Effects of adolescent morphine exposure on brain stimulation reward sensitivity and dopamine neuron excitability

Abstract

Adolescent substance abuse drives lasting changes in reward processing and decision making. To determine the effects of opioid treatment and withdrawal on reward sensitivity during adolescence, we used a rate-frequency (RF) intracranial self-stimulation (ICSS) paradigm in adolescent rats. On PD28, rats were implanted with stimulating electrodes in the medial forebrain bundle at the level of the lateral hypothalamus and trained twice-daily to perform a variant RF ICSS task tailored for adolescents. Morphine (5 mg/kg) or saline (0.5 ml/kg) was injected daily prior to the AM ICSS session from PD45–51 and we continued to perform sessions through PD60 to measure withdrawal. Rats were then trained on a standard adult RF ICSS task. All animals were tested from PD90–96 while undergoing daily morphine administration (10mg/kg) and for an additional 9 days of withdrawal. In adolescent controls, reward sensitivity was stable across baseline, injection, and post-injection sessions. Adolescent morphine administration resulted in a significant increase in reward sensitivity relative to controls followed by a reduction lasting for the entire 9-day withdrawal period. Re-exposure to morphine in adulthood resulted in a greater increase in sensitivity during administration and a more profound suppression in withdrawal relative to those that had not been exposed to morphine in adolescence. In a separate group of animals, withdrawal from adolescent morphine treatment resulted in reduced electrophysiological excitability of ventral tegmental area dopamine neurons that may contribute to lasting alterations in reward sensitivity. Such alterations following adolescent morphine exposure may drive susceptibility to maladaptive behaviors in adulthood.

1. Introduction

The endogenous opioid system plays a critical role in reward and reinforcement, contributing to the abuse potential of exogenous opioids. Opioid use disorder (OUD) has been proposed to result from an allostatic process where such use alters neurobiological set points of brain reward, stress, and executive control networks [1–5], ultimately resulting in a persistent, pathological, and compulsive cycle of opioid abuse [6,7]. While opioid receptors are widely expressed throughout the brain, they are particularly concentrated in mesolimbic pathways that subserve reward and reinforcement [8]. Overactivation of these pathways by drugs of abuse is thought to produce opponent processes that ultimately reduce reward function [2,4]. Indeed, depletion of dopamine (DA) in mesolimbic pathways is a common feature of withdrawal across multiple substances of abuse [9–11], serving as a risk factor for relapse.

Regulation of reward sensitivity undergoes a critical period of development during adolescence, in which maturation is dependent on experience, giving rise to the neural and behavioral patterns typically observed in adulthood [12,13]. During adolescence, activity in mesolimbic circuitry is heightened, with greater DA release in response to reward [14–17] and increased excitability of ventral tegmental area DA (VTA_DA) cell bodies [18]. Opioid use during adolescence may induce neuropathological changes of circuitry underlying reward and reinforcement behaviors [19,20], altering their developmental trajectory [21] and increasing long-term vulnerability to substance use disorders (SUD), including OUD.

For decades, research has used intracranial self-stimulation (ICSS) procedures to assay the effects of drugs of abuse on the functional sensitivity of brain reward systems. In ICSS, operant responses are reinforced by the delivery of electrical stimulation to the medial forebrain bundle (MFB) at the level of the lateral hypothalamus [22,23]. One mechanism thought to support ICSS is excitation of the mesolimbic pathway, driving VTA_DA neurons to release DA in downstream regions [14,24,25]. By varying the frequency of electrical stimulation delivered for each operant response and measuring the rate of self-delivery of stimulation, rate-frequency procedures allow for psychometric measurement of reward sensitivity [23,26]. ICSS rate-frequency estimation supports robust behavior without the need to induce a physiological need state for motivation [27]. While this behavior is stable for long periods when used in adulthood, the length of training (typically several weeks) has precluded its usefulness during the limited period of adolescence in rats [28].

Despite the critical role of the mesolimbic pathway in elevated long-term risk for SUDs, it can be challenging to study adolescent behavior in animal models, particularly for complex cognitive behaviors. However, patterns of human drug use indicate the importance of understanding this period. The onset and prevalence of substance use are most common during adolescence and young adulthood [29]. Most adolescents initiate opioid use through non-medical use of prescription opioids, with 14 % of high school students reporting use [30]. Adolescent opioid abuse has persistent long-term consequences [31], including up to 50 % of those misusing opioids in adolescence experiencing SUD symptoms in adulthood [29]. The consequences of such misuse are enormous individual and societal costs [32]. Thus, it is important to examine the effects of drug exposure on behavioral and neural function during adolescence as well as longitudinally.

Here, we tested the effects of chronic morphine administration on reward sensitivity during both adolescence and adulthood. We adapted the standard rate-frequency ICSS paradigm to probe reward sensitivity in the same animal first in adolescence, then longitudinally in adulthood. We hypothesized that administration of morphine in naïve animals, adult and adolescent, would increase reward sensitivity, and withdrawal would reduce it. Additionally, we hypothesized that re-exposure to morphine during adulthood would exacerbate the rewarding effects of morphine administration and the depressive effects of morphine withdrawal. Finally, to directly probe the effects of adolescent morphine withdrawal on the mesolimbic pathway, we performed whole-cell patch clamp electrophysiology to determine changes in the excitability of VTA_DA cell bodies. We predicted that VTA_DA neuron excitability would be significantly depressed during adolescent morphine withdrawal.

2. Methods

2.1. Subjects

Male (n = 25) and female (n = 23) experimentally naïve Long-Evans rats were pair housed in plastic cages for the duration of all experiments. Animals were kept in a temperature- and humidity-controlled vivarium on a 12-h light-dark cycle (7:00am to 7:00pm) and given ad libitum access to food and water. Experimental testing was conducted in a separate room during the light phase of the cycle. Animals were treated and monitored in accordance with guidelines set by the National Institutes for Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care Committee of the University of Illinois Chicago.

2.2. Timeline and test conditions

Effects of chronic morphine were first tested in adult animals (8 male, 7 female) to replicate established findings that chronic morphine treatment increases sensitivity to ICSS while withdrawal reduces it [33]. Surgeries to implant stimulating electrodes were performed after postnatal day (PD) 60. Following 7 days of postoperative recovery, animals performed 1 behavioral session each morning (AM) beginning with 7 shaping sessions (detailed below). After shaping, animals were trained on a standard rate frequency (RF) task until stable (~30 sessions). Testing occurred in 3 phases of a within-subjects design: baseline (BL), injection (INJ), and post-injection (POST). The final 4 sessions of RF training served as baseline sessions for each animal. During the 7 days of injection sessions, all animals were treated with morphine (10mg/kg, intraperitoneal (i.p.)) 20 min prior to the start of the RF session. Following discontinuation of morphine injections, POST RF sessions continued daily for 8 days (Fig. 1A).

Fig. 1. Experimental timelines.

(A) In the naïve adult morphine RF ICSS experiment, all testing was conducted after PD60. Once trained on the standard RF task, morphine (10mg/kg) was injected 20 min prior to the RF ICSS session for 7 days. Post-administration RF ICSS sessions continued for 8 additional days. (B) In adolescents, behavioral sessions of the abridged RF ICSS (aRF) task were conducted twice daily. Morphine (5mg/kg) or Control (Saline, 0.5ml/kg) injections were performed 20 min prior to the AM aRF ICSS session for 7 days beginning at PD45. Post-administration aRF ICSS sessions continued to PD60. (C) Subjects from the adolescent aRF ICSS experiment were then trained once daily on the standard RF ICSS task. Once fully trained, all subjects received morphine (10mg/kg) injections 20 min prior to the daily RF ICSS session for 7 days beginning at PD90. Post-administration RF ICSS sessions continued to PD108. (D) For adolescent post-morphine VTA DA whole cell patch clamp electrophysiology, surgeries to label VTA DA neurons were conducted on PD28. Seven daily Morphine (5mg/kg) or Control (Saline, 0.5ml/kg) injections were performed starting at PD45. Patch clamp electrophysiological was performed from PD53–55.

