Abstract
The paraventricular nucleus of the thalamus (PVT) is involved in drug addiction–related behaviors, and morphine is a widely used opioid for the relief of severe pain. Morphine acts via opioid receptors, but the function of opioid receptors in the PVT has not been fully elucidated. Here, we used in vitro electrophysiology to study neuronal activity and synaptic transmission in the PVT of male and female mice. Activation of opioid receptors suppresses the firing and inhibitory synaptic transmission of PVT neurons in brain slices. On the other hand, the involvement of opioid modulation is reduced after chronic morphine exposure, probably because of desensitization and internalization of opioid receptors in the PVT. Overall, the opioid system is essential for the modulation of PVT activities.
SIGNIFICANCE STATEMENT Opioid receptors modulate the activities and synaptic transmission in the PVT by suppressing the firing rate and inhibitory synaptic inputs. These modulations were largely diminished after chronic morphine exposure.
- firing
- morphine
- opioid receptor
- paraventricular nucleus of the thalamus
- synaptic transmission
- zona incerta
Introduction
The paraventricular nucleus of the thalamus (PVT) is a part of the dorsal midline thalamus and acts as a central hub that integrates cortical and subcortical inputs to regulate diverse behavioral responses (Kirouac, 2015; Millan et al., 2017). The PVT has diverse connections with many nuclei, including the hypothalamus, hippocampus, amygdala, and prelimbic cortex, and sends large projections to other regions involved in motivation and behavior regulation, such as the nucleus accumbens. Although the efferent projections are primarily glutamatergic, receptors for several neuromodulators and neuropeptides can be found in the PVT neurons, including serotonin, dopamine, norepinephrine, corticotropin-releasing hormone, orexin, and endogenous opioids (Kirouac, 2015; Barson et al., 2020). Studies have implicated the PVT in circadian rhythm, acute and chronic stress regulation, drug addiction–related behavior, attention processing, and decision-making (Iglesias and Flagel, 2021; Flagel, 2022).
Recently, the PVT has been identified as a key node in the neural circuits of drug addiction (Zhou and Zhu, 2019; Zhou et al., 2021). The PVT can be activated by acute exposure to cocaine, amphetamine, and morphine (Deutch et al., 1998; Zhu et al., 2016). PVT neurons projecting to the nucleus accumbens (NAc) shell are recruited during spontaneous or naloxone-precipitated morphine withdrawal. PVT mediates aversion and morphine-associated memories. Activation of the PVT→NAc pathway drives aversion in morphine withdrawal-induced conditioned place aversion tests (Zhu et al., 2016; Do-Monte et al., 2017; Keyes et al., 2020). Furthermore, the PVT→NAc pathway is both sufficient and necessary to drive aversion and heroin seeking following abstinence but not extinction (Giannotti et al., 2021). These findings are consistent with previous studies proposing that PVT neurons encode the salience of behaviorally relevant stimuli (Zhu et al., 2018; Choi et al., 2019), a proposal that suggests the PVT plays a fundamental role in behavioral control (Zhou and Zhu, 2019).
Opioids, such as morphine, are clinically effective analgesics, but they also induce euphoria and adaptive changes in reward circuits (Le Merrer et al., 2009). Morphine acts via G-protein-coupled opioid receptors to modulate presynaptic and postsynaptic ion channels (Lüscher and Slesinger, 2010; Nockemann et al., 2013) and disinhibit inhibitory control to modulate pain and reward (Zhang et al., 2014; Baimel and Borgland, 2015). Opioid receptors comprise three homologous G-protein-coupled receptors (GPCRs) known as μ-, δ- and κ-opioid receptors (MORs, DORs and KORs, respectively). Activation of opioid receptors inhibits neurons by activating inwardly rectifying potassium currents (Minami and Satoh, 1995; Brunton and Charpak, 1998; Ikeda et al., 2000), and opioid receptors are activated by endogenous opioid peptides under physiological conditions (Darcq and Kieffer, 2018). In addition, high expression of MOR and KOR has been found in midline thalamic nuclei, particularly the PVT (George et al., 1994; Mansour et al., 1994). The µ-opioid system in midline thalamic nuclei may be involved in ameliorating aversive or defensive behavioral states associated with stress, withdrawal, physical pain, or social rejection (Goedecke et al., 2019) and indeed modulates defense strategies against a conditioned fear stimulus in male mice (Bengoetxea et al., 2020). Intra-PVT infusion of a KOR agonist inhibits drug-seeking behavior (Marchant et al., 2010). KOR activation inhibits anterior PVT (aPVT) neurons in mice at different ages, particularly around puberty, suggesting a possible role for KOR in regulating aPVT-related brain functions, including the stress response and drug-seeking behavior, during adolescence (Chen et al., 2015). However, to date, it remains unclear how morphine affects the activity of PVT neurons and whether chronic morphine exposure alters this modulation.
In this study, we used patch-clamp recording to test the effects of morphine and opioid receptor agonists on the activities of PVT neurons and synaptic inputs to the PVT. We also examined the functions of opioid receptors in the PVT after chronic morphine treatment. Together, this study illustrates the modulatory role of opioids in the activities of PVT neurons.
Materials and Methods
Subjects
Male and female mice, age 8–12 weeks, were used in the experiments. Mice were maintained at 22–25°C under a 12 h light/dark cycle. All animal husbandry and experimental procedures in this study were approved by the Animal Care and Use Committees at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. C57BL/6 mice were obtained from Charles River Laboratories. GAD2-Cre (stock #010802, The Jackson Laboratory) were used in the current study.
Drugs
APV, CNQX, picrotoxin, DAMGO, naloxone, trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride (U50488), (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), and Tertiapin-Q were purchased from Tocris Bioscience.
Electrophysiological recording
Procedures to prepare acute brain slices and perform whole-cell recordings with optogenetic stimulation were similar to those described previously (Zhu et al., 2016). Briefly, mice were anesthetized with isoflurane and decapitated in the morning (light cycle). Brains were rapidly dissected, and coronal slices of 250–300 μm containing the PVT were prepared using a vibratome (VT-1000S, Leica) in an ice-cold choline-based solution containing the following (in mm): 110 choline chloride, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 1.3 NaH2PO4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO3, saturated with 95% O2 and 5% CO2. Slices were incubated in 36°C oxygenated artificial CSF (ACSF) containing the following (in mm): 125 NaCl, 2.5 KCl, 2 CaCl2, 1.3 MgCl2, 1.3 NaH2PO4, 1.3 Na-ascorbate, 0.6 Na-pyruvate, 25 glucose, and 25 NaHCO3) for at least 1 h before recording. Slices were transferred to a recording chamber and superfused with 2 ml min−1 artificial CSF. Patch pipettes (3–6 MΩ) were made of borosilicate glass (catalog #BF150-86-10, Sutter Instruments). For recording of action potential firing, the pipettes were filled with a K-based internal solution containing the following (in mm): 130 K-gluconate, 10 KCl, 10 HEPES, 1 EGTA, 2 Mg-ATP, 0.3 Na-GTP, 2 MgCl2, and 290 mOsm kg−1, adjusted to pH 7.3 with potassium hydroxide. For the postsynaptic current recording, pipettes were filled with a Cs-based low Cl− internal solution containing the following (in mm): 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314, 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-phosphocreatine, and 290 mOsm kg−1, adjusted to pH 7.3 with CsOH. In some experiments, the APV, CNQX, TTX, or picrotoxin blockers were applied by bath perfusion. Whole-cell voltage-clamp recordings were performed at room temperature (22–25°C) using a MultiClamp 700B amplifier and a Digidata 1550B system (Molecular Devices). Data were sampled at 10 kHz and analyzed using pClamp10 software (Molecular Devices). For the optogenetic experiments, a blue-light-emitting diode (470 nm, Thorlabs) controlled by digital commands from the Digidata 1550B was coupled to the microscope via a dual lamphouse adaptor (5-UL180, Olympus) to deliver photostimulation. To record light-evoked EPSCs and IPSCs, 2 ms, 0.5–2 mW blue light was delivered through the objective to illuminate the entire field of view. The membrane potential was held at −70 mV to record EPSCs and at 0 mV to record GABAA receptor-mediated IPSCs. Individual sweeps were separated by 15 s. Event analysis was performed using pClamp10 and AxoGraph 1.7.6 software, with a matching threshold of 2.8 was applied to minimize false-positives.
