Dysregulation of kappa opioid receptor neuromodulation of lateral habenula synaptic function following a repetitive mild traumatic brain injury

Abstract

Mild traumatic brain injury (mTBI) increases the risk of affective disorders, anxiety and substance use disorder. The lateral habenula (LHb) plays an important role in pathophysiology of psychiatric disorders. Recently, we demonstrated a causal link between mTBI-induced LHb hyperactivity due to excitation/inhibition (E/I) imbalance and motivational deficits in male mice using a repetitive closed head injury mTBI model. A major neuromodulatory system that is responsive to traumatic brain injuries, influences affective states and also modulates LHb activity is the dynorphin/kappa opioid receptor (Dyn/KOR) system. However, the effects of mTBI on KOR neuromodulation of LHb function are unknown. Here, we first used retrograde tracing in male and female Cre mouse lines and identified several major KOR-expressing and two prominent Dyn-expressing inputs projecting to the mouse LHb, highlighting the medial prefrontal cortex (mPFC) and the ventromedial nucleus of the hypothalamus (VMH) as the main LHb-projecting Dyn inputs that regulate KOR signaling to the LHb. We then functionally evaluated the effects of in vitro KOR modulation of spontaneous synaptic activity within the LHb of male and female sham and mTBI mice at 4 week post-injury. We observed sex-specific differences in spontaneous release of glutamate and GABA from presynaptic terminals onto LHb neurons with higher levels of presynaptic glutamate and GABA release in females compared to male mice. However, KOR effects on the spontaneous E/I ratios and synaptic drive ratio within the LHb did not differ between male and female sham and mTBI mice. KOR activation generally suppressed spontaneous glutamatergic transmission without altering GABAergic transmission, resulting in a significant but sex-similar reduction in net spontaneous E/I and synaptic drive ratios in LHb neurons of sham mice. Following mTBI, while responses to KOR activation at LHb glutamatergic synapses remained intact, LHb GABAergic synapses acquired an additional sensitivity to KOR-mediated inhibition where we observed a reduction in GABA release probability in response to KOR stimulation in LHb neurons of mTBI mice. Further analysis of percent change in spontaneous synaptic ratios induced by KOR activation revealed that independent of sex mTBI switches KOR-driven synaptic inhibition of LHb neurons (normally observed in sham mice) in a subset of mTBI mice toward synaptic excitation resulting in mTBI-induced divergence of KOR actions within the LHb. Overall, we uncovered the sources of major Dyn/KOR-expressing synaptic inputs projecting to the mouse LHb. We demonstrate that an engagement of intra-LHb Dyn/KOR signaling provides a global KOR-driven synaptic inhibition within the mouse LHb independent of sex. The additional engagement of KOR-mediated action on LHb GABAergic transmission by mTBI could contribute to the E/I imbalance after mTBI, with Dyn/KOR signaling serving as a disinhibitory mechanism for LHb neurons of a subset of mTBI mice.

Keywords: Dynorphin; Electrophysiology; Kappa opioid receptor; Lateral habenula; Mild traumatic brain injury; Synaptic transmission.

Figure 1.

Example labeled regions from KOR-Cre (left column) and Dyn-Cre mice with LHb injection with AAVrg-EF1α-DIO-EYFP. Scale bar: 200μm. Table displays relative gradient of labeling: - none; (+) sparse <5 EYFP-labeled cells; + medium 5–25 EYFP-labeled cells; high 25–50 EYFP-labeled cells; very high >50 EYFP-labeled cells within the sections through the named structure across Cortex, Striatum, Pallidum, Amygdala, Thalamus, Hypothalamus and Midbrain. Cg: cingulate cortex, PrL: prelimbic, IL: infralimbic, CP: caudate putamen, NAc: nucleus accumbens, VP: ventral pallidum, BNST: bed nucleus of stria terminalis, EP/GPi (entopeduncular nucleus/globus pallidus internus), BLA: basolateral amygdala. CeA: central amygdala, Ext. Amg: extended amygdala, aPVT: anterior paraventricular thalamus, AVPV: Anteroventral periventricular nucleus, LPO: lateral preoptic area, PVN: paraventricular nucleus of hypothalamus, LHA: lateral hypothalamus, VMH: ventromedial nucleus of the hypothalamus, PH: posterior hypothalamus, ZI: zona incerta, PAG: periaqueductal gray, RLi: rostral linear nucleus of the raphe, and IF: interfasicular nucleus, VTA: ventral tegmental area

Figure 2.. Effects of mTBI on spontaneous synaptic activity in LHb neurons in female mice.

