Complex opioid-driven modulation of glutamatergic and cholinergic neurotransmission in a GABAergic brain nucleus associated with emotion, reward, and addiction
- Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
Figures
Figure 1
Oprm1 gene and mu-opioid receptor (mOR) protein expression in the habenula–interpeduncular axis.
(a) 10X scRNAseq UMAP whole mouse brain showing all cellular classes (top left panel; 4.04 million cells) and Class 17 MH-LH Glut (right panel; corresponding to the medial habenula (mHb) and lateral habenula; 10.8K cells). Bottom panels focus on individual mHb cells (Subclass 145 MH; 8K cells) indicating log2 mRNA expression of Chat and Tac1. (b) Confocal image of a Tac1Cre:Ai9 mouse immunostained with anti-ChAT illustrating the distinct distribution of substance P (red) and cholinergic neurons (green) in dorsal and ventral mHb, respectively. (c) Conditional td-Tomato expression in cholinergic (left panels; ChatCre:Ai9) and SP neurons (right panels; Tac1Cre:Ai9) illustrating their spatial location within the mHb with their axonal outputs in the fasciculus retroflexus (fr) and largely non-overlapping terminal axonal arborization patterns in interpeduncular nucleus (IPN). (d) Profile of log2 mRNA expression of Oprm1 in Subclass 145 MH (top left panel). Corresponding dot and violin plots depicting three Chat (0632, 0634, and 0635) and one Tac1 (0633) supertype with relative expression of Slc17a7 (VGluT1) and Oprm1 in each corresponding supertype. In the violin plots, the Chat and Tac1 supertypes are color-coded blue and red, respectively. (e) Endogenous mOR expression throughout the mHb and IPN axis (left panels; red) assessed by immunocytochemistry. High-resolution airy scan images of mOR distribution in subdivisions of the IPN; rostral IPN (IPR), lateral IPN (IPC), and central IPN (IPC). Green fluorescence is DAPI-stained nuclei. (f) Densitometry analyses of mOR expression in the various subfields of IPN. Data are from two to four slices containing IPN taken from each of six mice aged P40–P60. Data depicted in (a) and (d) are from the publicly available Allen Brain cell Atlas (https://knowledge.brain-map.org/abcatlas). See methods for further details.
Figure 2
AMPAR-mediated synaptic transmission in lateral IPN (IPL) mediated by substance P neurons is inhibited by mu-opioid receptor (mOR) activation.
(a) Whole-cell voltage-clamp in adult (>p40) Tac1Cre:Ai32 mice (top left panel illustrating the axonal arborization of ChR2 expressing SP neuronal axons in IPN and the position of neuronal recording in IPL). Single voltage-clamp traces of light-evoked AMPAR EPSCs (bottom left panel; 470 nM light pulse; two stimulations at 20 Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (b, c) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPRs; n = 9 recorded IPL neurons from 7 mice).
Figure 3 with 1 supplement
AMPAR-mediated synaptic transmission in rostral IPN (IPR) mediated by medial habenula (mHb) cholinergic neurons is potentiated by mu-opioid receptor (mOR) activation.
(a) Whole-cell voltage-clamp in adult (>p40) ChatCre:Ai32 mice (top left panel illustrating the axonal arborization of ChR2 expressing cholinergic neuronal axons in IPN and the position of neuronal recording in IPR). Single voltage-clamp traces of light-evoked AMPAR EPSCs (bottom left panel; 470 nM light pulse; two stimulations at 20 Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (b, c) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPRs; n = 8 and 6 recorded IPR neurons from 7 and 5 mice for AMPAR EPSC amplitude and PPR, respectively). (d) Whole-cell voltage-clamp in adult (>p40) ChatChR2:SSTCre:Ai9 mice (top left panel illustrating the axonal arborization of ChR2 expressing cholinergic neuronal axons in IPN and the position of neuronal recording in RFP+ SST IPR neurons). Single voltage-clamp traces of light-evoked AMPAR EPSCs (bottom left panel; 470 nM light pulse; two stimulations at 20 Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (e, f) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding PPRs (n = 5 recorded IPR neurons from 4 mice). (g) Summary bar graph illustrating the effect of DAMGO (500 and 100 nM; n = 16 recorded IPR neurons from 14 mice and n = 9 recorded IPR neurons from 4 mice, respectively), met-enkephalin (5 µm; n = 7 recorded IPR neurons from 5 mice), morphine (10 µM; n = 5 recorded IPR neurons from 5 mice) and 500 nM DAMGO in presence of mOR antagonist 1 mM CTAP (n = 7 recorded IPR neurons from 2 mice). (h) Voltage-clamp trace examples (single light stimulus; blue bar; left panel) and time course (right panel) under baseline, DAMGO, and washout conditions in a recorded IPR neuron displaying no measurable baseline AMPAR EPSC in response to maximal light-evoked stimulation (470 nm; 6.9 mW/mm2). (i) Individual (gray filled and half-filled symbols for 500 nM DAMGO or 5 µM Met-enkephalin, respectively) and pooled (black filled symbols) data of light-evoked AMPAR EPSC amplitude during DAMGO application and washout (n = 6 and 3 recorded IPR cells from 6 and 3 mice for DAMGO and met-enkephalin application, respectively).