Morphology
The intracellular solution containing 0.1% Lucifer yellow (catalog #L0144, Sigma-Aldrich) was used to inject dye into PVT neurons for whole-cell recording. After recording, the brain slices were fixed in 4% paraformaldehyde overnight. To enhance the intensity and persistence of the fluorescence, anti-lucifer yellow antibody (rabbit, 1:500; catalog A5750, Invitrogen) was used for further staining, and the secondary antibody was conjugated with Alexa Fluor 488 (1:500, catalog #A-11008, Invitrogen). The labeled cells were imaged using a fluorescence microscope (BX53, Olympus).
Single-cell real-time PCR
At the end of each recording, the cytoplasm was aspirated into the patch pipette and ejected into a PCR tube. The single-cell real-time PCR (RT-PCR) protocol was designed to detect the presence of mRNAs encoding for opioid receptors. Preamplification and real-time PCR were performed with gene-specific TaqMan assays (Mm01188089_m1, catalog #4453320, Thermo Fisher Scientific; https://www.thermofisher.cn/taqman-gene-expression/product/Mm01188089_m1) using the Single Cell-to-CT Kit (catalog #4458237, Invitrogen) according to the protocol from the manufacturer. Amplification products were visualized by electrophoresis on a 2% agarose gel. Care was taken to minimize RNA degradation and contamination during the single-cell real-time PCR procedures.
Stereotaxic surgery
Adult mice were anesthetized with 2% isoflurane and placed in a stereotactic instrument (RWD). Microinjections were performed using a 33 gauge needle connected to a 10 μl Hamilton syringe. Virus was injected into the PVT (bregma, −1.0 mm; lateral, 0.3 mm; ventral, 3.0 mm, with a 5° angle from the center to the sides) and zona incerta (ZI; bregma, −1.0 mm; lateral, 0.7 mm; ventral, −4.4 mm). The target site was injected with 200 nl of purified and concentrated adeno-associated virus (AAV; 1012 IU/ml) with a slow injection rate (100 nl/min). The injection capillary was removed 5 min after the end of the injection. All mice were allowed to recover at least 3 weeks before electrophysiological recording. Histologic slides were examined blindly for EGFP or mCherry expression. Only the mice with virus infection at the correct site were selected for further analysis.
Immunostaining
Mice were anesthetized with pentobarbital sodium (0.8%) and perfused with 4% paraformaldehyde. Brains were postfixed overnight. Coronal sections of a 50 µm thickness were cut on a freezing microtome. Sections were incubated with primary antibodies for 24 h at 4°C. The primary antibodies were c-fos (rabbit, 1:1000; catalog #2250, Cell Signaling Technology), NeuN (mouse, 1:500; catalog #MAB377, Millipore), and MOR antibody (rabbit, 1:1000; catalog #24216, ImmunoStar). Secondary antibodies were conjugated to Alexa Fluor (1:500; Invitrogen). Sections were mounted in FluoroShield (Sigma-Aldrich). Images were captured using a 63× objective on a Zeiss LSM880 confocal microscope. Data were analyzed using ImageJ software.
Statistical analysis
Data are presented throughout as the mean ± SEM. Unless otherwise noted, male and female mice were used in all studies. No sex differences were observed for any of the parameters measured, and therefore data from male and female mice were pooled to increase statistical power. Electrophysiological data were analyzed using Student's t test and the ANOVA test. For all statistical comparisons, differences were considered as significant at p < 0.05.
Results
Opioid receptors modulate the activity of PVT neurons
To investigate the effects of opioid on the activity of PVT neurons, mice were injected intraperitoneally with morphine (10 mg/kg) to induce expression of the immediate early gene c-fos in the brain, which is a marker of recent neuronal activity (Zhu et al., 2016). After 90 min, mice were anesthetized and perfused with 4% formaldehyde, and then frozen brains were sectioned and immunostained with antibodies. Morphine injection increased the proportion of cells expressing c-fos in the PVT compared with saline injection (saline, 101.8 ± 11.57% vs morphine, 167.5 ± 13.75%, p = 0.0016, t = 3.676, df = 19, n = 2 mice each group, unpaired t test; Fig. 1A,B), indicating that morphine activates PVT neurons. Morphine acts via opioid receptors that couple to G-protein-gated inwardly rectifying potassium (GIRK) channels, inhibiting neuronal activity (Cruz et al., 2008; Kotecki et al., 2015; Rifkin et al., 2017). High expression of opioid receptors has been reported in PVT (George et al., 1994; Mansour et al., 1994). To investigate the functions of opioid receptors in PVT, we performed whole-cell patch-clamp recordings in brain slices. First, we recorded the action potential firing of PVT neurons. The firing rate was significantly reduced in most PVT cells after application of morphine (30 μm; ACSF, 5.06 ± 0.42 Hz, n = 16, vs morphine, 1.64 ± 0.60 Hz, n = 16, vs morphine + naloxone, 4.27 ± 0.55 Hz, n = 8; one-way ANOVA, F(1,16) = 19.63, p = 0.0002, followed by post hoc Tukey's test; Fig. 1C,E), which can be reversed by application of the opioid receptor antagonist naloxone (10 μm). The subtype of opioid receptors involved in the regulation of PVT neuronal activity was then determined. Three subtype agonists were used to record firings of PVT neurons, including the MOR agonist DAMGO (1 μm), the KOR agonist U50488 (1 μm), and the DOR agonist SNC80 (3 μm). DAMGO application significantly reduced the firing rate in most PVT cells, similar to the effect of morphine (ACSF, 5.17 ± 0.39 Hz, n = 16, vs DAMGO, 2.33 ± 0.77 Hz, n = 16, vs DAMGO + naloxone, 3.82 ± 0.30 Hz, n = 12; one-way ANOVA, F(1,19) = 22.04, p < 0.0001, followed by post hoc Tukey's test; Fig. 1D,F). However, the KOR agonist U50488 and the DOR agonist SNC80 had no effect on the firing rate of PVT neurons (ACSF, 5.92 ± 0.54 Hz vs U50488, 6.31 ± 0.59 Hz, n = 10, p = 0.4278, t = 0.83, df = 9; ACSF, 6.13 ± 0.61 Hz vs SNC80, 5.63 ± 0.60 Hz, n = 10, p = 0.1679, t = 1.5, df = 9, paired t test; Fig. 1G,H). These results suggest that the MOR is important in regulating the activities of PVT neurons.
