Mice aged 3–4 months were obtained from our breeding colonies at the University of California Los Angeles (UCLA). All experimental procedures were performed in accordance with the United States Public Health Service Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at UCLA. Every effort was made to minimize pain, discomfort, and the number of mice used. Animal housing conditions were maintained under a standard 12 h light/dark cycle (light cycle starting at 6 AM and ending at 6 PM) and at a temperature of 20–26°C. The animals had ad libitum access to food and water.

To examine the effects of opioids on direct and indirect pathway MSNs, D1- (n = 3) and A2A-Cre (n = 3) mice of either sex were injected bilaterally at stereotaxic coordinates, relative to Bregma (in mm): +1 A/P, ±2 M/L, -3.3 D/V (dorsal striatum) with 1 μl of an adeno-associated virus (AAV) containing GCaMP6f. After 2 weeks, the cortical tissue overlying the striatum was aspirated to allow implantation of a GRIN lens (1.8 mm, Edmund Optics, Barrington, NJ, USA) with dental cement (Supplementary Figures 1B,C). 1–2 weeks later, a baseplate to hold the open-source UCLA Miniscope (V3) was affixed to the skull with dental cement. For comparison, to examine the effects of opioids on cortical pyramidal neurons (CPNs), a separate group of mice (3 month-old) were injected with 0.5 μl of an AAV containing GCaMP6f expressed using either the pan-neuronal Syn promoter (n = 2 mice) or the more selective for excitatory neurons CaMKII promoter (n = 2 mice) at stereotaxic coordinates, relative to Bregma (in mm): +0.5 A/P, +1 M/L, and -0.6 D/V, corresponding to the motor M1 region (Supplementary Figure 1A). After 2 weeks, a thin layer of cortical tissue was aspirated to allow implantation of the GRIN lens (1.8 mm, see above). 1–2 weeks later a baseplate to hold the Miniscope was affixed to the skull.

Mice were placed in a cylindrical behavioral chamber (30 cm in diameter and 30 cm high) after a Miniscope was attached to the baseplate. They were allowed to acclimate to wearing the Miniscope and moving freely in the chamber (1 hr for three consecutive days). Both Ca²⁺ transients acquired with a V3 Miniscope and behavioral (webcam) data were collected using the Data Acquisition Software (DAQ, UCLA, Los Angeles, CA, USA) simultaneously for ∼5 min. Ca²⁺ transients were captured at 20 Hz with maximum exposure and gain. Behavioral videos were captured between 20 and 22 Hz due to the webcam frame rate. After baseline data were acquired, mice were injected with either oxycodone (3 mg/kg, IP) or saline as control. The final volume ranged between 0.2 and 0.3 ml. Recordings were taken at 5 and 15 min after oxycodone or saline administration, but mostly data from 15 min are reported. As we were only interested in the acute effects of systemic administration of oxycodone, each mouse was used only 1–2 times to avoid opioid receptor sensitization.

The number of mice, videos, and cells used for behavioral and Miniscopes data analyses are shown in Supplementary Table 1. Behavioral data were processed using the Python-based ezTrack video analysis pipeline (Pennington et al., 2019). Using the location tracing module, an animal’s center of mass (COM) was tracked across each session. A reference frame was defined, with each frame compared to the reference frame to locate the animal’s COM. The COM was saved, and the module created both traces and a heat map of the final output. Distance traveled was calculated in the software by measuring a specific distance in the module (in pixels) and converting this pixel distance to actual length (cm) based on the dimensions of the behavioral chamber. Animal movement was also analyzed manually through visual examination of each frame of the behavioral videos, receiving a score of 0 (non-movement) and 1 (movement). Lastly, the percentage of movement was calculated by dividing the number of movement frames by the total number of frames for each behavioral video.