Figure 3—figure supplement 1
AMPAR-mediated synaptic transmission in central IPN (IPC) mediated by medial habenula (mHb) cholinergic neurons is potentiated by mu-opioid receptor (mOR) activation.
(a) Whole-cell voltage-clamp in adult (>p40) ChatCre:Ai32 mice (top left panel illustrating the axonal arborization of ChR2 expressing cholinergic neuronal axons in IPN and the position of neuronal recording in IPC). Single voltage-clamp traces of light-evoked AMPAR EPSCs (bottom left panel; 470 nM light pulse; single stimulations, blue dash) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (b) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude (n = 8 recorded IPC neurons from 7 mice).
Figure 4
Mu-opioid receptor (mOR) activation increases fidelity of glutamatergic transmission mediated by medial habenula (mHb) cholinergic neurons to augment excitation:spike coupling in postsynaptic IPR neurons.
(a) Single example trace showing spontaneous action potential firing in cell-attached mode from a td-Tomato-positive ventral mHb neuron in the ChatCre:Ai9 mouse (top panel). Box plot and corresponding individual data of the spontaneous firing frequency of mHb cholinergic neurons (bottom panel; n = 27 recorded cells). (b) Single voltage-clamp traces of light-evoked (470 nm, five pulses delivered at 5 Hz; blue dashes) AMPAR EPSCs recorded from postsynaptic IPR neurons in adult ChatCre:Ai32 mice (p > 40) under baseline and following 500 nM DAMGO application (left panel). (c) Pooled data (shaded area denotes SEM) of peak amplitude for each given stimulus in the 5 Hz train during baseline and following 500 nM DAMGO application (n = 5 recorded IPR neurons from 5 mice). (d) Single current-clamp traces example (five consecutive overlaid sweeps) of light-evoked EPSP:action potential coupling (five stimuli at 5 Hz; blue dashes) under baseline, 500 nM DAMGO, and washout conditions (left panel). Corresponding single example time course plot depicting number of light-driven EPSP-evoked action potential (right panel). (e) Mean data showing percentage success over five consecutive traces of EPSP:action potential coupling for each given stimulus in the 5 Hz train under baseline (open symbols), 500 nM DAMGO (black symbols), and washout (gray symbols) conditions (n = 9 recorded IPR neurons from 7 mice).
Figure 5 with 1 supplement
Effect of mu-opioid receptor (mOR) activation on a novel functional SP neuronal-mediated evoked AMPAR EPSCs in IPR.