Morphine and the MOR agonist DAMGO reduce the firing rate of PVT neurons in brain slices. A, Immunostaining images showing that saline and morphine intraperitoneal injections induced robust expression of c-fos (green) in the PVT neurons (n = 2 mice per group). PVT areas are shown in yellow boxes. Scale bar, 500 μm. B, Normalized density of PVT projection neurons expressing c-fos. Morphine intraperitoneal injection (blue bar, n = 2 mice) induced more c-fos-positive (c-fos+) cells in the PVT, compared with saline intraperitoneal injection (gray bar, n = 2 mice; Saline, 101.8 ± 11.57% vs Morphine, 167.5 ± 13.75%, p = 0.0016, t = 3.676, df = 19, unpaired t test). C, Representative recording showing that morphine (30 μm) reduced action potential firing in the PVT, and the opioid receptor antagonist naloxone (10 μm) reversed this effect. D, Representative recording showing that the MOR agonist DAMGO (1 μm) reduced action potential firing in the PVT, and the opioid receptor antagonist naloxone (10 μm) reversed this effect. E, Morphine (30 μm) significantly decreased the firing rate of most PVT neurons (ACSF, 5.06 ± 0.42 Hz, n = 16 vs Morphine, 1.64 ± 0.60 Hz, n = 16 vs Morphine + Naloxone, 4.27 ± 0.55 Hz, n = 8; one-way ANOVA, F(1,16) = 19.63, p = 0.0002, followed by post hoc Tukey's test). Naloxone was not applied to all the cells. F, The MOR agonist DAMGO (1 μm) also significantly decreased the firing rate of most PVT neurons (ACSF, 5.17 ± 0.39 Hz, n = 16 vs DAMGO, 2.33 ± 0.77 Hz, n = 16 vs DAMGO + Naloxone, 3.82 ± 0.30 Hz, n = 12; one-way ANOVA, F(1,19) = 22.04, p < 0.0001, followed by post hoc Tukey's test). Naloxone was not applied to all the cells. G, The KOR agonist U50488 (1 μm) didn't change the firing rate of PVT neurons (ACSF, 5.92 ± 0.54 Hz vs U50488, 6.31 ± 0.59 Hz, n = 10, p = 0.4278, t = 0.83, df = 9, paired t test). H, DOR agonist SNC80 (3 μm) did not change the firing rate of PVT neurons (ACSF, 6.13 ± 0.61 Hz vs 5.63 ± 0.60 Hz, n = 10, p = 0.1679, t = 1.5, df = 9, paired t test). I, Examples of PVT neuron morphology with Lucifer yellow staining. Scale bar, 50 μm. J, Schematic of single-cell real-time PCR to test the gene oprm1 expression of the μ-opioid receptor in the PVT. GAPDH was used as an internal control; *p < 0.05, **p < 0.01, ***p < 0.001, N.S., Not significant.
Interestingly, a few cells had no apparent response to morphine or DAMGO. To investigate the difference between these opioid sensitive and insensitive neurons, we first examined the morphology of PVT neurons. Lucifer yellow CH dipotassium salt was added to the intracellular pipette solution during recording, and then brain slices were then immunostained with Lucifer yellow antibody to enhance the fluorescence. However, we did not see a clear difference in the morphology between these two groups of PVT neurons (examples are shown in Fig. 1I). Second, not all PVT neurons may express MORs. To investigate the difference in MOR expression in these two groups, we performed single-cell RT-PCR after recording. The single-cell RT PCR procedure is shown in Figure 1J. Both the cells that were sensitive to DAMGO and also the cells that showed no response to DAMGO expressed Oprm1 (the gene encoding MOR), suggesting that MORs were widely expressed in the PVT neurons. As the RT-PCR revealed the expression of MORs in the PVT neurons, hence the postsynaptic modulation, the insensitive neurons might reflect that the MORs were not functional or there might be some presynaptic mechanism to counteract it. Previous literature has reported that MORs could also be expressed presynaptically to regulate firing and that opioid receptors may be involved in regulating synaptic inputs to the PVT.
Opioid receptors modulate inhibitory synaptic inputs to the PVT
Opioid receptors have been reported to modulate synaptic transmission, particularly GABAergic inhibitory transmission (Fields and Margolis, 2015; Jiang et al., 2021). To assess the effects of opioid receptors on synaptic transmission in the PVT, we first recorded spontaneous EPSCs (sEPSCs) and IPSCs (sIPSCs). Bath application of morphine (30 μm) did not alter the frequency or amplitude of sEPSCs (Amplitude, ACSF, 11.09 ± 0.72 pA vs morphine, 10.45 ± 0.56 pA, n = 14, p = 0.444, t = 0.79, df = 13; Frequency, ACSF, 4.94 ± 0.59 Hz vs morphine, 4.53 ± 0.53 Hz, n = 14, p = 0.1824, t = 1.41, df = 13, paired t test; Fig. 2A–C) but decreased the frequency and amplitude of sIPSCs (Amplitude, ACSF, 13.30 ± 0.91 pA vs morphine, 11.89 ± 0.66 pA, n = 18, p = 0.0078, t = 3.01, df = 17; Frequency, ACSF, 4.03 ± 0.90 Hz vs morphine, 3.31 ± 0.68 Hz, n = 18, p = 0.0133, t = 2.76, df = 17, paired t test; Fig. 2D–F). Thus, opioid receptors can regulate inhibitory synaptic transmission in the PVT. To further investigate which subtype of opioid receptors contributes to the regulation of inhibitory inputs to the PVT, we recorded miniature IPSCs (mIPSCs) in the presence of APV (NMDA receptor antagonist, 50 μm), CNQX (AMPA receptor antagonist, 10 μm) and TTX (sodium channel blocker, 0.5 μm). Morphine (30 μm) also decreased the frequency and amplitude of mIPSCs in the PVT neurons (Amplitude, ACSF, 8.32 ± 0.54 pA vs morphine, 7.52 ± 0.54 pA, n = 10, p = 0.0015, t = 4.48, df = 9; Frequency, ACSF, 2.88 ± 0.31 Hz vs morphine, 2.34 ± 0.33 Hz, n = 10, p = 0.0028, t = 4.07, df = 9, paired t test; Fig. 3A,B). The MOR agonist DAMGO (1 μm) significantly reduced both the amplitude and frequency of mIPSCs in the PVT (Amplitude, ACSF, 9.57 ± 0.85 pA vs DAMGO, 8.71 ± 0.75 pA, n = 12, p = 0.0353, t = 2.4, df = 11; Frequency, ACSF, 2.66 ± 0.38 Hz vs DAMGO, 2.04 ± 0.41 Hz, n = 12, p = 0.0006, t = 4.73, df = 11, paired t test; Fig. 3C,D). The KOR agonist U50488 (1 μm) also significantly reduced the amplitude and frequency of mIPSCs in the PVT (Amplitude, ACSF, 11.48 ± 0.75 pA vs U50488, 10.02 ± 0.64 pA, n = 12, p = 0.0005, t = 4.9, df = 11; Frequency, ACSF, 4.35 ± 0.97 Hz vs U50488, 3.11 ± 0.70 Hz, n = 12, p = 0.0014, t = 4.26, df = 11, paired t test; Fig. 3E,F). There was no effect of the DOR agonist SNC80 (3 μm) on the mIPSCs (Amplitude, ACSF, 13.94 ± 1.07 pA vs SNC80, 13.42 ± 1.01 pA, n = 12, p = 0.0965, t = 1.82, df = 11; Frequency, ACSF, 4.95 ± 0.64 Hz vs SNC80, 4.75 ± 0.51 Hz, n = 12, p = 0.4612, t = 0.76, df = 11, paired t test; Fig. 3G,H). These results suggest that MOR and KOR are involved in the modulation of inhibitory synaptic inputs to the PVT.
Effects of morphine on spontaneous excitatory and inhibitory inputs to PVT neurons. A, Example traces showing spontaneous EPSCs before (black) and after (red) morphine (30 μm) application in brain slices. B, C, No differences were found in the amplitude and frequency of spontaneous EPSCs before and after morphine (30 μm) application (Amplitude, ACSF, 11.09 ± 0.72 pA vs Morphine, 10.45 ± 0.56 pA, n = 14, p = 0.444, t = 0.79, df = 13; Frequency, ACSF, 4.94 ± 0.59 Hz vs Morphine, 4.53 ± 0.53 Hz, n = 14, p = 0.1824, t = 1.41, df = 13, paired t test). D, Example traces showing spontaneous IPSCs before (black) and after (blue) morphine (30 μm) application in brain slices. E, F, Morphine (30 μm) reduced the amplitude and frequency of spontaneous IPSCs (Amplitude, ACSF, 13.30 ± 0.91 pA vs Morphine, 11.89 ± 0.66 pA, n = 18, p = 0.0078, t = 3.01, df = 17; Frequency, ACSF, 4.03 ± 0.90 Hz vs Morphine, 3.31 ± 0.68 Hz, n = 18, p = 0.0133, t = 2.76, df = 17, paired t test; *p < 0.05, **p < 0.01). N.S., Not significant.