Ca²⁺ transient activity from Miniscope video recordings was processed using MiniAn, an open-source Miniscope analysis pipeline (Dong et al., 2022). This pipeline performs background subtraction, rigid motion correction using the NormCorre algorithm, and source extraction using the constrained non-negative matrix factorization (CNMF) algorithm. MiniAn produces the estimated footprint, Ca²⁺ activity trace and the deconvolved activity trace of each neuron. To examine the same neurons across different recording sessions before and 15 min after drug administration we used the Python-based program Cross-reg, which is part of the MiniAn package. This software aligned the spatial footprints of the neurons and considered the neuron to be the same across the recordings if the footprints were within a five pixel threshold of movement between sessions. The Cross-reg program then outputs the neurons that are matched between sessions, and across all sessions in total. A threshold was manually set for each Ca²⁺ trace to identify peaks. The minimum threshold was set to the RMS of the trace and increased if the threshold did not appropriately remove noise. Neurons with non-physiological Ca²⁺ traces, e.g., continuous shifts of the baseline, also were manually removed. In addition, since after viral injection not only cell bodies but also the neuropil show fluorescence, we set up several criteria to select only what the program and our best estimates indicated high probability of detecting neuronal somata. These included a circular or ovoid shape, an area ranging from 200 to 400 μm² (average ∼294 ± 23 μm² from a random sample of n = 10 cells), and only objects exhibiting changes in fluorescence (Donzis et al., 2020). Thus, in contrast to fiber photometry, which detects mainly changes in the neuropil (Legaria et al., 2022), the Miniscopes allowed for more selective measurements of neuronal activity after elimination of spurious objects.

After the last Miniscope imaging recording, some mice were sacrificed and brain slices containing cerebral cortex and striatum (350 μm) were obtained for electrophysiological recordings and Ca²⁺ imaging in vitro using 2PLSM. Ca²⁺ imaging was performed using a Scientifica 2PLSM equipped with MaiTai laser (Spectra-physics). To image Ca²⁺ signals, the laser was tuned at 920 nm wavelength. The laser power measured at the sample was less than 30 mW with a 40 × water-immersion objective lens (Olympus, Tokyo, Japan). Ca²⁺ transients were sampled at a rate of 3.81 Hz using SciScan software (Scientifica, Uckfield, UK). Optical signal amplitudes were expressed as ΔF/F, where F represents the resting light intensity at the beginning of the optical trace, and ΔF represents the change in peak fluorescence during the biological signal. These values were then multiplied by 100 to obtain percentage values. ImageJ (NIH) was utilized to process raw fluorescence values, which were then exported to Excel (Microsoft, Redmond, WA, USA) and Clampfit software to calculate % ΔF/F.

The effects of oxycodone on membrane and synaptic properties were examined in labeled and unlabeled MSNs. Only the intact (non-aspirated) side was used. In a separate group of control mice not used for Miniscope recordings, whole-cell patch clamp recordings were obtained from unidentified MSNs. Coronal slices containing cerebral cortex and striatum were perfused with oxygenated ACSF and maintained in the chamber at room temperature. Whole-cell patch clamp recordings from MSNs were obtained in voltage clamp mode using Cs-methanesulfonate as the pipette solution, or in current clamp mode using K-gluconate as internal solution (Holley et al., 2022). After determination of basic membrane properties, including cell membrane capacitance, input resistance, and decay time constant, spontaneous synaptic activity was recorded. At -70 mV holding potential, most synaptic events are glutamatergic (excitatory post-synaptic currents, sEPSCs) and mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. At a holding potential of +10 mV, most events are GABAergic (inhibitory post-synaptic currents, sIPSCs) and mediated by GABA_A receptors. At this holding potential (+10 mV), after 3–5 min recording in control conditions, oxycodone (1 μM) was added to the external ACSF solution. The recording at this potential continued for at least 8 min and then the potential was reverted to -70 mV to examine the effects of oxycodone on glutamatergic activity. Measures of basic membrane properties used the Clampfit program and the frequency of spontaneous synaptic activity was calculated with the miniAnalysis program.

There were three levels of analysis in our study, mice, video recording sessions, and cells. Most animals were tested with oxycodone only once to prevent opioid receptor sensitization. One A2A mouse and one Syn mouse were tested twice to determine reproducibility of the response to the drug. The effects of oxycodone were assessed by comparing number of cells and their frequencies per video (session) analyzed. Values are expressed as mean ± SEM. Statistical significance was calculated using SigmaPlot (v. 14). Data were analyzed by Student’s t-tests, paired or unpaired, as well as one- and two-way ANOVAs with or without repeated measures (RM). Pairwise multiple comparison procedures used appropriate post-hoc tests. Differences between or among groups were considered significant if p < 0.05.