(a) High-resolution airy scan of the IPR in Tac1Cre:Ai32 mouse showing ChR2-expressing synaptic bouton-like structures (green) and endogenous ChAT expression (red). Right panels are magnified regions of the boxed area. Arrows indicate examples of non-overlap of TacCre:Ai32 boutons with ChAT. (b) High-resolution airy scan of the IPR in Tac1Cre:Ai32 mouse showing ChR2-expressing synaptic bouton-like structures (green) and endogenous VGlut1 expression via immunostaining (red). Right panels are magnified regions of the boxed area. Arrows indicate examples of expression of VGluT1 within TacCre:Ai32 bouton structures. (c) Comparison of light-evoked AMPAR EPSC peak amplitude in postsynaptic IPR neurons mediated by either cholinergic (ChatCre:Ai32 mice; n = 32 recorded IPR neurons) or substance P (Tac1Cre:Ai32 mice; n = 31 recorded IPR neurons) medial habenula (mHb) neurons at various arbitrary % LED (10–50% corresponding to an approximate power of 0.4–3.4 mW/mm2; right panel). (d) Whole-cell voltage-clamp in adult (>p40) Tac1Cre:Ai32 mice (top left panel illustrating the axonal arborization of ChR2-expressing SP neuronal axons in IPN and the position of neuronal recording in IPR). Single voltage-clamp traces of light-evoked AMPAR EPSCs (bottom left panel; 470 nM light pulse; two stimulations at 20 Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions (right panel). (e, f) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPRs; n = 8 recorded IPR neurons from 7 mice).
Figure 5—figure supplement 1
Mu-opioid receptor (mOR) activation imparts opposing effects on spontaneous AMPA EPSCs versus cholinergic neuron-mediated evoked AMPAR EPSCs in IPR.
(a) Single voltage-clamp traces (top panels) illustrating light-evoked AMPAR EPSCs mediated by cholinergic neurons and spontaneous EPSCs (sEPSCs) under baseline and following 10 µM DNQX application in an IPR neuron. Individual (gray symbols) and pooled data (black symbols) illustrating complete cessation of sEPSCs as assessed by frequency following 10 µM DNQX application (n = 8 recorded IPR neurons from 8 mice; bottom panel). (b) Single voltage-clamp traces under baseline, 500 nM DAMGO, and washout conditions of light-evoked AMPAR EPSCs mediated by medial habenula (mHb) cholinergic neurons (two stimuli at 20 Hz; blue dashes) and sEPSCs (left panels). Magnification of sEPSCs events (right panels) corresponding to the region of the traces in the left panels delineated by the black bars. (c, d) Individual (gray symbols) and mean (black symbols) data of sEPSC frequency and amplitude (n = 12 recorded IPR cells from 11 mice). (e) Box plot and individual data depicting the relative DAMGO-mediated percentage change from baseline of the light-evoked AMPAR EPSC peak amplitude versus AMPAR sEPSC frequency in each individual IPR neuron recorded (n = 12 recorded IPR cells from 11 mice).
Figure 6
Mu-opioid receptors (mORs) constitute a developmentally regulated molecular switch altering the salience of neurotransmission in IPR mediated by substance P versus cholinergic neurons.
(a) Confocal images of mOR protein expression in IPN during development (p10, p20, and p40). Densitometry analyses of mOR protein expression in IPR across development measurements taken from two slices containing IPR from each of two to four mice for each age. (b) Single examples of the time course of light-evoked AMPAR EPSC peak amplitudes mediated by medial habenula (mHb) cholinergic neurons following DAMGO application in postsynaptic IPR neurons at varying ages as indicated. (c) Individual data of the percent change of light-evoked cholinergic neuronal-mediated AMPAR EPSC peak amplitude elicited following DAMGO application across all ages tested (n = 44 recorded IPR neurons). (d) Single examples of the time course of light-evoked AMPAR EPSC peak amplitudes mediated by mHb substance P neurons following DAMGO application in postsynaptic IPR neurons at varying ages as indicated. (e) Individual data of the percent change of light-evoked SP neuronal-mediated EPSC peak amplitude elicited following DAMGO application across all ages tested. (f) Summary plot of the mean changes in the normalized AMPAR EPSC peak amplitude (% baseline) by DAMGO mediated by cholinergic and SP neurons binned at the following developmental epochs; p15–23 (postnatal), p24–34 (adolescent/pre-pubescent), p35–60 (adolescent/pubescent, sexual maturation) and >p60 (adult) (Brust et al., 2015). Total numbers of cells recorded = 39 and 21 cells from ChatCre:Ai32 and Tac1Cre:Ai32 mice, respectively. Error bars denote standard deviation of the mean. Note datapoints for ages >p40 in panels c and e are normalized data taken from recorded cells that were depicted in Figure 3b and Figure 5e as absolute peak amplitude changes, respectively.
