Differential modulation of pain and associated anxiety by GABAergic neuronal circuits in the lateral habenula

Abstract

Persistent pain frequently precipitates the development of anxiety disorders, yet the underlying mechanisms are not fully understood. In this study, we employed a mouse model that simulates trigeminal neuralgia and observed a marked reduction in the activity of GABAergic neurons in the lateral habenula (LHb), a critical region for modulating pain and anxiety. We utilized precise optogenetic and chemogenetic techniques to modulate these neurons, which significantly alleviated behaviors associated with pain and anxiety. Our investigations revealed an inhibitory pathway from the LHb GABAergic neurons to the posterior paraventricular thalamus. Activation of this pathway primarily mitigated pain-related behaviors, with minimal effects on anxiety. Conversely, interactions between GABAergic and glutamatergic neurons within the LHb were essential in alleviating both pain and anxiety following trigeminal nerve damage. Additionally, we identified that β-sitosterol interacts directly with LHb GABAergic neurons via the estrogen receptor α, providing dual therapeutic effects for both pain and anxiety. These findings highlight the critical role of reduced GABAergic neuronal activity in the LHb in the intersection of pain and anxiety, pointing to promising therapeutic possibilities.

Keywords: anxiety; neural circuit; pain.

Fig. 1.

Optogenetic activation of LHb GABAergic neurons alleviates pain-like and anxiety-like behaviors in pT-ION mice. (A) Diagram illustrating the pT-ION mouse model. (B) Schematic of the recording setup in acute slices. (C) Electrophysiological traces and summarized data from LHb GABAergic neurons in sham and pT-ION groups [two-tailed Student’s t test, sham, n = 6 cells from 6 mice (3♂+3♀); pT-ION, n = 6 cells from 6 mice (3♂+3♀); amplitude, t₁₀ = 4.744, P = 0.005; interevent intervals, t₁₀ = 0.1454, P = 0.9275]. (D) Traces showing evoked action potentials in LHb GABAergic neurons from sham and pT-ION mice [two-way ANOVA, sham, n = 5 cells from 5 mice (3♂+2♀); pT-ION, n = 6 cells from 6 mice (3♂+3♀); F_1,9 = 10.16, P = 0.0111]. (E and F) Schematics of surgical procedures and optogenetic stimulation protocol in pT-ION mice. (G) Image showing viral delivery for optogenetic targeting of LHb GABAergic neurons. (Scale bar, 200 μm). (H) ChR2-mediated stimulation significantly reduced mechanical allodynia in the orofacial V2 and V3 areas, and cold allodynia in the V3 area [two-way ANOVA followed by Sidak’s multiple comparisons test, DIO-mCherry, n = 12 mice (8♂+4♀); DIO-ChR2, n = 12 mice (7♂+5♀); V2: F_1.836,40.38 = 141.0, P < 0.001; V3 mechanical: F_1.949,42.87 = 164.6, P < 0.0003; V3 cold: F_2.294,50.47 = 83.51, P < 0.001]. (I) In the OFT, ChR2 stimulation increased the central time and central/total distance ratio, without affecting the total distance (two-way ANOVA followed by Sidak’s multiple comparisons test, central time: F_2,44 = 0.7464, P = 0.0107; central/total distance ratio: F_{1.737, 38.22} = 2.669, P = 0.0064; total distance: F_{1.599, 35.18} = 37.09, P = 0.8180). (J) In the EPM, ChR2 stimulation enhanced the percentage of time and distance in open arms, without altering the percentage of open-arm entries (two-way ANOVA followed by Sidak’s multiple comparisons test, time: F_1.427,31.40 = 14.07, P = 0.0165; distance: F_{1.461, 32.14} = 13.21, P = 0.0325; entries: F_{1.966, 43.24} = 17.35, P = 0.9309). All data are presented as mean ± SEM. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Activation of LHb^GABA→pPVT projections alleviates pain-like but not anxiety-like behaviors in pT-ION mice. (A and B) Schematic illustration and representative image showing the virus injection site and viral infection for anterograde tracing of LHb GABAergic neurons. (Scale bar, 200 μm). (C and D) Diagrams of virus injection and fiber-optic placement with sample electrophysiological traces of optically evoked inhibitory postsynaptic currents (oIPSC) and excitatory postsynaptic currents (oEPSC) in pPVT neurons [oIPSC, one-way ANOVA followed by Holm–Sidak’s multiple comparisons test, n = 3 neurons from 3 mice (3♂); F_{2, 4} = 15.04, Light OFF(Pre) vs. Light ON, P = 0.0168, Light OFF(Post) vs. Light ON, P = 0.0142; oEPSC, one-way ANOVA followed by Holm–Sidak’s multiple comparisons test, F_{2, 6} = 1.00, Light OFF(Pre) vs. Light ON, P > 0.9999, Light OFF(Post) vs. Light ON, P > 0.9999]. (E) Data summarizing the frequency and proportion of pPVT neurons responsive to optogenetic stimulation of LHb GABAergic neurons. (F and G) Surgical illustrations and experimental setup for chemogenetic activation of LHb GABAergic neuron terminals in the pPVT of pT-ION mice. (H) Representative image showing viral infection for in vivo chemogenetic studies on LHb GABAergic neurons. (Scale bar, 200 μm). (I) hM3Dq-CNO-mediated stimulation significantly reduced mechanical allodynia in the orofacial V2 and V3 areas, and cold allodynia in the V3 area [two-way ANOVA followed by Sidak’s multiple comparisons test, PBS, n = 8 mice (5♂+3♀); CNO, n = 9 mice (6♂+3♀); V2: F_1,15 = 18.94, P < 0.0001; V3 mechanical: F_1,15 = 14.45, P < 0.0001; V3 cold: F_1,15 = 44.38, P < 0.0001]. (J and K) Chemogenetic stimulation did not significantly affect anxiety-related measures in the OFT or EPM (two-tailed Student’s t test, OFT: central time, t₁₅ = 1.256, P = 0.2282; central/total distance ratio, t₁₅ = 1.129, P = 0.2766; total distance, t₁₅= 0.7639, P = 0.4568; EPM: open-arm time percentage, t₁₅ = 1.113, P = 0.2834; open-arm distance percentage, t₁₅ = 1.066, P = 0.3033; open-arm entries percentage, t₁₅ = 1.920, P = 0.0741). All data are presented as mean ± SEM. Significance levels: *P < 0.05, ***P < 0.001.