A-D show average and cumulative probability amplitude and frequency (inter-event interval) plots of sEPSCs and sIPSCs within the same LHb neurons from female sham (black filled round symbols) and mTBI (black open round symbols) mice at ~4 weeks following the injury. mTBI significantly shifted cumulative probability curves of sEPSC and sIPSC amplitude and frequency, resulting in an overall decreased presynaptic spontaneous excitatory and inhibitory transmission in LHb neurons while potentiating postsynaptic spontaneous synaptic activity in female mice. *p<0.05, **p<0.01, ****p<0.0001 by Kolmogorov–Smirnov tests. “(n)” in this and all following graphs represents the number of recorded cells/mice.

Figure 3.. Effects of mTBI on excitation/inhibition balance.

A, B, and C show the average and cumulative probability plots of the sE/I amplitude, frequency and excitatory synaptic drive in LHb neurons recorded from female sham (black filled round symbols) and mTBI (black open round symbols) mice. mTBI significantly shifted the distribution curves of sE/I frequency and synaptic drive ratios, resulting in an overall increased excitatory synaptic drive in LHb neurons of female mice. **p<0.01, ****p<0.0001 by Kolmogorov–Smirnov tests. D shows representative voltage-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs, recorded at −55mV) 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, 50pA/1 s).

Figure 4.. Effects of KOR activation by a selective KOR agonist (U50, 488) on sEPSC amplitude in LHb neurons in male and female sham and mTBI mice.

A-E show average and cumulative probability amplitude plots of sEPSCs within the same LHb neurons from male and female sham mice (black filled squares and circles representing data from males and females, respectively) and male and female mTBI mice (black open squares and circles representing data from males and females, respectively) before and after bath application of U50,488, 10μM (magenta filled squares and circles representing data from sham males and females, and magenta open squares and circles representing data from mTBI males and females, respectively) at ~4 weeks following the injury. In cumulative probability plots, data from sham males and females are shown by black filled squares and circles (before) and magenta filled squares and circles (after U50,488 application) and data from mTBI males and females are shown by black open squares and circles (before) and magenta open squares and circles (after U50,488 application). KOR activation significantly decreased postsynaptic spontaneous excitatory transmission in LHb neurons of male and female sham and mTBI mice. Three-way repeated measures ANOVA showed significant main effect of U50,488. *p<0.05, ***p<0.001, ****p<0.0001 by Kolmogorov–Smirnov tests.

Figure 5.. Effects of KOR activation by a selective KOR agonist (U50, 488) on sEPSC frequency in LHb neurons in male and female sham and mTBI mice.

A-E show average and cumulative probability frequency (inter-event interval) plots of sEPSCs within the same LHb neurons from male and female sham mice (black filled squares and circles representing data from males and females, respectively) and mTBI mice (black open squares and circles representing data from males and females, respectively) before and after bath application of U50,488, 10μM (magenta filled squares and circles representing data from sham males and females, and magenta open squares and circles representing data from mTBI males and females, respectively) at ~4 weeks following the injury. In cumulative probability plots, data from sham males and females are shown by black filled squares and cirles (before) and magenta filled squares and circles (after U50,488 application) and data from mTBI males and females are shown by black open squares and circles (before) and magenta open squares and circles (after U50,488 application). KOR activation significantly decreased presynaptic spontaneous excitatory transmission in LHb neurons of sham and mTBI male and female mice. Three-way repeated measures ANOVA showed significant main effects of sex and U50,488. ****p<0.0001 by Kolmogorov–Smirnov tests.