Figure 7 with 1 supplement
Kv1 channels constitute a molecular brake of nicotinic receptor-mediated signaling in the IPN.
(a) High-resolution airy scan images of IPR in ChatCre:Ai32 mice showing ChR2-expressing cholinergic boutons (green) and endogenous ChAT (red). Right panels are magnified regions of the boxed area in left panels. Arrows indicate faithful expression of ChAT within ChatCre:Ai32 boutons (Figure 5a). (b) Corresponding dot and violin plots illustrating the relative expression of Kcna-6 in Chat Supertypes only. (c) In situ hybridization for Kcna2 mRNA (top panel) and corresponding pseudo-colored expression level (bottom panel) illustrating bias toward ventral medial habenula (mHb). Data are from the Allen Brain Institute (https://mouse.brain-map.org/gene/show/16263). (d) Voltage-clamp example traces of light-evoked EPSCs mediated by cholinergic mHb neurons (left panel) under baseline, following 10 µM DNQX/100 µM DL-APV plus 50 µM 4-AP (top panel) or plus 100 nM dendrotoxin-α (bottom panel). (e) Voltage-clamp example traces of 5 Hz trains of light-evoked EPSCs (10 stimuli) mediated by cholinergic mHb neurons in the presence of 10 µM DNQX/100 µM DL-APV and 100 µM 4-AP (top panel) or DTX-α (bottom panel) in a ChatCre:Ai32 mouse. (f) Single light stimulus evoked EPSC in the presence of 10 µM DNQX/100 µM DL-APV and 100 µM 4-AP (top left panel) or DTX-α (bottom left panel) in the absence or presence of 2 µM DHβE. Box plot with individual data of the percentage inhibition of the light-driven EPSC peak amplitude mediated by cholinergic neurons in the presence of 10 µM DNQX/100 µM DL-APV/50 µM 4-AP or DTX-α (n = 9 recorded IPR neurons from 9 mice). (g) Box plot of the nicotine/AMPA (nAChR/AMPA) peak amplitude ratio within individual recorded IPR neurons percentage as measured under baseline (AMPA EPSC) and in the presence of 10 µM DNQX/50 µM DL-APV/100 µM 4-AP (nAChR EPSC; n = 15 recorded IPR neurons from 15 ChatCre:Ai32 mice) or 100 nM DTX-α (nAChR EPSC; n = 7 recorded IPR neurons from 5 ChatCre:Ai32 mice). nAChR/AMPA peak EPSC amplitude ratios were also performed in Chat-ChR2 (n = 6 recorded IPR neurons from 6 mice) and Tac1Cre:Ai32 mice (n = 6 recorded IPR neurons from 6 mice). Voltage-clamp example trace of light-evoked EPSCs under baseline and after addition of 10 µM DNQX/50 µM DL-APV/100 µM 4-AP in Chat-ChR2 (top right panel) and Tac1Cre:Ai32 (bottom right panel). (h) Voltage-clamp example trace of light-evoked nAChR-mediated EPSCs mediated by cholinergic mHb neurons under baseline and following application of 50 nM ambenonium (left panel) in a ChatChR2 mouse. Scatter plot of the individual (light red symbols) and pooled (red symbol) percentage change in nAChR EPSC peak amplitude versus EPSC charge (measured over the first 500 ms duration of the EPSC) in each individual recording (n = 7 recorded IPR neurons in 5 mice; right panel). Data in (b) and (c) are from the publicly available Allen Brain Cell Atlas (https://knowledge.brain-map.org/abcatlas) and the Allen Brain Map (Allen Brain; https://mouse.brain-map.org/gene/show/16263), respectively. See methods for further details.
Figure 7—figure supplement 1
Effect of DNQX on synaptic transmission mediated by medial habenula (mHb) cholinergic neurons onto IPN prior to or after mu-opioid receptor (mOR) activation and during high frequency stimulation.