A second group of animals (11 male, 10 female) was tested longitudinally from adolescence to adulthood [28,34]. These animals underwent surgery for stimulating electrode implantation on PD28. Following 7 days of postoperative recovery, animals performed sessions twice-daily (AM starting from 7:30–8AM, and PM starting from 4–4:30PM), beginning with 5 shaping sessions from PD35(AM)–37(AM). Following shaping, animals were trained on an abridged rate-frequency (aRF) ICSS task from PD37(PM)-42(PM). The four aRF sessions on PD43–44 served as the pre-treatment BL phase. From PD45–51, rats continued to perform two daily aRF sessions in the INJ phase, with half of the animals receiving an injection of morphine (5mg/kg, i.p.) 20 min prior to the start of the AM session. Control animals received daily injections of saline (0.5ml/kg, i.p.) 20 min before each AM session. For all animals, the PM session was conducted daily without drug administration preceding it. Following treatment, all animals continued to perform twice-daily POST sessions from PD52–60 under discontinuation of morphine/saline injections (Fig. 1B). From PD61–85, animals were trained on the standard RF task once-daily (AM). RF sessions from PD86–89 served as the pre-treatment BL phase. From PD90–96, all animals received an injection of morphine (10mg/kg, i.p.) 20 min prior to the start of the RF session. Following discontinuation of morphine injections, all animals continued to perform once-daily POST sessions of the RF task from PD97–105 (Fig. 1C).

A third group of animals (6 male, 6 female) was tested using ex vivo slice physiology experiments. Long-Evans rats expressing Cre recombinase under the control of the tyrosine hydroxylase promoter (TH:Cre⁺; [35]) underwent surgery on PD28 to label TH-positive neurons with Cre-dependent tdTomato in the VTA. From PD45–51, half of the animals received an injection of morphine (5mg/kg, i.p.), while control animals received an injection of saline (0.5ml/kg, i.p.) between 10 and 11 AM. Slice physiology was performed between PD53 and 56 (Fig. 1D).

2.3. Equipment

Behavioral testing was conducted in standard operant behavioral chambers equipped with a house light, speaker, retractable levers, and four infrared sources/detectors to measure locomotion in the chamber (Med Associates Inc.). Brain stimulation was generated by a programmable ICSS stimulator (Med Associates Inc.) and delivered via a bipolar stimulating electrode (PlasticsOne, Inc). Auditory stimuli were generated via a programmable audio generator (Med Associates Inc.).

2.4. Drugs

Morphine sulfate (10mg/ml) was administered at a dose of 5 mg/kg, intraperitoneal (i.p.) for adolescents or 10 mg/kg, i.p. for adults. Control saline injections were volumetrically matched (0.5ml/kg or 1ml/kg, i.p.). Doses were selected based on preliminary studies in our lab. In adults, we used fiber photometry to examine the effects of acute, escalating doses of morphine (2.5, 5, 7.5, and 10 mg/kg) on VTA_DA cell body activity and DA release in nucleus accumbens. The 10mg/kg dose elicited a robust increase in mesolimbic pathway activity [36]. In adolescent animals, the 10mg/kg dose resulted in adverse behavioral effects; 5mg/kg was selected to preserve rats’ ability to perform the aRF task.

2.5. Surgery

Rats were anesthetized with isoflurane (4 % induction, 2–3 % maintenance; SomnoSuite, Kent Scientific), injected with an analgesic (1mg/kg meloxicam (Mobic), subcutaneous (s.c.)), and placed in a stereotaxic instrument (Kopf Instruments). Depth of anesthesia was measured by pinching the animal’s toe and observing for withdrawal reflex periodically throughout the procedure. All measurements were made relative to Bregma [37].

For behavioral experiments, animals were implanted with a bipolar stimulating electrode targeting the MFB at the level of the LH (Adults: AP: −2.8, ML: ±1.7, DV: −7.8 mm relative to dura; Adolescents: AP: −2.5, ML: ±1.5, DV: −7.5 mm relative to dura). The electrode was secured to the skull by three stainless steel screws covered with Metabond (Parkell, Inc.) and dental acrylic. Electrode placements are shown in Fig. S1.

For slice electrophysiology experiments, TH:Cre+ Long Evans rats were unilaterally injected with a Cre-dependent td-Tomato (pAAV-FLEX-tdTomato; Addgene viral prep # 28,306-AAV1; 1 × 10¹³ vg/mL, diluted 1:2 in artificial cerebral spinal fluid (aCSF, Tocris) prior to injection) targeted to the VTA (AP: −4.5, ML: ± 0.7, DV: −6.5 mm).

Postoperative meloxicam (Mobic) and saline (1ml/kg, s.c.) were administered daily for two days following surgery. Postoperative monitoring of the incision site and subject body weight continued for a minimum of 10 days following surgery.

2.6. Behavioral testing

2.6.1. Shaping

Animals were shaped to lever press for stimulation on an FR1 schedule of reinforcement. Each session provided access to a lever for a period of 30 min. Each lever press delivered a 500 ms train of 100 Hz stimulation followed by a 500 ms timeout period. Amplitudes were initially set at 140 μA and were adjusted for each individual subject to the level between 80 and 250 μA that sustained greater than 50 lever presses per minute for adults, or 30 lever presses per minute for adolescents, for the first 15 min of the daily training session. Following shaping, individual stimulation amplitudes were held constant for the duration of all experimental manipulations.

2.6.2. Standard rate-frequency (RF) task

The Standard RF task measured the rate of self-administration of brain stimulation reinforcement over its full psychophysical range, from frequencies below the minimum to those above maximal, that support self-administration [23]. Each session consisted of five passes of 15 blocks, taking 75 min. A block began with the delivery of five, 500 ms non-contingent priming trains of rectangular bipolar stimulation pulses paired with a tone. Following the priming pulses, a lever extended, and subjects had access to 50 s of lever press contingent self-stimulation, where each lever press delivered a 500 ms train of stimulation and played an associated tone that scaled with stimulation frequency. A 5 s timeout period in which no stimulation was available preceded the start of the next block (Fig. 2A). The 15 frequencies ranged from 141 Hz to 28 Hz in 0.05-log10-Hz steps, with each paired with a corresponding auditory tone ranging from 3937 Hz to 100 Hz in 0.114-log10-Hz steps (Fig. 2B). Stimulation/tone frequencies were presented in descending order across blocks within each pass. Sessions of the standard RF task were conducted until animals attained stable performance, determined as theta values (threshold stimulation necessary to evoke responding, see below) within 10 % for at least three consecutive days (Fig. 2C).

Fig. 2. Rate-frequency ICSS Methods.

(A) Block design in all RF ICSS paradigms. Each block begins with houselight illumination and 5 priming pulses of 500 ms stimulation with paired auditory cue, followed by lever extension and cue light illumination for 50 s of self-stimulation on an FR1 schedule. Each block ends with a 5 s time-out period where no self-stimulation is available and the houselight is off. (B) Standard RF pass design, which consists of 15 blocks with descending stimulation frequencies from 141 to 28 Hz. Each RF session consists of 5 passes (C) Example of a standard RF session. The gray data points are from the first pass, which are not included in analysis. For each pass, three parameters are estimated from a fit (black curve) to describe sensitivity: Alpha (red triangle), the minimum frequency that supports maximal pressing rate; Theta (red circle), the minimum frequency to support pressing; and M50 (red square), the frequency that supports the mid-level of pressing from the linear portion of the curve. A leftward shift of the curve (decreased M50) indicates an increase in reward sensitivity, while a rightward shift (increased M50) indicates a decrease in reward sensitivity. (D) Abridged RF (aRF) pass design, which consists of 7 blocks with descending stimulation frequencies from 141 to 50 Hz. Each aRF session consists of 5 passes. (E) Example of an aRF session. The gray data points are from the first pass, which are not included in analysis. For each of the remaining passes, a linear curve is fit to lever presses as a function of frequency (black line) to determine the intercept, representing the number of lever presses for the median frequency (PR_M; red square). An upward shift of the curve (increased PR_M) indicates an increase in reward sensitivity, while a downward shift (decreased PR_M) indicates a decrease in reward sensitivity.