A single dose of oxycodone (3 mg/kg, IP) was selected for these studies. This is a relatively high dose more likely to replicate both analgesic and addictive properties of oxycodone in mice. In a previous study, we found that this dose induces hyperkinetic behavior and, when repeated for three consecutive days, it also induces locomotor sensitization, which does not occur in mice lacking MORs in striatal neurons from the direct pathway (D1 receptor-expressing) (Severino et al., 2020). Another study found that doses between 2 and 4 mg/kg (subcutaneous) produce maximal analgesia starting at ∼3 min after systemic injection (Nasseef et al., 2019).

One week following baseplate implantation the mice were allowed to acclimate to the recording chamber and to the Miniscope. They quickly adapted to both and the weight of the Miniscope did not perturb the ability of the mice to move and explore freely (Figure 1A). Before oxycodone administration, mice explored the behavioral arena, reared, and groomed normally. After oxycodone administration, typical signs of MOR activation occurred. These included the typical Straub tail starting ∼3 min after injection, increased locomotion after 5–6 min, and circling behavior (Figure 1B). In addition, the mice moved swiftly in a tiptoeing manner. Rearing episodes occurred at least once before oxycodone, whereas they were completely absent after oxycodone.

To determine the time course of oxycodone effects, as a first approximation, we took videos before (Pre), and at 5 and 15 min after systemic injection of oxycodone. We noticed that, while behavioral changes (e.g., hyperkinesia), were already apparent 5 min after oxycodone, changes in Ca²⁺ activity were more progressive and effects were more pronounced at 15 min compared with 5 min after oxycodone (Supplementary Figure 2). Thus, for subsequent statistical analyses we compared only Pre and 15 min after oxycodone or saline. This time coincides with maximal effects of oxycodone, based on pharmacokinetics in rodents (Huang et al., 2005; Nasseef et al., 2019).

We first measured the total amount of time the mouse displayed in movement versus non-movement before and 15 min after saline or oxycodone, regardless of Cre expression (D1- or A2A-Cre) or brain region injected with the Ca²⁺ reporter (striatum or cortex) (n = 5 sessions for saline and n = 8 for oxycodone). For that purpose, we determined the number of frames where movement was detected and divided by the total number of frames to obtain a percentage of movement. The duration of each frame was 45–50 ms based on webcam framerate. There was a statistically significant interaction between treatment and time (two-way ANOVA, F_1,22 = 31.9, p < 0.001). The percent of time moving increased significantly after oxycodone administration (Pre versus 15 min, p < 0.001, Holm–Sidak post-hoc t-test), whereas after saline, if anything, there was a non-significant decrease in movement (Pre versus 15 min, p = 0.08, Holm–Sidak post-hoc t-test) (Figure 1C). The total distance mice traveled before and 15 min after oxycodone administration also was determined. Similar to percent movement, there was a statistically significant interaction between treatment and time (two-way ANOVA, F_1,22 = 4.37, p = 0.048). The distance traveled increased significantly after oxycodone administration (Pre versus 15 min, p = 0.01, Holm–Sidak post-hoc t-test), whereas after saline there was practically no change in distance traveled (Pre versus 15 min, p = 0.66, Holm–Sidak post-hoc t-test). As for velocity, the difference in the mean values between Pre and post injection conditions was statistically significant when the different treatments were taken into consideration (two-way ANOVA, F_1,22 = 4.5, p = 0.045). Thus, the velocity significantly increased after oxycodone (Pre versus 15 min, p = 0.003, Holm–Sidak post-hoc t-test), whereas it remained constant 15 min after saline injection (Pre versus 15 min, p = 0.95, Holm–Sidak post-hoc t-test). Effects began to subside 30 min after oxycodone injection (not shown).

