Involvement of Lateral Habenula Dysfunction in Repetitive Mild Traumatic Brain Injury-Induced Motivational Deficits

Abstract

Affective disorders including depression (characterized by reduced motivation, social withdrawal, and anhedonia), anxiety, and irritability are frequently reported as long-term consequences of mild traumatic brain injury (mTBI) in addition to cognitive deficits, suggesting a possible dysregulation within mood/motivational neural circuits. One of the important brain regions that control motivation and mood is the lateral habenula (LHb), whose hyperactivity is associated with depression. Here, we used a repetitive closed-head injury mTBI model that is associated with social deficits in adult male mice and explored the possible long-term alterations in LHb activity and motivated behavior 10-18 days post-injury. We found that mTBI increased the proportion of spontaneous tonically active LHb neurons yet decreased the proportion of LHb neurons displaying bursting activity. Additionally, mTBI diminished spontaneous glutamatergic and GABAergic synaptic activity onto LHb neurons, while synaptic excitation and inhibition (E/I) balance was shifted toward excitation through a greater suppression of GABAergic transmission. Behaviorally, mTBI increased the latency in grooming behavior in the sucrose splash test suggesting reduced self-care motivated behavior following mTBI. To show whether limiting LHb hyperactivity could restore motivational deficits in grooming behavior, we then tested the effects of Gi (hM4Di)-DREADD-mediated inhibition of LHb activity in the sucrose splash test. We found that chemogenetic inhibition of LHb glutamatergic neurons was sufficient to reverse mTBI-induced delays in grooming behavior. Overall, our study provides the first evidence for persistent LHb neuronal dysfunction due to an altered synaptic integration as causal neural correlates of dysregulated motivational states by mTBI.

Keywords: DREADD; LHb, mild traumatic brain injury; electrophysiology; lateral habenula; mTBI.

FIG. 1.

Effects of mild traumatic brain injury (mTBI) on lateral habenula (LHb) spontaneous neuronal activity. Pie charts and representative traces of voltage-clamp cell-attached recordings (A; V = 0 mV, sham, n = 68/11; mTBI, n = 65/14) and current-clamp whole-cell recordings (B; I = 0 pA, sham, n = 30/11; mTBI, n = 27/14) of spontaneous neuronal activity across sham and mTBI mice. Comparison of the percent distributions of silent (black), tonic (blue), or bursting (red) LHb neurons increased their tonic LHb neuronal activity while decreased their bursting activity following mTBI. *p < 0.05, **p < 0.01 by chi-squared tests; n represents the number of recorded cells/mice.

FIG. 2.

Effects of mild traumatic brain injury (mTBI) on spontaneous synaptic activity in lateral habenula (LHb) neurons. (A) shows representative voltage-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs; recorded at -55 mV) and spontaneous inhibitory postsynaptic currents (sIPSCs; recorded at +10 mV) within the same LHb neurons in sham (left, black) and mTBI (right, red) mice (calibration bars, 50 pA/5 sec). (B-E) show average and cumulative probability amplitude and frequency (inter-event interval) plots of sEPSCs and sIPSCs within the same LHb neurons from sham and mTBI mice at 2 weeks following the injury. mTBI significantly shifted cumulative probability curves of sEPSC and sIPSC amplitude and frequency, resulting in an overall decreased spontaneous excitatory and inhibitory transmission in LHb neurons. ****p < 0.0001 by Kolmogorov–Smirnov tests; n in this and all following graphs represents the number of recorded cells/mice.

FIG. 3.

Effects of mild traumatic brain injury (mTBI) on excitation/inhibition balance. (A), (C), and (E) show the histograms of spontaneous excitatory/inhibitory (sE/I) amplitude, frequency and synaptic drive ratios in lateral habenula (LHb) neurons recorded from sham and mTBI mice. (B), (D), and (F) show the average and cumulative probability plots of the sE/I amplitude, frequency and synaptic drive in LHb neurons recorded from sham and mTBI mice. mTBI significantly shifted the distribution curves of sE/I amplitude, frequency and synaptic drive ratios, resulting in an overall increased excitatory synaptic drive in LHb neurons. ****p < 0.0001 by Kolmogorov–Smirnov tests; n represents the number of recorded cells/mice.