Fig. 3.

Optogenetic activation of LHb glutamatergic neurons induces pain-like and anxiety-like behaviors in sham mice. (A and B) Schematic depiction of virus injection, fiber-optic implantation sites, and the experimental design for optogenetic activation of LHb glutamatergic neurons in sham mice. (C) Activation via ChR2 in LHb glutamatergic neurons triggered mechanical allodynia in the orofacial V2 [two-way ANOVA followed by Sidak’s multiple comparisons test, EYFP, n = 8 mice (8♂); ChR2, n = 8 mice (8♂); F_1,14 = 1.320, P = 0.0029] and V3 areas (F_1,14 = 0.3504, P = 0.0070), and cold allodynia in the V3 area (F_1,14 = 18.05, P = 0.0094). (D) This optogenetic activation also resulted in a decrease in central time (F_1,14 = 8.481, P = 0.0049) and central/total distance ratio (F_1,14 = 4.270, P = 0.0004) in the OFT, while total distance remained unaffected (F_1,14 = 4.336, P = 0.9968). (E) In the EPM, ChR2 activation reduced open-arm time percentage (F_1,14 = 2.083, P = 0.0004) and open-arm distance percentage (F_1,14 = 3.407, P = 0.0004), but did not significantly alter open-arm entries percentage (F_1,14 = 2.608, P = 0.2353). (F and G) Diagram of experimental operations and sample traces of oIPSC and oEPSC from LHb glutamatergic neurons. (H and I) Data detailing the frequency and proportion of LHb glutamatergic neurons responding to optogenetic activation of LHb GABAergic neurons (oIPSC, one-way ANOVA followed by Holm–Sidak’s multiple comparisons test, n = 3 neurons from 3 mice (3♂); F_{2, 4} = 15.04, Light OFF(Pre) vs. Light ON, P = 0.0390, Light OFF(Post) vs. Light ON, P = 0.0101; oEPSC, one-way ANOVA followed by Holm–Sidak’s multiple comparisons test, F_{2, 6} = 1.00, Light OFF(Pre) vs. Light ON, P > 0.9999, Light OFF(Post) vs. Light ON, P > 0.9999]. All data are presented as mean ± SEM. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

β-sitosterol alleviates pain-like and anxiety-like behaviors by directly activating LHb GABAergic neurons in pT-ION mice. (A) Sample electrophysiological traces show the effects of β-sitosterol, both alone and combined with the ESRα antagonist AZD9496, on LHb GABAergic neurons in acute slices from pT-ION mice. (B–D) Application of β-sitosterol resulted in increased spike frequency [Two-way ANOVA followed by Dunnett’s multiple comparisons test, n = 3 neurons from 3 mice (3♂); F_3,88 = 9.830; β-sitosterol vs. Baseline, P < 0.0001; β-sitosterol vs. Washout, P = 0.003; β-sitosterol vs. β-sitosterol+AZD9496, P = 0.001], a decreased rheobase (One-way ANOVA followed by Holm–Sidak’s multiple comparisons test, F_3,6 = 25.00; β-sitosterol vs. Baseline, P = 0.0054; β-sitosterol vs. Washout, P = 0.0026; β-sitosterol vs. β-sitosterol+AZD9496, P = 0.0004), and a shifted RMP (F_1.126,2.252 = 9.081; β-sitosterol vs. Baseline, P = 0.0151; β-sitosterol vs. Washout, P = 0.0321; β-sitosterol vs. β-sitosterol+AZD9496, P = 0.1296), and (E) direct microinjection of β-sitosterol into the LHb significantly reduced mechanical and cold allodynia in the orofacial V2 and V3 areas [two-way ANOVA followed by Sidak’s multiple comparisons test, Vehicle, n = 11 mice (11♂); β-sitosterol, n = 11 mice (11♂); V2: F_1,20 = 28.30, P < 0.0001; V3: F_1,20 = 22.15, P = 0.0010; cold allodynia: F_1,20 = 31.75, P < 0.0001]. (F) This intervention also increased central time and the ratio of central to total distance in the OFT (two-tailed Student’s t test, central time: t₂₀ = 4.117, P = 0.0005; central/total distance ratio: t₂₀ = 4.027, P = 0.0007), while the overall distance remained unchanged (t₂₀ = 1.564, P = 0.1335). (G) In the EPM, microinjection of β-sitosterol enhanced the percentage of time spent in the open arms, distance traveled in the open arms, and entries into the open arms (two-tailed Student’s t test, time: t₂₀ = 2.930, P = 0.0083; distance: t₂₀ = 3.176, P = 0.0048; entries: t₂₀ = 2.177, P = 0.0416). All data are presented as mean ± SEM. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.