Activation of opioid receptors reduces the inhibitory transmission of PVT neurons. A, Example traces showing the mIPSCs before (black) and after (blue) morphine (30 μm) application. mIPSCs were recorded in the presence of APV (50 μm), CNQX (10 μm), and TTX (0.5 μm). B, Morphine (30 μm) reduced the amplitude (left) and frequency (right) of mIPSCs (Amplitude, ACSF, 8.32 ± 0.54 pA vs morphine, 7.52 ± 0.54 pA, n = 10, p = 0.0015, t = 4.48, df = 9; Frequency, ACSF, 2.88 ± 0.31 Hz vs morphine, 2.34 ± 0.33 Hz, n = 10, p = 0.0028, t = 4.07, df = 9, paired t test). C, Example traces showing the mIPSCs before (black) and after (red) the application of the MOR agonist DAMGO (1 μm). D, DAMGO (1 μm) reduced the amplitude (left) and frequency (right) of mIPSCs (Amplitude, ACSF, 9.57 ± 0.85 pA vs DAMGO, 8.71 ± 0.75 pA, n = 12, p = 0.0353, t = 2.4, df = 11; Frequency, ACSF, 2.66 ± 0.38 Hz vs DAMGO, 2.04 ± 0.41 Hz, n = 12, p = 0.0006, t = 4.73, df = 11, paired t test). E, Example traces showing the mIPSCs before (black) and after (green) application of the KOR agonist U50488 (1 μm). F, U50488 (1 μm) also reduced the amplitude (left) and frequency (right) of mIPSCs (Amplitude, ACSF, 11.48 ± 0.75 pA vs U50488, 10.02 ± 0.64 pA, n = 12, p = 0.0005, t = 4.9, df = 11; Frequency, ACSF, 4.35 ± 0.97 Hz vs U50488, 3.11 ± 0.70 Hz, n = 12, p = 0.0014, t = 4.26, df = 11, paired t test). G, Example traces showing the mIPSCs before (black) and after (purple) application of the DOR agonist SNC80 (3 μm). H, No differences were found after SNC80 (3 μm) application (Amplitude, ACSF, 13.94 ± 1.07 pA vs SNC80, 13.42 ± 1.01 pA, n = 12, p = 0.0965, t = 1.82, df = 11; Frequency, ACSF, 4.95 ± 0.64 Hz vs SNC80, 4.75 ± 0.51 Hz, n = 12, p = 0.4612, t = 0.76, df = 11, paired t test; *p < 0.05, **p < 0.01, ***p < 0.001). N.S., Not significant.
KOR modulates inhibitory synaptic transmission from the ZI to the PVT
To explore the source of the inhibitory inputs to the PVT, we performed retrograde tracing in transgenic mice. Cre-dependent retro-AAV-DIO-EGFP was injected into the PVT of GAD2-Cre mice (Fig. 4A). With this tracing strategy, only GABAergic neurons that project to the PVT are labeled with GFP. After 2–3 weeks of infection, we sectioned the whole brains of these mice and examined the distribution of GFP fluorescence. A few brain areas were found to have intense GFP-expressing neurons (Fig. 4B), including the suprachiasmatic nucleus (SCN), the ZI, and the dorsal raphe. The ZI is an inhibitory subthalamic region with extensive connections throughout the brain. Studies have demonstrated diverse functions of the ZI in processing sensory information, regulating behavior, mediating motivational states, and participating in neural plasticity (Wang et al., 2020). Projections from ZI to PVT can reliably produce rapid and substantial eating (Zhang and van den Pol, 2017). MOR and KOR are expressed in the ZI of rats and mice (DePaoli et al., 1994; George et al., 1994; Mansour et al., 1994; Jenab et al., 1995), but little is known about the function of opioid receptors in the ZI to PVT pathway.
The KOR agonist U50488 reduces inhibitory synaptic transmission from the ZI to the PVT. A, Schematic illustration of the viral approach for retrograde tracing used in GAD2-Cre transgenic mice. B, Representative image of retrograde tracing and EGFP expression in the upstream regions (green). Scale bars: top, 2 mm; bottom, 500 μm. cRt, Caudal reticular thalamus; DR, dorsal raphe. C, Schematic illustration of the optogenetic approach used to test synaptic transmission from the ZI to the PVT. D, Example traces of oIPSCs induced by a single light pulse (470 nm, 2 ms) and PVT neurons recorded when holding at ∼0 mV. No current was evoked when held at −70 mV (top). These oIPSCs could be blocked by the GABAA receptor antagonist picrotoxin (100 μm; bottom). E, Example traces of oIPSCs induced by a single light pulse (470 nm, 2 ms) before (top) and after (bottom) TTX (1 μm) and 4-AP (1 mm) application. F, The amplitude of oIPSCs is not changed by TTX (1 μm) and 4-AP (1 mm) in PVT neurons (ACSF, 339.0 ± 74.10 pA vs TTX + 4-AP, 344.2 ± 54.05 pA, n = 8, p = 0.8725, t = 0.17, df = 7, paired t test). G, Example traces showing the oIPSCs before and after DAMGO (1 μm; top) and U50488 (1 μm) application (bottom). H, I, The amplitude and PPR of oIPSCs were not different before and after DAMGO (1 μm) application (Amplitude, ACSF, 164.7 ± 34.95 pA vs DAMGO, 185.0 ± 42.65 pA, n = 13, p = 0.2109, t = 1.32, df = 12; PPR, ACSF, 1.02 ± 0.08 vs DAMGO, 1.01 ± 0.071, n = 13, p = 0.861, t = 0.18, df = 12, paired t test). The PPR was elicited by two consecutive light pulses (470 nm, 2 ms) with an interval of 100 ms. J, K, The amplitude of oIPSCs was reduced after U50488 (1 μm) application, but there was no difference for the paired-pulse ratio (Amplitude, ACSF, 134.0 ± 21.08 pA vs U50488, 105.3 ± 20.16 pA, n = 11, p = 0.0123, t = 3.05, df = 10; PPR, ACSF, 1.02 ± 0.05 vs U50488, 1.12 ± 0.12, n = 11, p = 0.3107, t = 1.07, df = 10, paired t test; *p < 0.05). N.S., Not significant.
To test whether opioid receptors regulate GABAergic inputs from the ZI to the PVT, ZI neurons were transduced with AAV-DIO-ChR2-mCherry (Fig. 4C), and optogenetic experiments were performed in the PVT brain slice 3–4 weeks after surgery. A brief pulse of blue light (470 nm, 2 ms) evoked robust optic IPSCs (oIPSCs) in PVT neurons when clamped at 0 mV (Fig. 4D, top), which can be blocked by the GABAA receptor antagonist picrotoxin (100 μm; Fig. 4D, bottom). Light stimulation did not evoke any detectable EPSCs when clamped at −70 mV (Fig. 4D, top). The oIPSCs were preserved in the presence of TTX (1 μm) and 4-AP (1 mm), suggesting that the ZI to PVT input is monosynaptic (ACSF, 339.0 ± 74.10 pA vs TTX and 4-AP, 344.2 ± 54.05 pA, n = 8, p = 0.8725, t = 0.17, df = 7, paired t test; Fig. 4E,F). We found that DAMGO (1 μm) had no effect on the amplitude and paired-pulse ratio (PPR) of oIPSCs from ZI to PVT (Amplitude, ACSF, 164.7 ± 34.95 pA vs DAMGO, 185.0 ± 42.65 pA, n = 13, p = 0.2109, t = 1.32, df = 12; PPR, ACSF, 1.02 ± 0.08 vs DAMGO, 1.01 ± 0.071, n = 13, p = 0.861, t = 0.18, df = 12, paired t test; Fig. 4H,I). The KOR agonist U50488 (1 μm) significantly reduced the amplitude of oIPSCs but did not alter the PPR (Amplitude, ACSF, 134.0 ± 21.08 pA vs U50488, 105.3 ± 20.16 pA, n = 11, p = 0.0123, t = 3.05, df = 10; PPR, ACSF, 1.02 ± 0.05 vs U50488, 1.12 ± 0.12, n = 11, p = 0.3107, t = 1.07, df = 10, paired t test; Fig. 4J,K). Thus, KOR activation suppressed the GABAergic inputs to the PVT, suggesting that KOR can modulate the inhibitory transmission in the ZI to PVT pathway. Surprisingly, the PPR at ZI synapses to PVT neurons was not altered. As opioid receptors are both presynaptically and postsynaptically located in the PVT, it is possible that MOR or KOR agonists act on both the presynaptic and postsynaptic receptors and counteract the change in PPR.