2.6.3. . Abridged rate-frequency (aRF) task

In adolescence, animals were tested on an adaptation of the Standard RF test paradigm to simplify the task such that training and testing could be completed within time constraints of rat adolescence [18]. In this task, the response to the full range of ICSS stimulation frequencies was measured using a subset of fewer, more discriminable frequencies. Each session consisted of 5 passes, and each pass consisted of 7 blocks, taking 35 min. Individual blocks were structured as described in the RF task but used an abridged set of frequencies. In the RF task, the 15 frequencies elicit a sigmoidal behavioral function, with a central span that shows a linear rate-frequency relationship. Within each pass of the aRF task, 7 descending frequencies were used to include the maximum (141 Hz), a linear portion (100, 89, 79, 71, and 63 Hz), and a sample of the low range (50 Hz) (Fig. 2D). These frequencies were selected to span the majority of the range of frequencies in the RF task, preserving the linear portion of the curve, while not oversampling the high and low ends where behavior is typically asymptotic at a ceiling or floor. Because session durations were shorter in the aRF task, two separate test sessions could be conducted each day (AM and PM). Twice-daily sessions were necessary to complete training by PD45, in advance of chronic morphine exposure during adolescence, with 5 shaping and 11 aRF training sessions completed in 8 days. While the primary interest was on the effects of morphine administration, the ability to test twice daily in adolescent animals allowed us to include sessions under the effect of morphine and hours after administration. It was not practical to test twice daily in adults due to the longer duration of standard RF sessions.

2.6.4. Motor behavior

To quantify potential effects of morphine administration and withdrawal on motor activity, we measured maximum lever press rate, total lever presses, and locomotion. By convention, behavior from the first pass was excluded (see 2.8). Maximum lever press rate was the maximum rate in a single block from each session, which cannot exceed 50 in the 50 s self-administration portion of any given block. Total lever presses was the sum of all lever presses from passes 2–5 of a session. The maximum total lever presses possible was fewer in the aRF task (1400) compared with the RF task (3000) due to the reduced number of frequencies tested (7 vs. 15). Locomotion was scored as the total number of beam breaks in the last pass of each session.

2.7. Histology

To validate electrode placement, following completion of all experiments, rats were deeply anesthetized with isoflurane. To mark the stimulation site, current (100 mA) was passed for 4 s through each electrode with a lesion-marking device (Ugo Basile, Comero, Verese Italy). Rats were then transcardially perfused with 0.9 % NaCl followed by 10 % buffered formalin solution (HT501320, Sigma Aldrich). Brains were removed and stored in a solution of 20 % sucrose in formalin for a minimum of 24 h at a temperature of 4° C. Brain tissue containing electrode tracks was sliced into 50 μm sections on a freezing stage microtome (SM2010R, Leica Biosystem) and stored in cryoprotectant at −20° C. Sections were mounted onto glass slides, air dried, and cover slipped prior to visualization. Anatomical boundaries were defined by the Paxinos and Watson rat brain atlas [37] and rats with electrode placements in the MFB at the level of the lateral hypothalamus were included in analyses (Fig. S1).

2.8. Analysis of behavior

All analyses were conducted in R v4.4.2 [38] using the tidyverse package v2.0.0 [39]. Figures were made with ggplot2 v3.5.1 [40] and ggpubr v0.6.0 [41]. Prior to analysis, RF data were processed according to established criteria [23]. The first pass of each session, when responding for stimulation is more variable, was excluded. In addition, if an animal failed to press during a trial, but the number of lever presses in the trials preceding and following the “0″ were greater than 20 percent of the maximum rate, the missing value was interpolated by averaging the preceding and following trial lever pressing values. Additionally, any time an animal failed to press during the first trial of a pass (i.e., the highest frequency), the “0” was replaced with the maximum presses for that pass. Any passes with fewer than 20 total lever presses in the standard RF task or 10 total lever presses in the abridged RF task were excluded from analysis.

2.8.1. Standard RF task

For each pass, the least-squares best fit of response rate across stimulation frequencies was calculated using the drc package v3.0.1 [42]. From this function, we determined values for three parameters - theta, M-50, and alpha – that describe the fit for each subject under baseline and treatment conditions. Theta, the theoretical threshold frequency that sustains initial responding, is determined as the point at which this line crossed the x-axis (red circle, Fig. 2C), representing the frequency above which animals lever press for stimulation. Alpha is the minimal frequency that elicits maximal responding (red triangle, Fig. 2C) and M-50 reflects the frequency that supports half-maximal responding (red square, Fig. 2C). Maximal lever pressing rates for a trial and mean total pass lever pressing were analyzed to measure potential motor effects and overall task engagement. Subject values for all valid passes within a session were averaged.

2.8.2. Abridged RF task

Because fewer frequencies were tested in the abridged task, the drc package v3.0.1 was not appropriate to fit adolescent behavior. Instead, behavior was centered at the median frequency (79 Hz) and the least-squares line that best fit rate of ICSS pressing across all 7 frequencies was estimated. From this function, we determined the slope and the intercept at the median (red dashed line, Fig. 2E) for each subject and session. The intercept provided the rate of pressing at the median frequency (PR_M), and shifts in sensitivity were quantified as changes in PR_M in the Abridged RF task.

2.8.3. Behavior across test phases

In general, linear mixed effects models (LMMs) were used to analyze shifts in reward sensitivity and indices of motor behavior across sexes and across experimental phases, BL, INJ, and POST, for each experiment, with the POST phase divided into POST_EARLY (sessions 8–10) and POST_LATE phases (sessions 11–15). Additional analyses were then conducted to evaluate variation from session-to-session within each phase. Separate models were specified for each phase, with session-level effects modeled independently for experiments in naïve adults and adolescents, or as interactions with group for the pre-exposed adult experiment. LMMs provide a test of the null hypothesis that the factor in question does not affect the measured behavior. It further offers a way to combine data across subjects to adjust for differences between subjects and allows the model to fit the factor of interest. The fitted regression coefficients (β) with 95 % confidence intervals (CI) furnish an estimate of each factor’s effect, and standardized effect size (d) allows comparison across conditions. LMMs were fitted using the glmmTMB package v1.1.10 [43]. Post hoc analyses in each group were conducted to explore interaction effects using the emmeans package v1.10.5 [44] for joint hypothesis testing and pairwise comparisons. Model parameters were extracted with the packages parameters v0.24.0 [45] and performance v0.13.0 [46], and effect sizes were calculated with the package effectsize v0.8.9 [47]. Statistical significance was determined at a threshold of p < 0.05.

To determine the effects morphine administration and withdrawal on reward sensitivity and motor behavior in naïve adults, LMMs of M50, maximum lever press rate, total lever presses, and locomotion were fit with fixed effects of phase (BL, INJ, POST_EARLY, POST_LATE), and sex (male, female), and random intercepts by subject. Model fit was evaluated by comparison to a null model containing only the fixed effect of sex and random intercepts by subject.

To determine the effects morphine administration and withdrawal on reward sensitivity and motor behavior during adolescence, LMMs were fit separately for each group (saline, morphine) to PR_M, maximum lever press rate, total lever presses, and locomotion with fixed effects of phase (BL, INJ, POST_EARLY, POST_LATE), session time (AM, PM), and sex (male, female), an interaction between phase and session time, and random intercepts by subject. Model fit was evaluated by comparison to a null model containing only the fixed effect of sex and random intercepts by subject.

To determine the effects of morphine administration and withdrawal on reward sensitivity and motor behavior during adulthood in animals pre-exposed during adolescence, LMMs were fit with random intercepts by subject and fixed effects including factors for phase (BL, INJ, POST_EARLY, POST_LATE), pre-exposure group (adolescent control (ADOL_CON), adolescent morphine (ADOL_MOR)), and sex (male, female). Model fit was evaluated using likelihood ratio tests by comparing the full model to a null model containing only sex and random intercepts for subject.

To compare effects of different drug treatment during adolescence (control, morphine) or of morphine treatment in adulthood between groups with different histories of exposure in adolescence, LMMs were fit to percent change in PR_M or M50 values with fixed effects of phase (three levels: INJ, POST_EARLY, POST_LATE), group (ADOL_CON, ADOL_MOR), sex (male, female), an interaction between phase and group, and random intercepts by subject. Model fit was evaluated by comparison to a null model containing only the fixed effect of sex and random intercepts by subject.

