Chronic morphine treatment induces sex- and synapse-specific cellular tolerance on thalamo-cortical mu opioid receptor signaling

Abstract

How cellular adaptations give rise to opioid analgesic tolerance to opioids like morphine is not well understood. For one, pain is a complex phenomenon comprising both sensory and affective components, largely mediated through separate circuits. Glutamatergic projections from the medial thalamus (MThal) to the anterior cingulate cortex (ACC) are implicated in processing of affective pain, a relatively understudied component of the pain experience. The goal of this study was to determine the effects of chronic morphine exposure on mu-opioid receptor (MOR) signaling on MThal-ACC synaptic transmission within the excitatory and feedforward inhibitory pathways. Using whole cell patch-clamp electrophysiology and optogenetics to selectively target these projections, we measured morphine-mediated inhibition of optically evoked postsynaptic currents in ACC layer V pyramidal neurons in drug-naïve and chronically morphine-treated mice. We found that morphine perfusion inhibited the excitatory and feedforward inhibitory pathways similarly in females but caused greater inhibition of the inhibitory pathway in males. Chronic morphine treatment robustly attenuated morphine presynaptic inhibition within the inhibitory pathway in males, but not females, and mildly attenuated presynaptic inhibition within the excitatory pathway in both sexes. These effects were not observed in MOR phosphorylation-deficient mice. This study indicates that chronic morphine treatment induces cellular tolerance to morphine within a thalamo-cortical circuit relevant to pain and opioid analgesia. Furthermore, it suggests this tolerance may be driven by MOR phosphorylation. Overall, these findings improve our understanding of how chronic opioid exposure alters cellular signaling in ways that may contribute to opioid analgesic tolerance.NEW & NOTEWORTHY Opioid signaling within the anterior cingulate cortex (ACC) is important for opioid modulation of affective pain. Glutamatergic medial thalamus (MThal) neurons synapse in the ACC and opioids, acting through mu opioid receptors (MORs), acutely inhibit synaptic transmission from MThal synapses. However, the effect of chronic opioid exposure on MThal-ACC synaptic transmission is not known. Here, we demonstrate that chronic morphine treatment induces cellular tolerance at these synapses in a sex-specific and phosphorylation-dependent manner.

Keywords: circuit: anterior cingulate cortex; opioid; synapse; thalamus; tolerance.

Graphical abstract

Figure 1.

Morphine inhibited direct and feedforward inhibitory signaling at MThal-ACC terminals. A: examples of acute brain slices showing viral expression of AAV2-ChR2 at the injection site (MThal) and axonal projections to the ACC. B: cartoon illustration depicting selective optogenetic activation of MThal-ACC terminals with patch-clamp recordings performed in ACC layer V pyramidal neurons. C: representative traces showing oIPSCs and oEPSCs during baseline conditions (gray), perfusion of morphine (3 μM, orange), and perfusion of naloxone (1 μM, black) in a slice from a drug-naïve male mouse. Summary of baseline oIPSC (D) and oEPSC (E) amplitudes recorded from male (filled data points) and female (open data points) mice (oIPSC: N = 13, n = 20, t₁₉ = 5.023, P < 0.0001, ratio paired t test; oEPSC: N = 13, n = 20, t₁₉ = 5.813, P < 0.0001, ratio paired t test). Connecting lines denote baseline and morphine recordings from the same cell. F: inhibition of oIPSCs by perfusion of morphine recorded from male (filled data points) and female (open data points) mice (males: 40.45 ± 9.31% of baseline; N = 6; n = 9; females: 62.34 ± 7.75% of baseline; N = 8; n = 11; t₁₈ = 1.824, P = 0.0849; unpaired t test). G: inhibition of oEPSCs by perfusion of morphine recorded from male (filled data points) and female (open data points) mice (males: 80.24 ± 6.40% of baseline; N = 6; n = 9; females: 72.22 ± 4.59% of baseline; N = 8; n = 11; t₁₈ = 1.043, P = 0.3110; unpaired t test). Lines and error bars represent means ± SE. ****P < 0.0001. ACC, anterior cingulate cortex; MThal, medial thalamus; oEPSC, optically evoked excitatory postsynaptic current; oIPSC, optically inhibitory postsynaptic current. n = number of cells and N = number of animals.