Figure 6.. Effects of KOR activation by a selective KOR agonist (U50, 488) on sIPSC amplitude in LHb neurons in male and female sham and mTBI mice.

A-E show average and cumulative probability amplitude plots of sIPSCs within the same LHb neurons from male and female sham mice (black filled squares and circles representing data from males and females, respectively) and mTBI mice (black open squares and circles representing data from males and females, respectively) before and after bath application of U50,488, 10μM (magenta filled squares and circles representing data from sham males and females, and magenta open squares and circles representing data from mTBI males and females, respectively) at ~4 weeks following the injury. In cumulative probability plots, data from sham males and females are shown by black filled squares and circles (before) and magenta filled squares and circles (after U50,488 application) and data from mTBI males and females are shown by black open squares and circles (before) and magenta open squares and circles (after U50,488 application). KOR activation did not alter postsynaptic spontaneous GABAergic transmission in LHb neurons of sham and mTBI male and female mice.

Figure 7.. Effects of KOR activation by a selective KOR agonist (U50, 488) on sIPSC frequency in LHb neurons in male and female sham and mTBI mice.

A-E show average and cumulative probability frequency (inter-event interval) plots of sIPSCs within the same LHb neurons from male and female sham mice (black filled squares and circles representing data from males and females, respectively) and mTBI mice (black open squares and circles representing data from males and females, respectively) before and after bath application of U50,488, 10μM (magenta filled squares and circles representing data from sham males and females, and magenta open squares and circles representing data from mTBI males and females, respectively) at ~4 weeks following the injury. In cumulative probability plots, data from sham males and females are shown by black filled squares and circles (before) and magenta filled squares and circles (after U50,488 application) and data from mTBI males and females are shown by black open squares and circles (before) and magenta open squares and circles (after U50,488 application). KOR activation significantly decreased presynaptic spontaneous GABAergic transmission in LHb neurons of mTBI male and female mice. Three-way repeated measures ANOVA showed significant main effects of sex and U50,488. **p<0.01, ****p<0.0001 by Kolmogorov–Smirnov tests.

Figure 8.. Effects of KOR activation on excitation/inhibition balance in male and female sham and mTBI mice.

Figure shows the average and cumulative probability plots of the sE/I amplitude, frequency and excitatory synaptic drive ratios in LHb neurons recorded from male and female sham mice (left panels, A-C, filled symbols) or male and female mTBI mice (right panel, D-F, open symbols) before (black) and after U50,488 bath application (magenta). In the average plots square symbols represent data from males, and round symbols show data from females. In cumulative probability plots, data from males and females are shown as triangle symbols. KOR stimulation significantly decreased sE/I amplitude, frequency or excitatory synaptic drive in LHb neurons of male and female sham mice while mTBI mice did not show any alterations in these measurements by KOR stimulation. *p<0.05, ****p<0.0001 by Paired Student’s t-tests and Kolmogorov–Smirnov tests.

Figure 9.. Effects of KOR activation by a selective KOR agonist (U50, 488) on percent change from baseline of spontaneous synaptic activity measurements in LHb neurons of male and female sham and mTBI mice.

A-G show KOR-driven percent changes in sEPSC amplitude, sEPSC frequency, sIPSC amplitude, sIPSC frequency, sE/I amplitude ratio, sE/I frequency ratio and synaptic drive ratio from male and female sham and mTBI mice (magenta filled squares and circles representing data from sham males and females, and magenta open squares and circles representing data from mTBI males and females, respectively) at ~4 weeks following the injury. Two-way repeated measures ANOVA only showed a trend for main effect of mTBI in percent change of synaptic drive ratios.

Figure 10.. Effects of mTBI on KOR-mediated neuromodulation of percent change of synaptic drive ratios within the LHb.

A shows the pie charts of pooled data across male and female mice in sham and mTBI groups with either KOR-mediated increase (excitatory) or decrease (inhibitory) in percent change of synaptic drive ratios. Purple represents KOR-mediated excitation and black represents KOR-mediated inhibition within the LHb (left, sham: n=12/12, right, mTBI: n=14/14), *p<0.05 by Chi squared tests.