FIG. 4.

Effects of mild traumatic brain injury (mTBI) at lateral habenula (LHb) glutamatergic synapses. (A) shows representative AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) traces from sham (black) and mTBI (red) mice (calibration bars, 30 pA/5 sec). Graph shows average and cumulative probability plots of mEPSC (B) amplitude, (C) charge transfer (area under the curve), (D) frequency (inter-event interval), and (E) decay time constants (Tau) in sham and mTBI mice at two weeks following the injury. Mild TBI significantly decreased the average charge transfer and Tau decay of mEPCSs and shifted the cumulative probability curves of mEPSC frequency and tau decay, suggesting mTBI-induced decreases in the probability of presynaptic glutamate release onto LHb neurons and changes in the kinetics of AMPAR mEPSCs. *p < 0.05, ****p < 0.0001 by unpaired Student's t-tests or Kolmogorov–Smirnov tests.

FIG. 5.

Effects of mild traumatic brain injury (mTBI) at lateral habenula (LHb) GABAergic synapses. (A) shows representative GABA_AR-mediated miniature inhibitory postsynaptic current (mIPSC) traces from sham (black) and mTBI (red) mice (calibration bars, 50 pA/5 sec). Graph shows average and cumulative probability plots of mIPSC (B) amplitude, (C) charge transfer (area under the curve), (D) frequency (inter-event interval), and (E) decay time constants (Tau) in sham and mTBI mice at 2 weeks following the injury. Mild TBI significantly shifted the cumulative probability curves of mIPSC amplitude, charge transfer and frequency, suggesting mTBI-induced decreases in postsynaptic GABA_AR function accompanied by increases in the probability of presynaptic GABA release onto LHb neurons. ****p < 0.0001 by Kolmogorov–Smirnov tests.

FIG. 6.

Effects of mild traumatic brain injury (mTBI) on sucrose splash and preference tests. (A, B) Mild TBI significantly increased the latency to start grooming without an overall change in total grooming time in the sucrose splash test (Sham: n = 8/group; mTBI: n = 9/group). C) mTBI did not alter sucrose preference in sucrose preference test (Sham: n = 10/group; mTBI: n = 10/group). Unpaired Student's t test, **p < 0.01.

FIG. 7.

Effects of chemogenetic inhibition of lateral habenula (LHb) glutamatergic neurons in sucrose splash test in sham and mTBI mice. A shows representative image of a coronal section of LHb with bilateral injection of AAV-CamkIIa-hM4D (Gi)-mCherry (Gi-DREADD, purple) in the LHb (300 μm). (B) shows sample current clamp recording of action potentials from a Gi-DREADD expressing LHb glutamatergic neuron at baseline (black trace), 15 min (middle, purple trace) and 30 min (top, purple trace) after bath application of the DREADD-specific agonist, JHU37160 (100 nM), in LHb slice from a Gi-DREADD mouse demonstrating the effectiveness of the Gi-DREADD strategy in chemogenetic inhibition of LHb glutamatergic neurons (calibration bars, 100 pA or 50mV/5 sec). (C, D) show sham (n = 3/group) and mTBI (n = 7-8/group) mice injected with control virus (AAV-CamKII-eGFP) into the LHb 4 weeks prior behavioral testing that received intraperitoneal injections of saline or 0.3 mg/kg JHU37160 30 min before sucrose splash tests. Latencies to grooming and total grooming time were measured following sucrose splash in sham and mTBI control mice. Mild TBI increased grooming latencies with no effects of JHU37160 on behavior in sham or mTBI control mice. (E, F) show sham (n = 8-9/group) and mTBI (n = 6-8/group) mice injected with Gi-DREADD into the LHb that received intraperitoneal injections of either saline or 0.3 mg/kg JHU37160 30 min before sucrose splash tests. Latencies to start grooming and total grooming time were measured following sucrose splash in sham and mTBI Gi-DREADD mice. Mild TBI-induced increases in grooming latencies were reversed by chemogenetic inhibition of LHb glutamatergic neurons. Two-way analysis of variance; *p < 0.05, ***p < 0.001.