Figure 2.

Morphine altered the excitatory to inhibitory synaptic balance at MThal-ACC projections. A: representative traces showing oIPSCs and oEPSCs during baseline conditions (gray), perfusion of morphine (3 μM, orange), and perfusion of naloxone (1 μM, black) in a cell recorded from a drug-naïve male mouse. B: summary of oEPSC and oIPSC raw amplitudes during baseline (gray) and perfusion of morphine (orange) in cells recorded from male mice (oEPSC: t₈ = 2.873, P = 0.0207, N = 6, n = 9; male oIPSC: t₈ = 3.951, P = 0.0042, N = 6, n = 9, multiple ratio paired t tests). C: representative traces showing oIPSCs and oEPSCs during baseline conditions (gray), perfusion of morphine (orange), and perfusion of naloxone (black) in a slice from a drug-naïve female mouse. D: summary of oEPSC and oIPSC raw amplitudes during baseline (gray) and perfusion of morphine (orange) in cells recorded from female mice (oEPSC: t₁₀ = 5.467, P = 0.0003, N = 8, n = 11; oIPSC: t₁₀ = 3.965, P = 0.0027, N = 8, n = 11, ratio paired t tests). E: baseline amplitudes of oEPSCs and oIPSCs recorded from male (filled data points) and female (open data points) mice (male oEPSC: 356.7 ± 55.7 pA; male oIPSC: 1018.0 ± 151.4 pA; female oEPSC: 314.9 ± 29.8 pA; female oIPSC: 854.7 ± 176.6 pA; N = 6–8, n = 9–11 for each group; main effect of current type: F_1,18 = 25.60, P < 0.0001; main effect of sex: F_1,18 = 0.6492, P = 0.4309; sex × current type interaction: F_1,18 = 0.2623, P = 0.6148; repeated-measures two-way ANOVA; male oEPSC vs. oIPSC: P = 0.0029; female oEPSC vs. oIPSC: P = 0.0065; Šidák’s multiple comparisons test). F: summary graph showing inhibition of oEPSCs and oIPSCs by perfusion of morphine in male (filled data points) and female (open data points) mice (male oEPSC in morphine: 80.24 ± 6.40% of baseline; male oIPSC in morphine: 40.45 ± 9.31% of baseline; female oEPSC in morphine: 72.22 ± 4.59% of baseline; female oIPSC in morphine: 62.34 ± 7.75% of baseline; N = 6–8, n = 9–11 for each group; main effect of current type: F_1,18 = 14.41, P = 0.0013; main effect of sex: F_1,18 = 0.8174, P = 0.3779; sex × current type interaction: F_1,18 = 5.227, P = 0.0346; male oEPSC vs. oIPSC: P = 0.0013, female oEPSC vs. oIPSC: P = 0.4744, Šidák’s multiple comparisons test). G: summary of log₂-transformed E/I ratios during baseline conditions and perfusion of morphine in male (filled data points) and female (open data points) mice (N = 6–8, n = 9–11 for each group; main effect of condition: F_1,18 = 13.99, P = 0.0015; main effect of sex: F_1,18 = 0.1579, P = 0.6958; sex × condition interaction: F_1,18 = 5.504, P = 0.0306; repeated-measures two-way ANOVA; male baseline vs. morphine: P = 0.0013; female baseline vs. morphine: P = 0.5270; Šidák’s multiple comparisons test). Connecting lines denote recordings from the same cell. n = number of cells and N = number of animals. *P < 0.05, **P < 0.01, ***P < 0.001. ACC, anterior cingulate cortex; MThal, medial thalamus; oEPSC, optically evoked excitatory postsynaptic current; oIPSC, optically inhibitory postsynaptic current.