2.9. VTA DA neuron electrophysiology

2.9.1. VTA DA whole cell patch clamp recordings

Whole cell patch clamp recordings of VTA DA neurons were made in rats treated with morphine or saline from PD45–51 in a separate group of animals (Fig. 1D). Recordings were conducted during the POST_EARLY phase (2–5 days post-injection, between PD52 and 55) in current clamp to assess excitability of DA neurons. Animals were deeply anesthetized with isoflurane and transcardially perfused with ice-cold oxygenated aCSF (in mM: 30 NaCl, 2.5 KCl, 2.0 CaCl2, 1.2 MgSO4, 1.25 NaH2PO4, 25 NaHCO3, and 10 dextrose [305–310 mOsm]). 240 μm reverse horizontal acute brain slices were then made using a Leica VTS 1200 (Leica Biosystems), where they were transferred to warmed (32° C) and oxygenated aCSF for recovery (30 min). The VTA was visualized under 10X, and patch clamp recordings were made in current clamp under 40X using K-Gluconate based internal solution (in mM: 135 K-gluconate, 2.0 MgCl2, 4.0 Na2ATP, 0.4 Na-GTP and 10 Na-phosphocreatine, 10 HEPES (pH adjusted to 7.3 with KOH, 285–290 mOsm)). DA neurons were identified by fluorescence. Access resistance was monitored over the course of recordings and cells that were unstable or above 25 megaohms were excluded from analysis. To assess DA neurons resting membrane potential (RMP), the holding current applied to maintain the patched cell at −70 mV was briefly removed (seconds) to allow the patched cell to reach its RMP and then holding current was reapplied for subsequent recordings to assess rheobase and excitability. To examine the ability of VTA DA neurons to produce action potentials, a series of square 10 pA current steps were applied to patched cells and the number of spikes were counted.

2.9.2. Analysis of whole cell patch clamp recordings

Analysis was performed in Clampfit (Axon pClamp). Data were exported to R v4.4.2 [38] for statistical analysis. To determine the effects of adolescent morphine administration on VTA DA neurons, parametric statistics were used to compare between groups. Independent two-sample t-tests were used to assess differences in RMP, rebound spike count, action potential threshold, input resistance, and rheobase. To evaluate effects on excitability, a 2-way ANOVA was performed to evaluate the effects of current step and group on spike count. Statistical significance was set at p < 0.05.

2.10. Data Availability

All data and complete reporting of all statistical analyses, including overall fit of LMMs, are available at: https://zenodo.org/records/15564482.

3. Results

3.1. Sex differences across experiments

All data fit by LMMs included sex as a fixed effect in the model and evaluated overall model fit by including sex and individual subject intercept in the null model. No interactions of sex and experimental phase/session were observed, so sex was included only as a fixed effect to account for any potential sex differences in the final models reported. The main effects of sex are included in the tables associated with each figure, but because there were not interactions, data combined between males and females are plotted with the size of any main effects noted. Sex was only found to affect sensitivity to ICSS in naïve adult animals, with females showing greater sensitivity (i.e. lower frequency of M50) overall (β = −18.23, CI: −31.79 to −4.68, p = 0.008, d = −0.797) and to a similar degree in each phase (BL: β = −16.64, CI: −31.51 to −1.76, p = 0.028, d = −0.851; INJ: β = −16.63, CI: −30.86 to −2.4, p = 0.022, d = −0.726; POST_EARLY: β = −16.99, CI: −31.29 to −2.69, p = 0.020, d = −0.878; POST_LATE: β = −18.77, CI: −33.93 to −3.6, p = 0.015, d = −0.912). In naïve adult animals, females also overall showed higher levels of beam breaks, a gross measurement for locomotor behavior, than their male conspecifics (β = 20.88, CI: 9.65 to 32.1, p < 0.001, d = 1.071) and to a similar degree in each phase (BL: β = 18.57, CI: 7.52 to 29.62, p = 0.001, d = 1.137; INJ: β = 19.99, CI: 7.6 to 32.37, p = 0.002, d = 1.052; POST_EARLY: β = 17.38, CI: 7.62 to 27.15, p < 0.001, d = 1.107; POST_LATE: β = 22.63, CI: 9.64 to 35.62, p = 0.001, d = 1.142).

For animals tested in adolescence, there were not sex differences in sensitivity to ICSS. There was a main effect on locomotion, in which control females showed greater locomotion than the control males (β = 10.99, CI: 5.03 to 16.96, p < 0.001, d = 0.974). This difference was observed to a similar degree in each phase (BL: β = 7.93, CI: −0.7 to 16.56, p = 0.072, d = 0.768; INJ: β = 10.51, CI: 4.1 to 16.93, p = 0.001, d = 0.967; POST_EARLY: β = 10.5, CI: 3.62 to 17.37, p = 0.003, d = 0.944; POST_LATE: β = 10.56, CI: 5.28 to 15.83, p < 0.001, d = 0.914). There were no sex differences in locomotion in the morphine treated group in adolescence. No differences in locomotion were observed between males and females treated with morphine.

In adults treated with morphine following adolescent saline or morphine treatment, there again were not sex differences in ICSS sensitivity, but there was significantly higher locomotor activity in female animals (β = 8.63, p = 0.044, d = 0.490), which was only present in the INJ phase (β = 11.61, p = 0.039, d = 0.557). No other analyses identified sex differences. It is plausible that a higher number of beam breaks were detected from female animals because of their smaller body size.

3.2. Effects of morphine administration on RF performance in naïve adult animals

To replicate prior work showing increased sensitivity to ICSS due to chronic morphine administration in adulthood, naïve adult rats were shaped to determine the minimum amplitude of current that supported ICSS and then trained to stability on the RF task. Final amplitudes ranged from 80 to 250 μA. Examples of typical behavior for a single subject are shown for each phase of the study in Fig. 3A–C. We compared RF behavior across BL, INJ, and POST conditions, with POST sessions divided into POST_EARLY (8–10) and POST_LATE (11–15) phases. A total of 15 subjects were included in the analysis. Due to missed electrode placement, 2 subjects were excluded.

Fig. 3. Chronic morphine administration and withdrawal modulates RF ICSS M50 and locomotion in naïve adult rats.

Representative example RF ICSS sessions during (A) Baseline (BL), (B) Morphine administration (INJ), and C) Early post-morphine (POST) administration withdrawal. (D) Average M50 values across experiment phase. Asterisks indicate significant post hoc comparisons between phases. (E) Change in M50 from BL for each session. Decreases reflect greater sensitivity to ICSS, while increases reflect reductions. Asterisks indicate sessions with significant difference from BL M50 (dotted line at 0) in post hoc comparisons. (F) Average locomotor behavior, measured as the number of beam breaks during the final block of each session. Asterisks indicate significant post hoc comparisons between phases. (G) Session-to-session changes in locomotor activity were compared to BL activity. Asterisks indicate sessions with significant difference from BL locomotion (dotted line at 0) in post hoc comparisons. Averages are plotted ± SE *p < 0.05; **p < 0.01; ***p < 0.001.

3.2.1. Sensitivity

In naïve adult rats, morphine administration affected sensitivity to ICSS, quantified as M50, the median preferred stimulation frequency (Fig. 2C). Overall, M50 was significantly affected by experiment phase and sex (X²(248) = 74.50, p < 0.001; Fig. 3D). Mean M50 decreased significantly from the BL phase (M = 89.9 Hz) to the INJ phase (M = 73.2 Hz; β = −13.23, CI: −17.71 to −8.73, p < 0.001, d = −0.578), indicating a leftward shift in the rate-frequency function. By contrast, in the POST_EARLY phase, M50 values increased significantly relative to baseline to a mean of 96.2 Hz (β = 6.07, CI: 0.97 to 11.16, p = 0.020, d = 0.265), indicating a rightward shift in the rate-frequency function and a decrease in reward sensitivity. In the POST_LATE phase, M50 values were not significantly different from baseline with a mean of 93.9 Hz (β = 4.03, CI: −0.48 to 8.5, p = 0.080, d = 0.176). Post hoc multiple comparisons between phases revealed that M50 values during INJ were significantly lower than BL (t(248) = 5.77, p < 0.001, d = 1.007), POST_EARLY (t(248) = −7.75, p < 0.001, d = 1.469), and POST_LATE (t(248) = −7.93, p < 0.001, d = 1.314) phases. POST_EARLY M50 values were significantly greater than baseline (t(248) = −2.33, p = 0.020, d = 0.462), but not significantly different than POST_LATE M50 values (t(248) = 0.82, p = 0.414, d = 0.155). In naïve adults, there was an overall effect of sex on M50 values, with females showing higher sensitivity, as detailed in 3.1.