Chronic morphine exposure reduced the inhibition of firing by MOR
Opioids are currently the most effective drugs for pain relief. However, they are also rewarding, and their repeated use can lead to dependence and addiction (Fields and Margolis, 2015). Addiction is a complex, relapsing disorder in which abused drugs hijack, overstimulate, and compromise reward-processing systems and associated networks (Darcq and Kieffer, 2018). Activation of the PVT can induce aversion and contribute to opioid withdrawal (Zhu et al., 2016). In our results, acute morphine could modulate the activities of PVT neurons. To investigate the functions of opioid receptors after chronic morphine treatment, mice were rendered opiate dependent by daily intraperitoneal injections of morphine in their home cage with doses escalating from 10 to 50 mg per kg body weight (Zhu et al., 2016; Fig. 5A). Control mice were intraperitoneally injected with the same volume of saline. On day 7, whole-cell recording was performed in brain slices, and the activities of PVT neurons before and after MOR agonist application were tested. Representative recordings are shown in Figure 5, D and E. DAMGO application significantly reduced the firing rate of PVT neurons in the saline-treated mice (ACSF, 2.91 ± 0.33 Hz vs DAMGO, 0.64 ± 0.34 Hz vs DAMGO + naloxone, 2.53 ± 0.37 Hz; n = 12; one-way ANOVA, F(1,14) = 22.93, p = 0.0001, followed by post hoc Tukey's test; Fig. 5B), and this effect was largely suppressed in the morphine-treated mice (ACSF, 4.79 ± 0.36 Hz vs DAMGO, 3.66 ± 0.61 Hz vs DAMGO + naloxone, 4.73 ± 0.34 Hz; n = 15; one-way ANOVA, F(1,18) = 5.998, p = 0.0184, followed by post hoc Tukey's test; Fig. 5C).
Chronic morphine treatment attenuates the inhibitory effects of DAMGO on the firing rate of PVT neurons, which is partly mediated by GIRK channels. A, Experimental schedule for chronic morphine treatment, morphine intraperitoneal injection for 5 consecutive days with a concentration gradient. Patch-clamp recording on day 7. B, Action potential firings were recorded in the PVT after chronic saline treatment, and DAMGO (1 μm) significantly reduced the firing rate in saline-treated mice (ACSF, 2.91 ± 0.33 Hz vs DAMGO, 0.64 ± 0.34 Hz vs DAMGO + Naloxone, 2.53 ± 0.37 Hz; n = 12; one-way ANOVA, F(1,14) = 22.93, p = 0.0001, followed by post hoc Tukey's test). C, Action potential firings were recorded in the PVT after chronic morphine treatment, and the reduction in firing rate was attenuated compared with that in saline-treated mice (ACSF, 4.79 ± 0.36 Hz vs DAMGO, 3.66 ± 0.61 Hz vs DAMGO + Naloxone, 4.73 ± 0.34 Hz; n = 15; one-way ANOVA, F(1,18) = 5.998, p = 0.0184, followed by post hoc Tukey's test). D, Representative recording showing that DAMGO (1 μm) reduced action potential firing in the PVT of the saline-treated mice and that naloxone (10 μm) reversed this effect. E, Representative recording showing that DAMGO (1 μm) could not significantly reduce firing in the morphine-treated mice. F, Experimental schedule for morphine exposure was morphine intraperitoneal injection for 5 consecutive days with a concentration gradient. Two hours after the injection on day 5, the mouse was anesthetized and decapitated for preparation of brain slices. G, Experimental schedule for naloxone-precipitated withdrawal was morphine intraperitoneal injection for 5 consecutive days with a concentration gradient. Two hours after the injection on day 5, the mouse was intraperitoneally injected with naloxone (5 mg/kg). Ten to 15 min later, the mouse was anesthetized and decapitated for preparation of brain slices. H, Action potential firings were recorded in the PVT of morphine-exposed mice. DAMGO (1 μm) did not significantly reduce the firing rates (ACSF, 3.03 ± 0.25 Hz vs DAMGO, 2.25 ± 0.44 Hz vs DAMGO + Naloxone, 2.91 ± 0.22 Hz; n = 9; one-way ANOVA, F(1,12) = 2.339, p = 0.1472, followed by post hoc Tukey's test). I, Action potential firings were recorded in the PVT of naloxone-precipitated withdrawal mice. DAMGO (1 μm) could not significantly reduce the firing rates, which is similar to that in morphine-treated mice (ACSF, 3.59 ± 0.33 Hz vs DAMGO, 2.71 ± 0.56 Hz vs DAMGO + Naloxone, 3.73 ± 0.30 Hz; n = 6; one-way ANOVA, F(1,6) = 6.134, p = 0.0466, followed by post hoc Tukey's test). J, Suppression ratio for firing in the saline-treated, morphine-treated, morphine exposure, and naloxone-precipitated withdrawal groups. Percentage of change in the firing rate was calculated by dividing the data in drug divided by that in ACSF (Saline treatment, 0.18 ± 0.09 Hz/Hz, n = 12 vs Morphine treatment, 0.74 ± 0.10 Hz/Hz, n = 15 vs Morphine exposure, 0.75 ± 0.12 Hz/Hz, n = 9 vs Naloxone-precipitated withdrawal, 0.74 ± 0.13 Hz/Hz, n = 6; one-way ANOVA, F(2,14) = 7.797, p = 0.0063, followed by post hoc Dunnett's test). K, Example traces of GIRK currents induced by DAMGO (3 μm) in the saline-treated mice (black), morphine-treated mice (red), morphine-exposed mice (blue), and naloxone-precipitated withdrawal mice (orange). U50488 (3 μm) could not induce GIRK currents in the saline-treated mice (green). L, There was no difference in the GIRK currents between the saline-treated and morphine-treated mice and no difference between the saline-treated and morphine-exposed mice. The GIRK currents were much smaller in the naloxone-precipitated withdrawal mice than that in the saline-treated mice (Saline treatment, 19.09 ± 4.03 pA, n = 9 vs Morphine treatment, 22.67 ± 3.29 pA, n = 11 vs Morphine exposure, 15.32 ± 4.45 pA, n = 7 vs Naloxone-precipitated withdrawal, 2.82 ± 1.96 pA, n = 6; one-way ANOVA, F(2,16) = 4.714, p = 0.0288, followed by post hoc Dunnett's test). M, Example recording showing that DAMGO (1 μm) hyperpolarized the membrane potential and abolished firing, but the GIRK channel antagonist Tertiapin-Q (1 μm) could not fully reverse this effect in wild-type mice. N, Tertiapin-Q (1 μm) could not reverse the firing rates (ACSF, 3.28 ± 0.33 Hz vs DAMGO, 0.18 ± 0.18 Hz vs DAMGO + Tertiapin-Q, 0.60 ± 0.56 Hz, n = 5; one-way ANOVA, F(1,5) = 27.05, p = 0.0028, followed by post hoc Tukey's test). O, DAMGO (1 μm) significantly decreased the membrane potential, but the GIRK channel antagonist Tertiapin-Q (1 μm) could not fully reverse it (ACSF, −40.29 ± 1.93 mV vs DAMGO, −49.27 ± 2.22 mV vs DAMGO + Tertiapin-Q, −43.39 ± 1.11 mV, n = 5; one-way ANOVA, F(1,6) = 11.82, p = 0.0118, followed by post hoc Tukey's test; *p < 0.05, **p < 0.01, ***p <0.001). N.S., Not significant.
