AMPAR-mediated synaptic transmission in lateral IPN mediated by mHB substance P neurons is inhibited by mOR activation

(a) 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. (b) Conditional td-Tomato expression in cholinergic (left panels; ChATcre:Ai9) and SP neurons (right panels; Tac1Cre:Ai9) illustrating their axonal outputs via the fasciculus retroflexus (fr) and largely non-overlapping axonal arborization patterns in IPN. (c) 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 are DAPI stained nuclei. (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 IPL). Single voltage-clamp traces of light evoked AMPAR EPSCs (bottom left panel; 470nM light pulse; 2 stimulations at 20Hz, blue dashes)) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (e,f) Individual (grey filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPR; n=5 recorded IPL neurons from 5 mice).

AMPAR-mediated synaptic transmission in rostral IPN mediated by mHB cholinergic neurons is potentiated by mOR activation

(a) Whole-cell voltage-clamp in adult (>p40) ChATCre: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; 470nM light pulse; 2 stimulations at 20Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions. (b,c) Individual (grey filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPR; n=8 recorded IPR neurons from 7 mice). (d) Summary bar graph illustrating the effect of DAMGO (500nM and 100nM; n=8 recorded IPR neurons from 7 mice and n=9 recorded IPR neurons from 4 mice, respectively), met-enkephalin (5μm; n=7 recorded IPR neurons from 5 mice), morphine (10μM; n=5 recorded IPR neurons from 5 mice) and 500nM DAMGO in presence of mOR antagonist 1mM CTAP (n=4 recorded IPR neurons from 2 mice). (e) 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 (470nm; 6.9mW/mm2). (f) Individual (gray filled and half-filled symbols for 500nM DAMGO or 5μM Met-enkephalin respectively) and pooled (black filled symbols) data of light-evoked AMPAR EPSC amplitude during DAMGO application and washout (n = 4 and 3 recorded IPR cells from 4 and 3 mice for DAMGO and met-enkephalin application, respectively).

mOR activation increases fidelity of glutamatergic transmission mediated by 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 (470nm, 5 pulses delivered at 5Hz; blue dashes) AMPAR EPSCs recorded from postsynaptic IPR neurons in adult ChATCre:Ai32 mice (p>40) under baseline and following 500nM DAMGO application (left panel). (c) Pooled data (shaded area denotes SEM) of peak amplitude for each given stimulus in the 5Hz train during baseline and following 500nM DAMGO application (n = 5 recorded IPR neurons from 5 mice). (d) Single current-clamp traces example (5 consecutive overlaid sweeps) of light-evoked EPSP:action potential coupling (5 stimuli at 5 Hz; blue dashes) under baseline, 500nM 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 5 consecutive traces of EPSP:action potential coupling for each given stimulus in the 5Hz train under baseline (open symbols), 500nM DAMGO (black symbols) and washout (grey symbols) conditions (n = 7 recorded IPR neurons from 6 mice).

mOR activation imparts opposing effects on evoked versus spontaneous AMPAR EPSCs mediated by mHB cholinergic neurons and demonstration of functional SP neuronal mediated evoked AMPAR EPSCs in IPR.

(a) Single voltage-clamp traces (top panels) illustrating light-evoked AMPAR EPSCs mediate by cholinergic neurons and spontaneous EPSCs (sEPSCs) under baseline and following 10μM DNQX application in an IPR neuron. Individual (grey symbols) and pooled data (black symbols) illustrating complete cessation of sEPSCs as assessed by frequency following 10μM DNQX application (n = 8 recorded IPR neurons from 8 mice; bottom panel). (b) Single voltage-clamp traces under baseline, 500nM DAMGO and washout conditions of light-evoked AMPAR EPSCs mediated by mHb cholinergic neurons (2 stimuli at 20Hz; 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 (grey 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). (g) 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. (h) 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. (i) Comparison of light-evoked AMPAR EPSC peak amplitude in postsynaptic IPR neurons mediated by either cholinergic (ChATCre:Ai32 mice; n = 32 recorded IPR neurons) and substance P (Tac1Cre:Ai32 mice; n = 31 recorded IPR neurons) mHB neurons at various arbitrary % LED (10–50% corresponding to an approximate power of 0.4 – 3.4 mW/mm2; right panel).

mORs constitute a developmentally regulated molecular switch altering the salience of neurotransmission in IPR mediated by substance P (SP) versus cholinergic neurons

(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 IPR). Single voltage-clamp traces of light evoked AMPAR EPSCs (bottom left panel; 470nM light pulse; 2 stimulations at 20Hz, blue dashes) and time course of peak amplitudes (right panel) under baseline, during application of 500 nM DAMGO and washout conditions (right panel). (b,c) Individual (grey filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude and corresponding paired pulse ratios (PPR; n=7 recorded IPR neurons from 7 mice). (d) Confocal images of mOR protein expression in IPN during development (p5-p40). (e) Single examples of the time course of light-evoked AMPAR EPSC peak amplitudes mediated by mHB cholinergic neurons following DAMGO application in postsynaptic IPR neurons at varying ages as indicated. (f) Pooled data of the percent change of light-evoked cholinergic neuronal mediated EPSC peak amplitude elicited following DAMGO application across all ages tested (n = 44 recorded IPR neurons). (g) 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. (h) Pooled data of the percent change of light-evoked SP neuronal mediated EPSC peak amplitude elicited following DAMGO application across all ages tested. (i) 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)91. Error bars denote standard deviation of the mean. Note panels f,g and h,i contain normalized data from the same recorded cells depicted in Figs. 2b and 5b as absolute peak amplitude changes, respectively.