To determine session-to-session changes in sensitivity, separate models were fit for each phase. During BL, M50 did not significantly vary as a function of session and sex (X²(52) = 3.72, p = 0.294). There were no significant effects of session. For the remaining phases, session-to-session changes were tested relative to BL. In the INJ phase, M50 depended on session and sex (X²(126) = 33.82, p < 0.001), with values from sessions 1, 4, 5, 6, and 7 significantly lower than BL (all p < 0.05; Fig. 3E). Likewise, M50 varied according to session and sex in the POST_EARLY phase (X²(97)= 10.26, p = 0.017), with sessions 8 and 10 significantly higher relative to baseline (both p < 0.05). In the POST_LATE phase, M50 did not vary overall as a function of session and sex (X²(126) = −0.57, p = 0.726). The increased sensitivity to ICSS under morphine treatment in naïve adults was also associated with a reduction in the maximum rate of lever pressing per trial from a mean of 46.8 per trial in BL to 29.9 in INJ, which resulted in a reduced total number of lever presses per session as well, from 1018.1 to 819.7 (Fig. S2).

3.2.2. Locomotion

To assay potential generalized drug effects, locomotion in each phase was measured as the number of beam breaks across four sensors in the operant chamber in the final block of each RF pass. During BL, the average final block beam break count was 23.7. General locomotion depended on experimental phase and sex (X²(278) = 32.81, p < 0.001), with females showing overall higher beam break counts (see 3.1). Compared with BL, INJ and POST_EARLY phases both produced significant decreases in locomotion (INJ: M = 17.3, β = −6.41, CI: −10.14 to −2.69, p = 0.001, d = −0.329; POST_EARLY: M = 17.1, β = −6.63, CI: −11.17 to −2.09, p = 0.004, d = −0.340; Fig. 3F). Locomotion returned to baseline levels in the POST_LATE phase (M = 26.4, β = 2.76, CI: −1.23 to 6.74, p = 0.175, d = 0.141). Post hoc comparisons between phases revealed that in addition to being significantly lower than BL, locomotion in the INJ and POST_EARLY phases were significantly lower than in the POST_LATE phase (INJ vs POST_LATE: t(278) = −5.16, p < 0.001, d = −0.781; POST_EARLY vs POST_LATE: t(278) = −4.24, p < 0.001, d = −0.799).

From session-to-session in the BL phase, locomotion did not depend on session and sex (X²(53) = 2.69, p = 0.442), although more beam breaks were detected in females (see 3.1). During the INJ phase, locomotion depended on session and sex (X²(154) = 24.29, p = 0.001) and was significantly reduced relative to BL in sessions 1 through 4 (all p < 0.05; Fig 3G), then returning to BL levels. In the POST_EARLY phase, there also was a significant effect of session and sex (X²(98) = 22.13, p < 0.001), with significantly reduced locomotion again during sessions 8 and 9 relative to BL (both p < 0.05). Locomotion did not depend on session and sex in the POST_LATE phase (X²(126) = 3.94, p = 0.558), though more beam breaks were again detected from females (see 3.1). Because the same pattern of activity from session-to-session was observed in males and females (no interactions between session and sex), all subjects are plotted together in Fig. 3G, acknowledging that the main effect of sex contributes to variability.

3.3. Effects of morphine administration on adolescent aRF performance

3.3.1. Sensitivity

To determine the effects of adolescent morphine administration on reward sensitivity, rats were implanted with stimulating electrodes on PD28 and shaped to determine the minimum amplitude of current that supported ICSS from days PD35–37. Final amplitudes ranging from 130 to 250 μA were used for testing. Examples of typical behavior for a single subject are shown for each phase of the study in Fig. 4A–C. A total of 21 subjects were included in the analysis. Due to missed electrode placement and/or damage to the implant, 6 subjects were excluded.

Fig. 4. Chronic morphine administration and withdrawal during adolescence alters aRF ICSS PR_M and locomotion.

Representative example of aRF ICSS sessions for morphine treatment group during (A) Baseline (BL), (B) Morphine administration (INJ), and C) Post-morphine (POST) administration withdrawal. (D) Average PR_M values in Control animals across experiment phase. Increases reflect greater sensitivity to ICSS, while decreases reflect reductions. Hash marks indicate significant post hoc comparisons between phases. (E) Average PR_M values in Morphine-treated animals across experiment phase. Asterisks indicate significant post hoc comparisons between phases. (F) Change in PR_M from BL for each session. Hash marks and asterisks indicate sessions with significant PR_M difference from BL (dotted line at 0) in post hoc comparisons for Control and Morphine-treated animals, respectively. Control animals are shown in blue and Morphine-treated in red. (G) Average locomotion in Control animals across experiment phase. No significant differences were found in post hoc comparisons. (H) Average locomotion in Morphine-treated animals across experiment phase. Asterisks indicate significant post hoc comparisons between phases. (I) Change in locomotion from BL for each session. Same conventions as F. (J) For each phase, the percent change from BL in AM sessions was calculated for PR_M and compared between Control and Morphine-treated animals. Ampersands indicate significant post hoc comparisons between group for each phase. (K) Percent change from BL for last block locomotion between Control and Morphine-treated animals. Same conventions as J. Averages are plotted ± SE. Control: ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 Morphine: *p < 0.05; **p < 0.01; ***p < 0.001 Control vs. Morphine: ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001.

Sensitivity to ICSS was quantified as the number of presses at 79 Hz, the median frequency of the aRF range (PR_M; Fig. 2E). An increase in PR_M reflects greater sensitivity to ICSS (more presses at the median frequency), corresponding to a leftward shift of the standard RF curve tested in adulthood (reduced M50 frequency). Likewise, decreases in PR_M, corresponding to a rightward shift of the standard RF curve (elevated M50 frequency), indicate reduced sensitivity. In the adolescent control group, PR_M was modeled by experimental phase, session time, and sex (X²(276) = 22.22, p = 0.002). In this model, session time (AM or PM) did not affect PR_M (β = −0.05, CI: −1.19 to 1.08, p = 0.931, d = −0.022), nor did sex (β = −0.07, CI: −1.89 to 1.76, p = 0.944, d = −0.029), or interaction between any phases and session time (all p > 0.05). During the BL phase, mean PR_M at baseline of AM sessions was 4.8. PR_M then significantly decreased relative to BL for all phases (INJ: M = 3.9, β = −1.15, CI: −2.04 to −0.25, p = 0.012, d = −0.504; POST_EARLY: M = 3.5, β = −1.51, CI: −2.53 to −0.49, p = 0.004, d = −0.664; POST_LATE: M = 3.6, β = −1.46, CI: −2.4 to −0.52, p = 0.002, d = −0.642; Fig. 4D). Follow up tests revealed no significant differences between post-BL phases in AM (all p > 0.05) or PM sessions (all p > 0.05, PM sessions shown in Fig. S3). For behavior during the BL phase, follow up tests of day-to-day performance indicated no significant effect of day, session time, and sex (X²(29) = 1.21, p = 0.750). In the INJ phase, the difference in PR_M from BL depended on day, session time, and sex (X²(135) = 30.72, p = 0.010; blue symbols, Fig. 4F), with PR_M in AM sessions significantly lower on PD48–51 than baseline (all p < 0.05). No effects of day, session time, or sex were observed in the POST_EARLY (X²(74) = 13.39, p = 0.063) or POST_LATE (X²(103) = 16.87, p = 0.205) phases.