(a, b) Voltage-clamp example traces of light-evoked AMPAR EPSCs mediated by cholinergic mHb neurons and time course of peak amplitude under baseline, 10 µM DNQX and 10 µm DNQX plus 500 nM DAMGO conditions. (c) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude under baseline, 10 µM DNQX and 10 µm DNQX plus 500 nM DAMGO conditions (n = 7 recorded IPR neurons from 5 mice). (d) Voltage-clamp example traces single stimulus light-evoked AMPAR EPSCs mediated by cholinergic mHb neurons stimulation in the presence and absence of 10 µM DNQX (left panels). Traces from the same cell in response to trains of light 1 ms, 25 stimulations given at 50 or 25 Hz; right panels. (e) Pooled data of light-evoked responses in response to single stimulation (black) and during 50 and 25 Hz trains (gray and open symbols, respectively; n = 5 recorded cells from 2 mice). (f, g) Voltage-clamp example trace of single light-evoked AMPAR-mediated EPSCs mediated by cholinergic mHb neurons and time course of peak amplitude under baseline, 500 nM DAMGO, and 500 nM DAMGO plus 10 µm DNQX conditions. (h) Individual (gray filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude under baseline, 500 nM DAMGO, and 500 nM DAMGO plus 10 µm DNQX conditions (n = 5 recorded IPR neurons from 5 mice).
Figure 8
Mu-opioid receptor (mOR) potentiates nAChR EPSC amplitude revealing an interplay between opioid and cholinergic systems in the habenulo-interpeduncular axis.
(a) Voltage-clamp example traces of light-evoked nAChR EPSCs (two stimuli at 20 Hz; blue dashes) mediated by cholinergic medial habenula (mHb) neurons (left panel) and time course of peak amplitude (right panel) under baseline, 500 nM DAMGO, and 500 nM DAMGO plus 2 µm DHβE conditions. (b) Individual and mean (red filled symbols) data of nAChR EPSC peak amplitude. (c, d) Voltage-clamp example traces of simultaneous electrical (lightning symbol) and light-evoked (blue dash) nAChR EPSCs and time course of peak amplitude (open and filled symbols representing electrical and light-evoked peak amplitude of nAChR EPSCs, respectively) under baseline, 500 nM DAMGO, and 500 nM DAMGO plus 2 µm DHβE conditions. (e) Percentage change in electrical and light-evoked nAChR EPSC peak amplitude elicited by 500 nM DAMGO in each individual recorded IPR neuron (n = 6 recorded neurons from 6 mice). (f) Confocal images of endogenous mOR protein expression in IPN of WT and homozygote Oprm1 KO mice. (g) Voltage-clamp example traces of light-evoked AMPA and nAChR EPSCs (two stimuli at 20 Hz; blue dashes) mediated by cholinergic mHb neurons (top and bottom left panels, respectively) and time course of peak amplitude (right panels) under baseline, 500 nM DAMGO, 1 µM baclofen, and washout conditions in ChatChR2: Oprm1KO mice. (h) Individual and mean data of light-evoked AMPAR (black symbols; n = 4 from 4 mice) and nAChR (red symbols; n = 3 from 3 mice) peak amplitude in response to DAMGO and baclofen application in ChatChR2: Oprm1KO mice. Note in two of the four AMPAR EPSC recordings, washout of the baclofen effect was not performed. (i) Summary box plot with individual data of the percentage normalized changes (log2) of AMPAR and nAChR peak amplitudes in response to mOR activation mediated by SP and cholinergic neurons in various subdivisions of IPN tested. Note that the normalized data in this panel are replotted from the absolute peak EPSC amplitude changes mediated by mOR agonists in Figure 2b, Figure 3b, f, Figure 5e and Figure 7b and Figure 3—figure supplement 1b.
Author response image 1
Comparison of the mOR (500nM DAMGO) mediated potentiation on evoked (a) AMPAR and (b) nAChR EPSCs in IPR between male and female mice.
Author response image 2
Sterotaxic viral injection into mHB pf Tac1Cre mice taken from Allen Brain connectivity atlas (Link to Connectivity Atlas for mHb SP neuronal projection pattern).
Author response image 3
Comparison of the mOR (500nM DAMGO) mediated potentiation on evoked AMPAR (a) and nAChR (b)EPSCs in IPR between ChATCre:Ai32 and ChATChR2 mice.
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Complex opioid-driven modulation of glutamatergic and cholinergic neurotransmission in a GABAergic brain nucleus associated with emotion, reward, and addiction
eLife 14:RP106062.
https://doi.org/10.7554/eLife.106062.3