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 (cf. Figure 4g). (b) In situ hybridization for KCNA2 mRNA (top panel) and corresponding pseudo-colored expression level (bottom panel) illustrating bias towards ventral mHb. Data are from the Allen Brain Institute (https://mouse.brain-map.org/gene/show/16263). (c) Voltage-clamp example traces of light-evoked EPSCs mediated by cholinergic mHb neurons (left panel) under baseline, following 10μM DNQX/100μM DL-APV plus 50μM 4-AP (top panel) or plus 100nM dendrotoxin-α (bottom panel). (d) Voltage-clamp example trace of light-evoked EPSCs mediated by cholinergic mHb neurons in the presence of 10μM DNQX/100μM DL-APV/100μM 4-AP. Light delivered at 5Hz for 10 stimuli under baseline conditions (top panel) or 1 stimulus in the absence or presence of 2μM DHβE (bottom panel). (e) Box plot with individual data of the percentage inhibition of the light-driven EPSC peak amplitude mediated by cholinergic neurons in the presence of 10μM DNQX/100μM DL-APV/50μM 4-AP (n = 7 recorded IPR neurons from 7 mice). (f) Voltage-clamp example trace of light-evoked EPSCs under baseline and after addition of 10μM DNQX/50μM DL-APV/100μM 4-AP in ChAT-ChR2 (top panel) and Tac1Cre:ai32 (bottom panel). (g) Box plot with individual data of the nicotine/AMPA peak amplitude ratio within individual recorded IPR neurons percentage as measured under baseline (AMPA EPSC) and in the presence of 10μM DNQX/50μM DL-APV/100μM 4-AP (nAChR EPSC; n = 15 recorded IPR neurons from 15 ChATCre:Ai32 mice) or 100nM dendrotoxin-α (nAChR EPSC; n = 3 recorded IPR neurons from 3 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). (h) Voltage-clamp example trace of light-evoked nAChR-mediated EPSCs mediated by cholinergic mHb neurons under baseline and following application of 50nM ambenonium (left panel). Scatter plot of the individual (grey symbols) and pooled (red symbol) percentage change in nAChR EPSC peak amplitude versus EPSC charge (measured over the first 500ms duration of the EPSC) in each individual recording (n = 4 recorded IPR neurons in 4 mice; right panel).

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 (2 stimuli at 20Hz; blue dashes) mediated by cholinergic mHb neurons (left panel) and time course of peak amplitude (right panel) under baseline, 500nM DAMGO and 500nM DAMGO plus 2μm 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, 500nM DAMGO and 500nM DAMGO plus 2μm DHβE conditions. (e) Percentage change in electrical and light-evoked nAChR EPSC peak amplitude elicited by 500nM DAMGO in each individual recorded IPR neuron (n = 6 recorded IPR 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 (2 stimuli at 20Hz; blue dashes) mediated by cholinergic mHb neurons (top and bottom left panels, respectively) and time course of peak amplitude (right panels) under baseline, 500nM DAMGO, 1μM 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 2 of the 4 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 data in this panel are replotted from the absolute peak EPSC amplitude changes mediated by mOR agonists in Figs. 1e, 2b, 5a and 7b.

mOR induced potentiation of light-driven EPSCs by mHb cholinergic neurons are mediated exclusively by AMPARs.

(a) Voltage-clamp example traces of light-evoked AMPAR EPSCs mediated by cholinergic mHb neurons (left panel) and time course of peak amplitude (right panel) under baseline, 10μM DNQX and 10μm DNQX plus 500nM DAMGO conditions. (c) Individual (grey filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude under baseline, 10μM DNQX and 10μm DNQX plus 500nM DAMGO conditions (n = 4 recorded IPR neurons from 4 mice). (c) Voltage-clamp example trace of single light-evoked AMPAR mediated EPSCs mediated by cholinergic mHb neurons (left panel) and time course of peak amplitude (right panel) under baseline, 500nM DAMGO and 500nM DAMGO plus 10μm DNQX conditions. (d) Individual (grey filled symbols) and mean (black filled symbols) data of AMPAR EPSC amplitude under, baseline, 500nM DAMGO and 500nM DAMGO plus 10μm DNQX conditions. (n = 3 recorded IPR neurons from 3 mice).