In the morphine administration group, PR_M was significantly fit by a model that included phase, session time, and sex (X²(325) = 51.51, p < 0.001). Average PR_M during AM BL sessions was 8.8. Unlike control animals, PR_M in INJ phase AM sessions of morphine-treated animals did not differ from BL performance (M = 11.2; β = 1.76, CI: −1.13 to 4.65, p = 0.232, d = 0.256; Fig. 4E). In the INJ phase, the interaction of phase with session time approached, but did not reach, significance (β = −3.91, CI: −7.93 to 0.1, p = 0.056, d = 0.567). PR_M in both POST phases was lower than BL (POST_EARLY:M = 6.0, β = −3.31, CI: −6.62 to −0.01, p = 0.050, d = −0.480; POST_LATE: M = 4.9, β = −4.65, CI: −7.62 to −1.68, p = 0.002, d = −0.675). In addition, sensitivity in POST phases was lower than that of INJ (INJ vs POST_EARLY: t(325) = 3.82, p < 0.001, d = 0.872; INJ vs POST_LATE: t(325) = 5.90, p < 0.001, d = 1.102). No significant difference was observed between the early and late POST phases (t(325) = 0.98, p = 0.327, d = 0.230).

When examining day-to-day performance, PR_M did not depend on day, session time, or sex in the BL phase (X²(36) = 3.05, p = 0.384). However, the difference in PR_M from BL to INJ was significantly fit by a model that included day, session time, and sex (X²(156) = 32.95, p = 0.005; red symbols, Fig. 4F). Relative to BL, PR_M significantly increased on PD51 in the AM session (β = 7.64, CI: 2.65 to 12.64, p = 0.003, d = 0.932), and significantly decreased on PD51 in the PM session (β = −9.76, CI: −16.63 to −2.88, p = 0.005, d = −1.190). During POST_EARLY, PR_M did not depend on day, session time, or sex (X²(89)= 11.75, p = 0.109), but in the POST_LATE phase, performance depended on both day and time (X²(130) = 40.18, p < 0.001). PR_M in AM sessions was significantly reduced relative to BL on all days in the POST phase (all p < 0.01). By the end of the POST phase, ICSS sensitivity remained reduced relative to BL. Changes in maximum lever pressing rate and total lever pressing consistent with changes in PR_M were observed (Fig. S4).

To compare the effect of morphine and control treatments across groups, percent change from BL values were calculated for each subject by phase. Overall, percent change PR_M was significantly fit by a model that included group, phase, and sex (X²(529) = 29.76, p = 0.002). The difference in percent change of PR_M between groups (Morphine M = 41.2 % vs. Control M = −15.1 %) in the INJ phase was significant (t (529) = −2.22, p = 0.027, d = 0.655; Fig. 4J). There were no differences between the groups in the POST_EARLY (t(529) = −0.50, p = 0.619, d = 0.178) or POST_LATE (t(529) = 0.28, p = 0.782, d = 0.087) phases.

3.3.2. Locomotion

In the control group, the only effect on locomotion during the last block of each aRF pass was that of sex. The mean number of beam breaks during the BL phase was 22.8. The overall model significantly fit locomotion with factors for phase, session time, and sex (X²(347) = 15.07, p = 0.035), but the only main effect was sex (see 3.1). No main effects of phase, session time, or interactions between phase and session Time (all p > 0.05; Fig. 4G). From session-to-session, the only significant effect was a reduction of beam breaks from the AM to PM session on PD59 (p < 0.05; blue symbols, Fig. 4I). As in Fig. 3, males and females are plotted together in Fig. 4.

In the morphine administration group, locomotion was significantly fit by a model that included phase, session time, and sex (X²(385) = 152.71, p < 0.001). In BL, locomotion count was 24.8. During INJ, locomotion increased significantly relative to BL in the AM sessions following morphine administration (M = 37.4, β = 12.55, CI: 7.75 to 17.35, p < 0.001, d = 0.771; Fig. 4H). Additionally, there was a significant interaction between phase and session time during INJ, reflecting a decrease in locomotion relative to BL in PM session (M = 19.6, β = −12.02, CI: −18.81 to −5.23, p = 0.001, d = 0.739; Fig. S3). Follow up tests across phases indicated a significant decrease in locomotion in the AM sessions between INJ and both POST_EARLY (M = 22.0, t(385) = 7.27, p < 0.001, d = 1.513) and POST_LATE (M = 23.7, t(385) = 8.03, p < 0.001, d = 1.347) phases.

From day-to-day, locomotion depended on day, session time, and sex in the adolescent morphine group during BL (X²(37) = 11.38, p = 0.023), with less activity in the PM sessions (β = −6.95, CI: −11.61 to −2.29, p = 0.003, d = −0.571). In the INJ phase, day, session time, and sex significantly affected locomotion (X² = 117.51, p < 0.001; red symbols, Fig. 4I). On PD46–51, last block locomotion was significantly higher than baseline in the AM sessions (all p <0.05). In the PM sessions, last block locomotion was significantly lower than baseline on days PD46 and PD48–51 (all p < 0.05). In both POST phases, locomotor activity was significantly fit by a model that included day, session time, and sex (POST_EARLY: X²(99) = 21.78, p = 0.003; POST_LATE: X²(159) = 28.32, p = 0.008), in which only a main effect of session time was observed (POST_EARLY: β = −5.69, CI: −9.92 to −1.47, p = 0.008, d = −0.473; POST_LATE: β = −5.69, CI: −10.53 to −0.85, p = 0.021, d = −0.443).

The percent change in locomotion from BL was calculated to compare between groups as in 3.3.1. Overall, the inclusion of group, phase, and sex significantly improved model fit for percent change locomotion values (X²(657) = 176.51, p < 0.001). Adolescents treated with morphine showed higher locomotion counts than controls in the INJ and POST_LATE phases (INJ: Morphine M = 76.5 % vs. Control M = −8.3 %; t(657) = −6.63, p < 0.001, d = 1.683; POST_LATE: Morphine M = 14.4 % vs. Control M = −15.3 %; t(657) = −2.10, p = 0.036, d = 0.552; Fig. 4K).

3.4. Effects of re-exposure to morphine on RF performance in adulthood

All animals tested in adolescence were treated with morphine in adulthood to test the effect of re-exposure in adulthood on reward sensitivity. Following the completion of POST phase testing in adolescence, rats began training on the standard RF task (Fig. 1C). From PD90–96, all rats received morphine injection (10mg/kg, i.p.). We compared RF behavior across phases in adults who had been in the control or morphine adolescent treatment groups (Fig. 5A–C for morphine re-exposure example). A total of 21 subjects were included in the analysis.

Fig. 5. Adolescent morphine experience amplifies morphine-driven effects on RF ICSS M50 and locomotion during adulthood.

Representative example of RF ICSS sessions for morphine re-exposure group during (A) Baseline (BL), (B) Morphine administration (INJ), and (C) Post-morphine (POST) administration withdrawal. (D) Average M50 values across experiment phase in animals that had been in the Control group as adolescents (ADOL_CON). Decreases reflect greater sensitivity to ICSS, while increases reflect reductions. Hash marks indicate significant post hoc comparisons for a given session. (E) Average M50 values across experiment phase in animals that had been in the Morphine group as adolescents (ADOL_MOR). Asterisks indicate significant post hoc comparisons across phases. (F) Change in M50 from BL for each session. Hash marks and asterisks indicate sessions with significant M50 difference from BL (dotted line at 0) in post hoc comparisons for ADOL_CON and ADOL_MOR animals, respectively. Ampersands indicate significant post hoc comparisons between group for a given session. ADOL_CON animals are shown in blue and ADOL_MOR in red. (G) Average locomotion in ADOL_CON animals across experiment phase. No significant differences were found in post hoc comparisons. (H) Average locomotion in ADOL_MOR animals across experiment phase. Asterisks indicate significant post hoc comparisons between phases. (I) Change in locomotion from BL for each session. Same conventions as F. (J) For each phase, the percent change from BL was calculated for M50 and compared between ADOL_CON and ADOL_MOR animals. Ampersands indicate significant post hoc comparisons between group for each phase. (K) Percent change from BL for last block locomotion between ADOL_CON and ADOL_MOR animals. Same conventions as J. Averages are plotted ± SE. ADOL_CON: ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 ADOL_MOR: *p < 0.05; **p < 0.01; ***p < 0.001 ADOL_CON vs. ADOL_MOR: ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001.