As the recordings were made 2 d after the last morphine injection (Fig. 5A), the animal could be in a state of spontaneous withdrawal. To discriminate between desensitization effects because of morphine exposure and spontaneous withdrawal, we performed recordings from chronic morphine exposure mice in which the brain slices were prepared 2 h after the last morphine injection on day 5 (Fig. 5F). DAMGO did not reduce the firing rates in the morphine exposure group (ACSF, 3.03 ± 0.25 Hz vs DAMGO, 2.25 ± 0.44 Hz vs DAMGO + naloxone, 2.91 ± 0.22 Hz; n = 9; one-way ANOVA, F(1,12) = 2.339, p = 0.1472, followed by post hoc Tukey's test; Fig. 5H). In addition, we examined the effects of DAMGO on firing when animals were in a state of naloxone-precipitated withdrawal. Mice were intraperitoneally injected with naloxone (5 mg/kg) 2 h after the last morphine injection, and 10–15 min later, the animals were anesthetized and decapitated for preparation of brain slices (Zhu et al., 2016; Fig. 5G). DAMGO also did not reduce the firing rates in these naloxone-precipitated withdrawal mice (ACSF, 3.59 ± 0.33 Hz vs DAMGO, 2.71 ± 0.56 Hz vs DAMGO + naloxone, 3.73 ± 0.30 Hz; n = 6; one-way ANOVA, F(1,6) = 6.134, p = 0.0466, followed by post hoc Tukey's test; Fig. 5I). Furthermore, the degree of suppression, as quantified by suppression ratios (ratios between the firing rate in ACSF and DAMGO conditions), was similar in the morphine treatment, morphine exposure, and naloxone-precipitated withdrawal groups, and all changed less than in the saline treatment group (saline treatment, 0.18 ± 0.09 Hz/Hz, n = 12, vs morphine treatment, 0.74 ± 0.10 Hz/Hz, n = 15, vs morphine exposure, 0.75 ± 0.12 Hz/Hz, n = 9, vs naloxone-precipitated withdrawal, 0.74 ± 0.13 Hz/Hz, n = 6; one-way ANOVA, F(2,14) = 7.797, p = 0.0063, followed by post hoc Dunnett's test; Fig. 5J). As the reduction in DAMGO inhibition was already present on day 5 in the morphine exposure group, without any withdrawal effect, the reduction in firing rate on day 7 in the morphine treatment group was more likely because of morphine exposure rather than spontaneous opioid withdrawal.
Opioid receptors are members of the GPCR family, and they can activate GIRK channels via G-proteins. Activation of GIRK channels induces membrane hyperpolarization of the neurons via K+ efflux and reduces neuronal excitability (Ikeda et al., 2002; Rifkin et al., 2017). Because opioids act through MORs that couple to GIRK channels, we asked whether the reduced inhibition of firing rate by DAMGO is because of the reduced MOR coupling to GIRK after chronic morphine treatment. We recorded GIRK currents induced by DAMGO (3 μm) when the membrane potential was clamped at −60 mV. DAMGO application induced robust outward GIRK currents in saline-treated control mice (Fig. 5K, black). DAMGO application also induced significant GIRK currents in morphine-treated and morphine-exposed mice, and the amplitudes were similar to those in saline-treated mice (Fig. 5K,L). However, DAMGO could not induce obvious GIRK currents in the naloxone-precipitated withdrawal mice (saline treatment, 19.09 ± 4.03 pA, n = 9, vs morphine treatment, 22.67 ± 3.29 pA, n = 11, vs morphine exposure, 15.32 ± 4.45 pA, n = 7, vs naloxone-precipitated withdrawal, 2.82 ± 1.96 pA, n = 6; one-way ANOVA, F(2,16) = 4.714, p = 0.0288, followed by post hoc Dunnett's test; Fig. 5K,L). Thus, the results showed that DAMGO-induced GIRK currents were reduced in naloxone-precipitated withdrawal mice, suggesting a decoupling of MOR and GIRK induced by naloxone-precipitated withdrawal.
To confirm the contribution of GIRK channels to the DAMGO inhibition, we used a GIRK channel antagonist, Tertiapin-Q, to reverse DAMGO-induced inhibition of PVT neurons. As we have shown previously, DAMGO suppressed the firing and hyperpolarized PVT neurons (Fig. 5M). Application of Tertiapin-Q (1 μm) did not restore the action potential firing (ACSF, 3.28 ± 0.33 Hz vs DAMGO, 0.18 ± 0.18 Hz vs DAMGO + Tertiapin-Q, 0.60 ± 0.56 Hz, n = 5; one-way ANOVA, F(1,5) = 27.05, p = 0.0028, followed by post hoc Tukey's test; Fig. 5N) but partially reversed the hyperpolarization of the membrane potential (ACSF, −40.29 ± 1.93 mV vs DAMGO, −49.27 ± 2.22 mV vs DAMGO + Tertiapin-Q, −43.39 ± 1.11 mV, n = 5; one-way ANOVA, F(1,6) = 11.82, p = 0.0118, followed by post hoc Tukey's test; Fig. 5O). These results suggest that GIRK channels contribute to membrane potential hyperpolarization but not to the firing suppression effect of DAMGO on PVT neurons. We did not see any GIRK currents induced by the KOR agonist U50488 (3 μm; Fig. 5K, green).
Prolonged use of opioids leads to a reduction in their effectiveness, known as tolerance, and research has been devoted to elucidating the molecular basis of desensitization (Marie et al., 2006). The MOR mediates both presynaptic inhibition and postsynaptic neuromodulatory effects of endogenous opioid peptides (Kieffer and Evans, 2009; Corder et al., 2018; Darcq and Kieffer, 2018). The mechanism underlying postsynaptic MOR desensitization is based on ligand-induced phosphorylation of the MOR cytoplasmic tail by GPCR kinases followed by receptor internalization (Gainetdinov et al., 2004; Just et al., 2013; Williams et al., 2013; Yousuf et al., 2015; Arttamangkul et al., 2018; Jullié et al., 2020). To investigate whether MOR is internalized in PVT neurons after chronic morphine exposure, MOR antibody was used to show the distribution of MOR, and NeuN antibody was used to show the soma of the neurons (Fig. 6A). Radius analysis shows the distribution of MOR from the center to the periphery of the PVT cells (n = 8 cells per group). In the chronic morphine treatment group, MORs were scattered in the cell body (cytoplasm), whereas in the saline treatment group, MORs were mostly distributed across the periphery (membrane; Cytoplasm area, morphine treatment, 47.67 ± 1.78 AU vs saline treatment, 36.39 ± 1.18 AU, n = 8; Membrane area, morphine treatment, 45.94 ± 0.43 AU vs saline treatment, 67.80 ± 0.73 AU, n = 8; AU: arbitrary units; two-way ANOVA followed by post hoc Tukey's test; drug treatment × cellular location, F(1,8) = 181.7, p < 0.0001; drug treatment, F(1,8) = 24.9, p < 0.01; cellular location, F(1,8) = 145.8, p < 0.0001; Fig. 6B,C). Thus, chronic morphine treatment induced the internalization of MORs in the PVT neurons and reduced the postsynaptic neuromodulatory effects of opioids.