Figure 3.

Chronic morphine treatment decreased the sensitivity of MThal-ACC terminals to subsequent morphine in a sex-specific manner. Representative traces showing oEPSCs and oIPSCs in layer V pyramidal neurons during baseline (gray), perfusion of morphine (3 μM, orange), and perfusion of naloxone (1 μM, black) in slices from drug-naïve (A) and chronically treated (B) male mice or drug naive (D) and chronically morphine-treated (E) female mice. C: summary of normalized oIPSC inhibition by perfusion of morphine in drug-naïve (black) and chronically treated (purple) male and female mice (male naïve: 40.45 ± 9.31% of baseline; male chronic morphine: 78.34 ± 8.48% of baseline; female naïve: 62.34 ± 7.75% of baseline; female chronic morphine: 71.78 ± 9.05% of baseline; N = 6–8, n = 9–11 for each group; main effect of treatment: F_1,35 = 5.452, P = 0.0254; main effect of sex: F_1,35 = 3.025, P = 0.0908; treatment × sex interaction: F_1,35 = 0.7823, P = 0.7823; male naïve vs. chronic morphine: P = 0.0103; female naïve vs. chronic morphine: P = 0.6729; Šidák’s multiple comparisons test. Representative traces showing oEPSCs and oIPSCs in layer V pyramidal neurons during baseline (gray), perfusion of morphine (3 μM, orange), and perfusion of naloxone (1 μM, black) in drug-naïve (A) and chronically treated (B) female mice. F: summary of normalized oEPSC inhibition by perfusion of morphine in drug-naïve (black) and chronically treated (purple) male and female mice (male naïve: 80.24 ± 6.40% of baseline; male chronic morphine: 94.59 ± 5.99% of baseline; female naïve: 72.22 ± 4.59% of baseline; female chronic morphine: 83.51 ± 5.12% of baseline; N = 6–8, n = 9–11 for each group; main effect of treatment: F_1,35 = 5.452, P = 0.0254; main effect of sex: F_1,35 = 3.025, P = 0.0908; treatment × sex interaction: F_1,35 = 0.7823, P = 0.7823; male naïve vs. chronic morphine: P = 0.1600; female naïve vs. chronic morphine: P = 0.2593; Šidák’s multiple comparisons test. G: summary of log₂-transformed E/I ratios during baseline conditions and perfusion of morphine in male (filled data points) and female (open data points) chronically treated mice (N = 6–8, n = 9–10 for each group; main effect of condition: F_1,17 = 0.319, P = 0.0531; main effect of sex: F_1,17 = 0.07398, P = 0.7889; sex × condition interaction: F_1,17 = 0.002425, P = 0.3973; repeated-measures two-way ANOVA). Connecting lines denote baseline and morphine recordings from the same cell. H: summary of log₂-transformed E/I ratios during the baseline condition in drug-naïve (black) and chronically treated (purple) male and female mice (N = 6–8, n = 9–11 for each group; main effect of treatment: F_1,35 = 1.179, P = 0.2850; main effect of sex: F_1,35 = 0.03806, P = 0.8464; treatment × sex interaction: F_1,35 = 0.5348, P = 0.5348; ordinary two-way ANOVA). I: schematic showing chronic morphine treatment paradigm. Morphine (80 mg/kg/day) was continuously administered via osmotic minipump for 7 days before recordings. Lines and error bars represent means ± SE. n = number of cells and N = number of animals. *P < 0.05. ACC, anterior cingulate cortex; MThal, medial thalamus; oEPSC, optically evoked excitatory postsynaptic current; oIPSC, optically inhibitory postsynaptic current.

Figure 4.