3.4.1. Sensitivity

To determine the effects of prior morphine treatment on adult exposure, RF behavior was compared across the animals tested previously as adolescent control (ADOL_CON) and morphine (ADOL_MOR) groups. For adult testing, sensitivity was again measured as M50, the frequency that supported half-maximal responding to the full range of RF frequencies, with decreases of M50 indicating increased sensitivity. We first analyzed sensitivity to RF ICSS across experimental phase. Because all rats received treatment with morphine, behavior was analyzed in a single model with factors for group, phase, sex, and an interaction between phase and group. Overall, sensitivity to ICSS across phases, measured as M50, was significantly fit by this model (X²(293) = 87.60, p < 0.001). Animals showed increased sensitivity to ICSS in the INJ (β = −5.30, CI: −9.42 to −1.18, p = 0.012, d = −0.532) and POST_EARLY (β = −5.88, CI: −9.87 to −1.89, p = 0.004, d = −0.590) phases. Significant interactions between phase and group revealed a greater increase in sensitivity in the ADOL_MOR group compared with ADOL_CON during INJ (β = −7.40, CI: −12.9 to −1.9, p = 0.008, d = −0.743), but a greater reduction in ADOL_MOR than ADOL_CON animals during POST_EARLY (β = 7.08, CI: 1.3 to 12.85, p = 0.016, d = 0.711). Follow up tests compared across phases within each group. In ADOL_CON animals average M50 was 106.9 Hz, which significantly decreased to 101.5 Hz in the INJ phase (t(293) = 2.52, p = 0.012, d = 0.643; Fig. 5D). M50 values remained lower through POST_EARLY (M = 101.1 Hz, t(293) = 2.89, p = 0.004, d = 0.713), but returned to BL in the POST_LATE (M = 106.1, t(293) = 0.63, p = 0.526, d = 0.139). In the ADOL_MOR group, average M50 at BL was 105.0 Hz (Fig. 5E). This group also showed a significant decrease in M50 from BL during INJ (M = 92.2 Hz, t(293) = 6.84, p < 0.001, d = 1.476). Relative to INJ, M50 values increased significantly in both the POST_EARLY (M = 106.0 Hz, t(293) = −6.47, p < 0.001, d = −0.923) and POST_LATE (M = 107.7 Hz, t(293) = −8.90, p < 0.001, d = −1.318) phases, but neither POST phase was significantly different than BL (POST_EARLY: t(293) = −0.56, p = 0.573, d = 0.554; POST_LATE: t(293) = −1.58, p = 0.114, d = 0.159).

Sensitivity to ICSS also varied from session-to-session. In each phase, performance was analyzed using a full model that accounted for effects of group, session, sex, and an interaction between group and session. During BL, no significant differences were observed (X²(68) = 8.61, p = 0.282). During the INJ phase sessions, M50 was significantly fit by the full model (X²(129) = 46.52, p < 0.001). Within each group, post hoc tests compared each INJ session to BL. In the ADOL_CON group, only session 2 was significantly lower than baseline (t(129) = 2.09, p = 0.038, d = 1.288; blue symbols, Fig. 5F). In the ADOL_MOR group, all seven morphine injections resulted in significantly lower M50 values relative to baseline (all p < 0.05, d range: 1.05 to 1.923; red symbols, Fig. 5F). Between groups, M50 values were significantly lower in ADOL_MOR compared to ADOL_CON in sessions 5 (t(129) = 2.71, p = 0.008, d = 1.751) and 7 (t(129) = 2.14, p = 0.034, d = 1.383). Across all POST sessions, sensitivity to ICSS was significantly fit by the full model (X²(212) = 32.43, p = 0.028). Within each group, post hoc tests compared each POST session to BL. In ADOL_CON animals, M50 remained significantly lower than BL on sessions 8 (t(212) = 2.87, p = 0.004, d = 1.022) and 9 (t(212) = 3.94, p < 0.001, d = 1.402). No individual sessions were significantly different than BL in the ADOL_MOR group (all p > 0.05). Between group tests revealed significantly higher M50 values in the ADOL_MOR group than ADOL_CON in the first two sessions after drug discontinuation (session 8: (t(212) = −2.53, p = 0.012, d = 1.335); session 9 (t(212) = −2.72, p = 0.007, d = 1.321). Changes in maximum lever pressing rate and total lever pressing consistent with changes in M50 were observed (Fig. S5).

To compare the effect of adult morphine treatment across groups with a history of control or morphine pre-exposure, percent change in M50 from BL values were calculated for each subject by phase. Overall, percent change of M50 values from BL were significantly fit by a model that included group, phase, sex, and an interaction between phase and group (X²(216) = 69.76, p < 0.001). While morphine administration reduced M50 values in both groups (ADOL_CON: M = −6.3 %; ADOL_MOR: M = −12.6 %), the decrease was significantly greater in the ADOL_MOR group (t(216) = 2.15, p = 0.033, d = 0.916; Fig. 5J). Further, during the POST_EARLY, the ADOL_MOR group demonstrated significantly higher M50 values relative to controls (ADOL_CON: M = −5.3 %; ADOL_MOR: M = 1.0 %; t(216) = −2.03, p = 0.044, d = −0.877). There were no differences between the groups in the POST_LATE phase (t(216) = −1.39, p = 0.165, d = −0.540). Together, adolescent pre-exposure to morphine resulted in greater sensitivity to ICSS reward, as well as stronger depressive effects of withdrawal during adulthood than those without a history of exposure.

3.4.2. Locomotion

Finally, we examined the effects of prior morphine treatment on locomotor activity elicited by adult exposure and withdrawal. Locomotor behavior was analyzed in the same way as sensitivity, using a single model with factors for group, phase, sex, and an interaction between phase and group. This full model significantly fit locomotion across experimental phases (X²(399) = 28.15, p < 0.001). Overall, there was a main effect of sex (see 3.1) and a significant interaction between phase and group during INJ (β = 10.15, CI: 2.7 to 17.6, p = 0.008, d = 0.576). Follow up post hoc tests showed no significant differences in beam breaks between any phases in the ADOL_CON group (all p > 0.05; Fig. 5G). ADOL_MOR animals showed more locomotion in the INJ phase (M = 26.1) relative to BL (M = 18.6, t(399) = −2.88, p = 0.004, d = −0.545), POST_EARLY (M = 13.4, t(399) = 4.39, p < 0.001, d = 0.914), and POST_LATE (M = 18.4, t(399) = 3.14, p = 0.002, d = 0.534; Fig. 5H) phases. Neither POST phase was significantly different than BL (both p > 0.05).

Session-to-session changes in each phase were tested with a model that included group, session, sex, and an interaction between session and group. During the BL phase, session-to-session locomotion was not significantly fit by this model (X²(73) = 5.07, p = 0.651). During the INJ phase, locomotion across sessions was improved by this full model (X²(212) = 25.40, p = 0.045). Post hoc tests showed that adults in the ADOL_CON group had reduced levels of locomotion only during session 1 (t(212) = 2.44, p = 0.016, d = 0.862; blue symbols, Fig. 5I). Locomotion of adults in the ADOL_MOR group during INJ was not significantly different from BL in sessions 1 through 4, but significantly higher in sessions 5 through 7 (all p < 0.05, red symbols, Fig. 5I). In the POST phases, locomotion was significantly fit by the full model (X²(240) = 37.12, p = 0.008). In the ADOL_CON group, locomotion was significantly lower than BL in sessions 11 (t(240) = 2.21, p = 0.028, d = 0.780) and 13 (t(240) = 2.55, p = 0.011, d = 0.945), while in the ADOL_MOR group, locomotion was significantly reduced in sessions 8, 9, and 11 (all p < 0.05).

To compare groups, the percent change in locomotion from BL was calculated (as in 3.3.1). Overall, the inclusion of group, phase, sex, and interaction between session and sex significantly improved model fit for percent change locomotion values (X²(318) = 35.40, p < 0.001). In the INJ phase, the increase in ADOL_MOR animals (M = 58.5 %) was significantly greater (t(318) = −4.68, p <0.001, d = −0.777; Fig. 5K) than the change in ADOL_CON (M = −13.8 %).