Chronic morphine exposure causes MOR internalization. A, Confocal images showing the distribution of MOR (red) in the cell bodies of the PVT after chronic saline and morphine treatment. NeuN antibody was used to visualize the cell body (green). Scale bar, 5 μm. B, Radius analysis showing the distribution of MOR from the center to the periphery of the PVT cells (n = 8 per group). MORs were scattered in the cell body (cytoplasm) in the chronic morphine group (red line), and MORs were mostly distributed in the periphery (membrane) in the saline group (black line). C, Quantification analysis shows that more MORs were distributed in the cytoplasm in the PVT cells of morphine-treated mice, corresponding to the blue box in B (Morphine treatment, 47.67 ± 1.78 AU vs Saline treatment, 36.39 ± 1.18 AU, n = 8) AU: arbitrary units). More MORs were distributed in the membrane area in the PVT cells of saline-treated mice, corresponding to the green box in B (Morphine treatment, 45.94 ± 0.43 AU vs Saline treatment, 67.80 ± 0.73 AU, n = 8; AU: arbitrary units; two-way ANOVA followed by post hoc Tukey's test; drug treatment × cellular location, F(1,8) = 181.7, p < 0.0001; drug treatment, F(1,8) = 24.9, p < 0.01; cellular location, F(1,8) = 145.8, p < 0.0001; ****p < 0.0001).
Chronic morphine exposure reduced the modulation of inhibitory inputs by MOR and KOR
Previously, we found that opioid receptors contribute to the modulation of inhibitory inputs to the PVT in wild-type mice. Could opioid receptors still modulate the inhibitory transmission in the PVT after chronic morphine treatment? DAMGO (1 μm) reduced the amplitude and frequency of mIPSCs in saline-treated mice (Amplitude, ACSF, 9.01 ± 0.82 pA vs DAMGO, 8.55 ± 0.82 pA, n = 11, p = 0.0459, t = 2.28, df = 10; Frequency, ACSF, 3.76 ± 0.85 Hz vs DAMGO, 3.52 ± 0.83 Hz, n = 11, p = 0.006, t = 3.47, df = 10, paired t test; Fig. 7A,C,D), consistent with what we found in wild-type mice. U50488 (1 μm) also decreased the amplitude and frequency of mIPSCs in saline-treated mice (Amplitude, ACSF, 8.30 ± 0.44 pA vs U50488, 7.44 ± 0.40 pA, n = 13, p = 0.0013, t = 4.19, df = 12; Frequency, ACSF, 4.01 ± 0.56 Hz vs U50488, 3.01 ± 0.44 Hz, n = 13, p = 0.0077, t = 3.19, df = 12, paired t test; Fig. 7B,E,F). Different from the control saline treatment group, the suppressive effects of DAMGO on mIPSCs were diminished after chronic morphine treatment (Amplitude, ACSF, 9.66 ± 0.79 pA vs DAMGO, 9.11 ± 0.66 pA, n = 12, p = 0.0794, t = 1.93, df = 11; Frequency, ACSF, 4.33 ± 0.78 Hz vs DAMGO, 3.81 ± 0.72 Hz, n = 12, p = 0.0544, t = 2.15, df = 11, paired t test; Fig. 7G,I,J). The effects of U50488 (1 μm) were also attenuated after chronic morphine treatment (Amplitude, ACSF, 8.31 ± 0.86 pA vs U50488, 8.05 ± 0.80 pA, n = 12, p = 0.266, t = 1.17, df = 11; Frequency, ACSF, 2.94 ± 0.56 Hz vs U50488, 2.63 ± 0.54 Hz, n = 12, p = 0.0495, t = 2.21, df = 11, paired t test; Fig. 7H,K,L). The modulation of inhibitory inputs by MOR and KOR were reduced in morphine-treated mice, suggesting that the involvement of MOR and KOR in PVT was reduced after chronic morphine exposure.
Chronic morphine exposure attenuates the suppressive effects of MOR and KOR agonists on inhibitory inputs to PVT neurons and also reduces the kappa opioid regulation in the ZI to PVT pathway. A, Example traces showing the mIPSCs before (black) and after (blue) the application of the MOR agonist DAMGO (1 μm) in the saline-treated mice. B, Example traces showing the mIPSCs before (black) and after (green) KOR agonist U50488 (1 μm) application in the saline-treated mice. C, D, DAMGO (1 μm) reduced the amplitude and frequency of mIPSCs in saline-treated mice (Amplitude, ACSF, 9.01 ± 0.82 pA vs DAMGO, 8.55 ± 0.82 pA, n = 11, p = 0.0459, t = 2.28, df = 10; Frequency, ACSF, 3.76 ± 0.85 Hz vs DAMGO, 3.52 ± 0.83 Hz, n = 11, p = 0.006, t = 3.47, df = 10, paired t test). E, F, U50488 (1 μm) reduced the amplitude and frequency of mIPSCs in saline-treated mice (Amplitude, ACSF, 8.30 ± 0.44 pA vs U50488, 7.44 ± 0.40 pA, n = 13, p = 0.0013, t = 4.19, df = 12; Frequency, ACSF, 4.01 ± 0.56 Hz vs U50488, 3.01 ± 0.44 Hz, n = 13, p = 0.0077, t = 3.19, df = 12, paired t test). G, Example traces showing the mIPSCs before (black) and after (red) DAMGO (1 μm) application in the morphine-treated mice. H, Example traces showing the mIPSCs before (black) and after (purple) U50488 (1 μm) application in the morphine-treated mice. I, J, DAMGO (1 μm) failed to alter the amplitude and frequency of mIPSCs after chronic morphine treatment (Amplitude, ACSF, 9.66 ± 0.79 pA vs DAMGO, 9.11 ± 0.66 pA, n = 12, p = 0.0794, t = 1.93, df = 11; Frequency, ACSF, 4.33 ± 0.78 Hz vs DAMGO, 3.81 ± 0.72 Hz, n = 12, p = 0.0544, t = 2.15, df = 11, paired t test). K, L, U50488 (1 μm) could not change the amplitude of mIPSCs after chronic morphine treatment (ACSF, 8.31 ± 0.86 pA vs U50488, 8.05 ± 0.80 pA, n = 12, p = 0.266, t = 1.17, df = 11, paired t test). U50488 (1 μm) reduced the frequency of mIPSCs (ACSF, 2.94 ± 0.56 Hz vs U50488, 2.63 ± 0.54 Hz, n = 12, p = 0.0495, t = 2.21, df = 11, paired t test), but the effects were attenuated in morphine-treated mice compared with saline-treated mice. M, The amplitude of oIPSCs was reduced after U50488 (1 μm) application in the saline-treated mice, but the paired-pulse ratio did not change (Amplitude, ACSF, 255.3 ± 21.94 pA vs U50488, 205.9 ± 19.36 pA, n = 20, p = 0.00007, t = 5.06, df = 19; PPR, ACSF, 0.91 ± 0.04 pA vs U50488, 0.91 ± 0.03 pA, n = 20, p = 0.7451, t = 0.33, df = 19, paired t test). N, The amplitude and PPR of oIPSCs didn't change after U50488 (1 μm) application in the morphine-treated mice (Amplitude, ACSF, 108.2 ± 13.85 pA vs U50488, 103.2 ± 14.03 pA, n = 19, p = 0.2403, t = 1.21, df = 18; PPR, ACSF, 1.08 ± 0.12 pA vs U50488, 1.19 ± 0.13 pA, n = 14, p = 0.196, t = 1.36, df = 13, paired t test; *p < 0.05, **p < 0.01, ****p <0.0001). N.S., Not significant.