After oxycodone injection, the overall fluorescence in the field of view was reduced significantly. The major contributor of this reduction was a decrease in the number of visible cells. In contrast, after saline injection only a small, non-significant decrease was detected (Figures 2A,B) and appeared to be correlated with less overall movement (see Figure 1C). When grouping together D1 and A2A MSNs, there was a statistically significant difference in the mean values between Pre and post injection conditions when the saline versus oxycodone treatments were taken into consideration (two-way ANOVA, F_1,26 = 10.6, p = 0.003). After oxycodone there was a statistically significant reduction in the number of visible neurons (p = 0.008, Holm–Sidak post-hoc t-test), whereas after saline there was no change (p = 0.1, Holm–Sidak post-hoc t-test).

Ca²⁺ transient frequency of striatal MSNs (D1 + A2A) remained unchanged before and after saline injections (Supplementary Figure 4). Further, before oxycodone and before or 15 min after saline injection, multi-neuronal Ca²⁺ activity was tightly associated with movement (Figures 3A–C and Supplementary Figures 3A, 4). To determine this association, we calculated the frequency of Ca²⁺ transients during epochs of movement versus non-movement. Movement was defined as a clear and measurable displacement of the body within the arena. Small head movements were not included in the movement category. Regardless of treatment, prior to injection the mice spent most of the time in non-movement compared with movement (79 ± 5% versus 21 ± 5%). During episodes of immobility, the frequency of Ca²⁺ transients was significantly reduced compared with episodes of movement (Supplementary Figure 3A). We then compared the effects of oxycodone on the frequency of Ca²⁺ transients Pre and 15 min after injection. There was an overall reduction in frequency after oxycodone (D1 + A2A, p = 0.0048, Student’s t-test). However, while the reduction in frequency observed in D1-MSNs was not statistically significant (p = 0.20, Student’s t-test), the reduction in A2A-MSNs Ca²⁺ transient frequency was statistically significant (p = 0.021, Student’s t-test) (Figure 3D). This suggests that the overall reduction in frequency of striatal neurons after oxycodone is driven primarily by MSNs constituting the A2A adenosine receptor-tagged indirect pathway. In agreement, even though the frequency of Ca²⁺ transients was not significantly reduced in D1-MSNs after oxycodone, their activity still maintained a positive correlation between number of Ca²⁺ transients during movement versus non-movement, this correlation was lost in A2A-MSNs (Supplementary Figure 3A). We also noticed that after oxycodone there was a dissociation between Ca²⁺ transient activity and behavior, which became more stereotyped compared to the more random behavior in Pre oxycodone conditions (see Figure 3B).

To determine if opioid administration causes generalized depression of neuronal activity and as a comparison with striatal changes, we also examined the effects of oxycodone on CPNs. Mice were injected with the Ca²⁺ indicator using a Syn (n = 2) or a CaMKII (n = 2) promoter to more selectively infect excitatory CPNs. The number of CPNs infected with the Ca²⁺ indicator was consistently higher than that in striatal MSNs. The overall frequency of Ca²⁺ transients also was consistently higher than that of MSNs. Similarly, the number of CPNs infected with the Syn promoter was higher than that of CPNs infected with the CaMKII promoter (Figure 4). As changes induced by oxycodone in CPNs infected with either promoter were similar, and considering that CPNs are the most abundant cell types in the cerebral cortex, data were analyzed separately or pooled together. Notably, the number of CPNs visualized Pre and 15 min after oxycodone injection did not change significantly when all CPNs (Syn + CaMKII) were considered (p = 0.577, Student’s t-test) or when separated (Syn p = 0.618, CaMKII p = 0.723, Student’s t-test), indicating that the depression of Ca²⁺ activity observed in striatum did not occur in the cortex (Figures 4A,B). More remarkably, the frequency of Ca²⁺ transients was significantly increased after oxycodone (all CPNs combined p = 0.003, Syn CPNs p = 0.028, and CaMKII CPNs p = 0.029, Student’s t-test) (Figure 5), suggesting that DLS is an unlikely substrate of behavioral hyperactivity after oxycodone. Interestingly, the correlation between Ca²⁺ transient activity and behavior was less evident in cortex compared to stratum. Thus, while the Ca²⁺ transient frequency was slightly reduced during non-movement compared to movement, this correlation was lost after oxycodone, during which the number of Ca²⁺ transients was practically equal during movement and non-movement (Supplementary Figure 3B). Some examples of individual MSNs and CPNs before and after oxycodone can be seen in Supplementary Figure 5. In addition, raw videos (without motion correction or background subtraction) illustrating effects of oxycodone in striatum and cortex are available in the Supplementary Videos 1–6.