Morphine tolerance at MThal-ACC terminals is eliminated in MOR phosphorylation-deficient mice. Representative traces showing oEPSCs and oIPSCs in layer V pyramidal neurons during baseline (gray), perfusion of morphine (3 μM, orange), and perfusion of naloxone (1 μM, black) in drug-naïve (A) and chronically treated (B) male 10 S/T-A mice. C: schematic representation of serine (S) and threonine (T) to alanine (A) phosphorylation site mutations on the MOR C-terminus in MOR 10 S/T-A mice. D: summary of oEPSC and oIPSC raw amplitudes during baseline (gray) and perfusion of morphine (orange) in cells recorded from male 10 S/T-A mice (oEPSCs: 0.94 ± 0.06 times baseline, t₉ = 1.130, P = 0.2875, N = 5, n = 10; ratio paired t test; oIPSCs: 0.59 ± 0.10 times baseline, t₉ = 2.887, P = 0.0180, N = 5, n = 10; ratio paired t test). E: summary of normalized oIPSC inhibition by perfusion of morphine in drug-naïve (black) and chronically treated (gray) male WT and 10 S/T-A mice (WT naïve: 40.45 ± 9.31% of baseline; WT chronic morphine: 78.34 ± 8.48% of baseline; 10 S/T-A naïve: 59.20 ± 9.85% of baseline; 10 S/T-A chronic morphine: 61.62 ± 8.23% of baseline; N = 5–7, n = 9–10 for each group; main effect of strain: F_1,34 = 0.01263, P = 0.9112; main effect of treatment: F_1,34 = 3.861, P = 0.0322; strain × treatment interaction: F_1,34 = 3.861, P = 0.0576; ordinary two-way ANOVA; WT naïve vs. chronic morphine: P = 0.0132; 10 S/T-A naïve vs. chronic morphine: P = 0.9765; Šidák’s multiple comparisons test). F: summary of normalized oEPSC inhibition by perfusion of morphine in drug-naïve (black) and chronically treated (gray) male WT and 10 S/T-A mice (WT naïve: 80.24 ± 6.40% of baseline; WT chronic morphine: 94.59 ± 5.99% of baseline; 10 S/T-A naïve: 94.24 ± 5.69% of baseline; 10 S/T-A chronic morphine: 97.85 ± 4.95% of baseline; N = 5–7, n = 9–10 for each group; main effect of strain: F_1,34 = 2.249, P = 0.1429; main effect of treatment: F_1,34 = 2.437, P = 0.1278; strain × treatment interaction: F_1,34 = 0.8710, P = 0.3573; ordinary two-way ANOVA). G: summary of log₂-transformed E/I ratios during baseline conditions and perfusion of morphine in drug-naïve and chronically treated 10 S/T-A mice (N = 5–6, n = 10 for each group; main effect of condition: F_1,18 = 13.87, P = 0.0016; main effect of treatment: F_1,18 = 0.7626, P = 0.3940; strain × treatment interaction: F_1,18 = 0.0845, P = 0.7746; repeated-measures two-way ANOVA; naïve baseline vs. morphine: P = 0.0217; chronic morphine baseline vs. morphine: P = 0.0512; Šidák’s multiple comparisons test). Connecting lines denote baseline and morphine recordings from the same cell. H: summary of log₂-transformed E/I ratios during the baseline condition in drug-naïve (black) and chronically treated (gray) WT and 10 S/T-A male mice (N = 5–7, n = 9–10 for each group; main effect of treatment: F_1,34 = 0.1732, P = 0.6899; main effect of strain: F_1,34 = 2.912, P = 0.0971; treatment × strain interaction: F_1,34 = 0.7255, P = 0.4003; ordinary two-way ANOVA). Lines and error bars represent means ± SE. n = number of cells and N = number of animals. *P < 0.05, **P < 0.01. ACC, anterior cingulate cortex; MOR, mu-opioid receptor; MThal, medial thalamus; oEPSC, optically evoked excitatory postsynaptic current; oIPSC, optically inhibitory postsynaptic current.