic neurons from the LPBN (top panel) and around the neuronal cell bodies in VTA (bottom panel). (H) Activation of ChR2 with 475-nm illumination in the VTA increased the immobility in the TST in the ChR2-expressing (i) but not in the non-ChR2-expressing control (ii) mice; n = 10, ^∗∗∗p < 0.001, paired t test. See also Figures S5 and S6.

Figure 6

Direct glutamatergic projection pathway linking Sp5C, LPBN, and VTA governs the chronic-pain-induced depression behaviors (A and B) Projections from the Sp5C to the LPBN identified through retrograde labeling. Retrobeads and AAV-DIO-GFP were injected in the LPBN of Vglut2-Cre mice (A). Co-staining with retrobeads and GFP is visible in multiple neurons in the Sp5C (B). (C) Schematic for the trans-monosynaptic retrograde projection tracing of the Sp5C-LPBN-VTA pathway. Viruses used, the injection sites, and the time of injection are indicated. The Cre-dependent constructs were injected into the Vglut2-Cre mice, thus restricting the expression to the glutamatergic neurons. (D) Visualization of the expression of tdTomato and GFP in the VTA, the LPBN, and the Sp5C. The tdTomato was prominently visible in the glutamatergic neuron somata in the LPBN (middle panel) and in presumed projection terminals (from LPBN) in the VTA (top panel, arrowheads); tdTomato was not detected in the Sp5C (bottom panel). This pattern indicates a restricted expression of the tdTomato in the glutamatergic neurons of the LPBN projecting to the VTA. GFP, injected into the VTA, was clearly expressed in the glutamatergic neuron somata in the Sp5C (bottom panel), indicating a monosynaptic connection between the glutamatergic neurons in the Sp5C and the LPBN that projected to the VTA. (E) Schematic for testing the effects of Sp5C-LPBN pathway inhibition on the depression behaviors by using optogenetic manipulation. (F) Schematic indicating viruses used and injection and illumination sites. The viruses (200 nL) were injected bilaterally to the Sp5C of the Vglut2-Cre mice at 21 days after the pIONT operation, and an optical fiber was implanted in the LPBN; a 590-nm laser light was used for NpHR stimulation. (G) Following Sp5C injection, NpHR was expressed at the terminals of Sp5C-LPBN-projecting glutamatergic neurons in the LPBN (visualized by eYFP fluorescence). (H) Summary data for the effects of inhibiting the glutamatergic projections from the Sp5C to the LPBN on the immobility time in the TST; illumination of the LPBN with a 590-nm laser reduced the immobility time in NpHR expression mice (i) but not in the control mice (ii); n = 8, ^∗∗∗p < 0.001, paired t test.