After completing the Miniscope recordings, some mice were sacrificed, the baseplate and lens removed, and the brains extracted and sliced to determine the location and extent of the viral injection. Visual and microscopic examination confirmed the location above the dorsal striatum, with no damage extending into this region. However, as in these mice the GCaMP6 viral injections were bilateral, only the intact side was used for slice electrophysiology (Supplementary Figure 1D). Most of the recordings were from fluorescent-positive cells (n = 7 from 6 different mice). In the case of D1- or A2A-Cre, if a cell was not fluorescent, it was considered as belonging to the other pathway, i.e., a negative cell from a D1-Cre animal was assumed to belong to the indirect pathway and vice versa. Data from Cre animals were pooled. In current clamp mode, oxycodone administration in brain slices caused a small membrane hyperpolarization (2–3 mV) in all but 1 MSN. This hyperpolarization was enough to cause an increase in inward rectification and reduction in membrane input resistance. As a consequence, cell excitability decreased in all but 1 cell (7 ± 2 APs in control conditions and 5.2 ± 1.6 after oxycodone, with 1 s duration pulses) (Figure 6A). The difference however, did not reach statistical significance (p = 0.17, paired t-test). Finally, similar to the previous voltage clamp recordings in intact animals, the spontaneous synaptic activity recorded at resting membrane potential was reduced significantly 10 min after oxycodone application (3.46 ± 0.4 Hz in control conditions and 2.48 ± 0.2 Hz after oxycodone, p = 0.024, paired t-test).

Electrophysiological data on the effects of bath application of oxycodone (1 μM) also were collected from intact WT mice (n = 5) not used for Miniscope imaging. Under infrared videomicroscopy, cells had the typical size, and shape of striatal MSNs (n = 10). In addition, basic membrane properties measured in voltage clamp also corresponded to those of MSNs and had an average cell membrane capacitance of 136 ± 13 pF, input resistance of 47.6 ± 4.3 MΩ, and a decay time constant of 2.3 ± 0.2 ms. The spontaneous synaptic currents recorded at -70 mV (putative EPSCs) were reduced after acute application of oxycodone (3.2 ± 0.4 Hz control and 2.0 ± 0.3 Hz after oxycodone, p = 0.004, paired t-test) (Figures 6B–D). At +10 mV, the average frequency of spontaneous synaptic currents (putative IPSCs) also was significantly reduced by acute application of oxycodone (3.1 ± 0.4 Hz in control conditions and 2.1 ± 03 Hz after the drug, p = 0.008, paired t-test). Overall, the electrophysiological data confirmed a generalized reduction of MSN excitability and synaptic inputs in DLS.

As oxycodone increased locomotor activity, we expected to observe an increase in Ca²⁺ transient activity in D1 MSNs. In order to confirm results from Miniscope imaging, some slices from D1-Cre mice also were used for 2PLSM imaging after in vivo recordings were completed. Because striatal MSNs in vitro do not fire APs spontaneously due to highly hyperpolarized resting membrane potentials, cells were activated with the K⁺ channel blocker 4-aminopyridine (4-AP, 100 μM). By blocking A-type K⁺ currents, 4-AP increases neurotransmitter release and induces spontaneous membrane oscillations. Alternatively, APs were evoked by intracellular injection of depolarizing currents. In all cases, oxycodone caused a reduction in both Ca²⁺ transient frequency (from 5.75 events to just 1/600 s, p = 0.05, paired t-test) and amplitude (52% reduction, p = 0.008, paired t-test) of MSNs in D1-Cre mice (n = 7 cells, in 6 slices from 3 D1-Cre mice) (Figure 7 and Supplementary Videos 7, 8).