Is the kappa opioid regulation of the ZI to PVT input sensitive to chronic morphine exposure? Optically evoked IPSCs were recorded from saline or morphine-treated mice. U50488 (1 μm) reduced the amplitude of oIPSCs from ZI to PVT in the saline treatment mice (ACSF, 255.3 ± 21.94 pA vs U50488, 205.9 ± 19.36 pA, n = 20, p = 0.00,007, t = 5.06, df = 19, paired t test; Fig. 7M, left), but this effect was reduced by chronic morphine treatment (ACSF, 108.2 ± 13.85 pA vs U50488, 103.2 ± 14.03 pA, n = 19, p = 0.2403, t = 1.21, df = 18, paired t test; Fig. 7N, left). As in wild-type mice, U50488 did not alter the PPR in both saline-treated and morphine-treated mice (saline treatment, ACSF, 0.91 ± 0.04 vs U50488, 0.91 ± 0.03, n = 20, p = 0.7451, t = 0.33, df = 19; morphine treatment, ACSF, 1.08 ± 0.12 vs U50488, 1.19 ± 0.13, n = 14, p = 0.196, t = 1.36, df = 13, paired t test; Fig. 7M,N). These results suggest that KOR is important in modulating inhibitory input from the ZI to the PVT and that chronic morphine exposure can disinhibit the ZI to PVT pathway.
Discussion
The PVT serves as a key node in the neural circuits that regulate addictive behaviors. In addition to the acute effects of drugs, PVT neurons are also recruited at different stages of the drug addiction cycle (Zhou and Zhu, 2019). In this study, we investigated the effects of morphine and opioid receptor agonists on the activities of PVT neurons. Bath-applied morphine and the MOR agonist DAMGO reduced the activities of PVT neurons in brain slices. Furthermore, MOR and KOR were also involved in modulating inhibitory inputs to the PVT. Prolonged morphine use decreased the contribution of opioid receptors in the PVT. We did not observe any involvement of DOR in the regulation of PVT activities. Our results showed that MOR and KOR are essential in the regulation of PVT activities.
Neurons in the PVT are primarily glutamatergic and receive GABAergic inputs from other nuclei, such as the ZI. We found that KOR can regulate the inhibitory projection from the ZI to the PVT. The ZI is also an integrative node for behavioral modulation and an inhibitory subthalamic region connecting with many brain areas (Wang et al., 2020). Recently, different action sequences based on the motivational level of novelty seeking have been revealed, and a circuit underlying curiosity and novelty-seeking behavior requires a subpopulation of medial ZI neurons (Ahmadlou et al., 2021). PVT neurons encode multiple salient features of sensory stimuli, including reward, aversion, and novelty, and weigh the valence between the positive and negative information (Zhu et al., 2018). We found that activation of opioid receptors in the terminals of ZI neurons reduced the inhibitory inputs to the PVT, which could disinhibit the activities of PVT neurons. Thus, the ZI to PVT pathway may be important for motivational behavior, and the opioid system in this pathway may influence behavioral responses to dynamic environmental contexts.
Opioid receptors are members of the GPCR family, and they can activate GIRK channels via G-proteins. Activation of GIRK channels induces membrane hyperpolarization of the neurons via K+ efflux and reduces neuronal excitability (Ikeda et al., 2002; Rifkin et al., 2017). We applied the MOR agonist DAMGO and the GIRK channel antagonist Tertiapin-Q to brain slices. DAMGO application decreased the firing rate and hyperpolarized the membrane potential of PVT neurons. Tertiapin-Q reversed the hyperpolarization induced by DAMGO but failed to restore the firing in most of the cells. We therefore proposed that activation of GIRK channels is the underlying mechanism for membrane potential hyperpolarization, but other mechanisms such as a decrease in calcium conductance may contribute to the reduction in the firing rate on MOR activation (Borgland, 2001), which requires future investigation.
Chronic morphine exposure induces adaptive phenomena such as tolerance and dependence. In tolerance, more morphine is required to achieve the initial effect, whereas dependence is manifested by the withdrawal syndrome induced by cessation of morphine exposure (Cruz et al., 2008). To distinguish between the effects of morphine tolerance and withdrawal, we compared the results between the morphine exposure group and the naloxone-precipitated withdrawal group. As in the morphine treatment group (2 d after the last morphine injection), the decrease in the firing rate induced by DAMGO was dramatically attenuated in both the morphine exposure and naloxone-precipitated withdrawal groups. Thus, these results suggest that the reduced inhibition of firing rate by DAMGO is because of chronic morphine exposure rather than spontaneous withdrawal.
We also found that DAMGO was able to induce robust GIRK currents with similar amplitudes in the saline-treated, morphine-treated, and morphine-exposed mice. MOR coupling to GIRKs was not desensitized by chronic treatment, suggesting that other intracellular downstream effectors such as adenylyl cyclase, voltage-gated Ca2+ channels, and others (Williams et al., 2001) play more important roles in MOR desensitization. However, DAMGO failed to evoke any apparent outward currents in the naloxone-precipitated withdrawal mice, suggesting that the coupling of MOR to GIRK channels was reduced in these mice. This result indicates that naloxone induces uncoupling of MORs and GIRKs. Naloxone might suppress functional GIRK channels, probably through a compensatory mechanism involving internalization and phosphorylation of GIRK channels (Hearing et al., 2013). These results were also consistent with previous report highlighting the importance of GIRK in the naloxone-precipitated morphine withdrawal (Cruz et al., 2008).
Desensitization of opioid receptors is thought to be required for tolerance and involves phosphorylation by kinases and uncoupling from G-proteins realized by arrestins (Marie et al., 2006). Internalization or endocytosis of GPCRs is another common way to regulate their activity by removing active receptors from the cell surface into the intracellular space. GPCR internalization is mediated by clathrin-coated pits, caveolae, and uncoated vesicles (Claing et al., 2002). Opioid receptors rapidly diffuse across the axon surface and externalize specifically at presynaptic terminals following ligand-induced activation (Jullié et al., 2020). In this study, the effects of opioid receptor agonists on the excitability and inhibitory inputs of PVT neurons were attenuated after chronic morphine exposure. These results may be because of desensitization and internalization of opioid receptors. The diminished effects of firing could also be caused by possible changes in opioid regulation of presynaptic inputs as our recordings were made in the absence of synaptic blockers.
A previous study demonstrated a morphine-induced increase in the firing rate of PVT neurons, which was only observed during the light cycle but not the dark cycle (McDevitt and Graziane, 2019). This study compared the firing rate between the saline-treated and morphine-treated mice, which reflects contributions from several factors including synaptic inputs, intrinsic excitability, and opioid and other neuromodulation. We did the recordings during the light cycle, but we applied opioid agonists and antagonists to the brain slices, directly probing the function of opioid receptors in the PVT. Our study also revealed the changes in opioid modulation of PVT activities induced by chronic morphine treatment.
Together, MOR and KOR contribute to the modulation of PVT activities and inhibitory synaptic inputs, and these effects can be reduced by chronic morphine exposure.
Footnotes
This work was supported by Ministry of Science and Technology of the People's Republic of China Science and Technology Innovation 2030—Major Project Grant 2021ZD0202103; National Natural Science Foundation of China Grants 31900809, 81922024, and 82171492; China Postdoctoral Science Foundation Grant 2019M653116; Science, Technology, and Innovation Commission of Shenzhen Municipality Grants RCJC20200714114556103, ZDSYS20190902093601675, and JCYJ20210324141201003; Guangdong Basic and Applied Basic Research Foundation Grant 2021A1515010729; and Guangdong Provincial Key Laboratory of Brain Connectome and Behavior Grant 2017B030301017. We thank Dr. Ming-Hu Han and Dr. Jianyuan Sun for comments on the manuscript
The authors declare no competing financial interests
- Correspondence should be addressed to Yingjie Zhu at yj.zhu1{at}siat.ac.cn