3.5. Effects of morphine administration on VTA DA cell physiology in adolescence

Since adolescents treated with morphine showed reductions in ICSS sensitivity following drug discontinuation and were more sensitive to morphine when re-exposed as adults, we examined whether this treatment altered neurophysiological properties of VTA_DA cells. VTA_DA neurons recorded from morphine-treated rats displayed significant hyperpolarization of RMP compared to controls (−53.6 vs −43.5 mV, t(13.78) = 3.18, p = 0.007, d = 1.508; Fig. 6A). Additionally, we observed that while VTA_DA neurons in controls exhibited spike(s) during the release from hold period (M = 3.6), spikes during release from hold were uncommon in the morphine treated group and occurred significantly less relative to the control group (M = 0.2, t(8.27) = 2.92, p = 0.019, d = 1.377; Fig. 6B Example trace recordings shown in Fig. 6C). To examine action potential characteristics, we maintained a −70 mV potential in current clamp and applied ramp current steps to measure rheobase and threshold of the first action potential that emerged. We observed significantly depolarized thresholds in the VTA_DA neurons of the morphine-treated group relative to control (−37.3 vs −43.7 mV, t(13.19) = −3.14, p = 0.008, d = −1.480; Fig. 6D), but no significant difference in input resistance (395.5 vs. 353.2 mOhms, t(11.24) = −0.47, p = 0.649, d = −0.234; Fig. 6E) or Rheobase (82.6 vs. 75.6 pA, t(14.99) = −0.35, p = 0.729, d = −0.170; Fig. 6F. Example traces shown in Fig. 6G) between groups.

Fig. 6. Adolescent morphine withdrawal decreases excitability of VTA DA neurons.

Whole cell patch clamp recordings of VTA DA neurons were conducted during the POST_EARLY phase (2–5 days post-injection, PD52–55). (A) Resting membrane potential (RMP) was significantly reduced in adolescent morphine-treated rats relative to controls. (B) During the release from hold period, spikes occurred significantly less frequently in the morphine-treated compared to the control group. (C) Example trace recordings by group. (D) Thresholds (mV) were significantly depolarized in the VTA DA neurons of the morphine-treated group relative to control. (E) No significant difference in input resistance (mOhms) was observed between groups. (F) No significant difference in rheobase (pA) was observed between groups. (G) Example rheobase traces between groups. (H) In response to a series of square 10 pA current steps, the number of spikes per current step was significantly reduced in morphine-treated rats relative to controls. Averages are plotted ± SE. ^&p < 0.05; ^&&p < 0.01.

To examine the ability of VTA_DA neurons to produce action potentials, a series of square 10 pA current steps were applied to patched cells (Fig. 6H, traces in Fig. 6I). We observed a small but significant reduction in action potential generation per current step in post-injection morphine-treated animals relative to controls (F(1, 248) = 4.04, p = 0.046, f = 0.13). Taken together these experiments suggest that following adolescent morphine administration, VTA_DA neuron excitability is reduced relative to saline-treated control animals.

4. Discussion

Adolescence is a unique developmental stage with heightened reward system activity. Opioid use during adolescence is linked to increased long term risk of adulthood OUD [29]. Since adolescence is a critical period for neural development [12,34], it is likely that adolescent opioid use drives maladaptive neurobiological changes that persist into adulthood. Here, we report the effects of morphine administration on reward systems during adolescence, as well as during and after morphine re-exposure in adulthood. Rate-frequency ICSS tasks were used to assay reward sensitivity prior to, during, and after morphine administration in naïve adult, adolescent, and morphine-experienced adult rats. To address possible mechanisms of shifts in reward-directed behavior, we also investigated the impact of adolescent morphine withdrawal on VTA_DA neuronal excitability.

We first replicated prior literature by examining the effects of morphine treatment on ICSS rate-frequency sensitivity in drug-naïve adults. During the injection phase, morphine administration facilitated reward sensitivity through the entire treatment period. When morphine was discontinued, reward sensitivity depressed in the early period, returning to baseline following ~3 days. Locomotion was significantly lower than baseline levels during both the injection and early post phases. More specifically, follow up tests showed that locomotion decreased in the first 4 days of the injection phase, then returned to baseline, suggesting behavioral tolerance even while sensitivity to ICSS remained elevated. In the early post-injection phase, reduced locomotion coincided with reduced reward sensitivity, with both returning to baseline during the late post-injection phase. Together, these results replicate prior literature [48–51] and indicate that chronic administration of a moderate dose of morphine in drug naïve adults modulates reward sensitivity and locomotion, but relatively transiently.

While rate-frequency ICSS tasks have been widely used in adult animals to assay shifts in reward sensitivity in response to drugs of abuse, our study is the first to our knowledge to adapt the standard RF ICSS task for adolescent rats. The aRF task enabled us to test the effect of chronic morphine exposure during mid to late adolescence [28,52], a peak period for substance use initiation, including opioid misuse [29,30]. In the saline-treated control group, task engagement declined mildly over time, with a slight reduction in sensitivity for all phases relative to baseline, but no differences between those phases and no changes in locomotor activity. Under morphine administration, the reduction in sensitivity to ICSS relative to baseline observed in saline controls did not occur. Sensitivity was significantly higher in morphine-treated animals than controls in the morning sessions directly following drug administration, returning to, or falling below, baseline in the afternoon sessions. Likewise, locomotion increased significantly after morphine administration in the morning sessions, then dropped below baseline levels in the afternoon sessions. During treatment, adolescents did not show evidence of tolerance as observed in drug-naïve adults.

After cessation of morphine administration, reward sensitivity significantly decreased relative to both baseline- and injection-phase levels and remained reduced throughout the entire post-administration period. Locomotion during withdrawal was lower than that observed during the injection phase but did not differ from baseline levels of activity. During withdrawal, there was a dissociation between reward sensitivity and locomotion, with behavioral effects apparent in ICSS sensitivity, but not locomotion. Further, the reduction in ICSS sensitivity during withdrawal from morphine in adolescence is in contrast to the adult naïve group, who recovered in a short time frame, suggesting potential longer-term changes in reward circuitry resulting from a single chronic exposure to morphine in adolescence. This result is seemingly contradictory to prior studies suggesting that adolescents are less sensitive to the aversive effects of withdrawal from nicotine [53–55] and ethanol [56,57], or of kappa opioid agonist treatment [58]. However, changes in ICSS are common across withdrawal from drug treatment and may serve as a more sensitive assay of withdrawal [59]. Here, both naïve adults and adolescents showed ongoing shifts in sensitivity to ICSS even when locomotor behavior was no longer affected.

To determine whether adolescent morphine exposure resulted in long-term effects on reward sensitivity, adolescent control and morphine-treated rats were re-tested in adulthood, with both groups receiving morphine. While both groups displayed greater reward sensitivity relative to baseline during morphine administration, the increase was significantly more pronounced in rats that had been pre-exposed during adolescence. Different effects were observed with locomotion as well. In adults receiving morphine for the first time, treatment initially suppressed locomotion, but the group showed potential tolerance in a return to baseline levels. In animals undergoing re-exposure, locomotion did not show the initial decline, but increased across daily sessions. Together, these results suggest that adolescent morphine exposure increases the facilitatory effects of morphine administration in adulthood. Such sensitization is elicited by several drugs of abuse, including cocaine, morphine, amphetamine, and nicotine [60,61].

In contrast, cessation of morphine administration in adult animals resulted in stronger reductions in reward sensitivity in animals re-exposed to morphine compared with those receiving morphine for the first time in adulthood. While the relative shift in reward sensitivity during withdrawal was smaller than the increase during administration, the disparity between the adolescent morphine and control groups may indicate that prior adolescent history of morphine exposure exacerbated the negative effects of morphine withdrawal during adulthood. Lasting effects of withdrawal from adolescent treatment have been previously demonstrated across multiple substances of abuse, including nicotine, cocaine, and morphine [62–65]. Additional studies will be needed to compare whether the magnitude of pre-exposure effects depend on the age of initial administration, as sensitization is well-established in adulthood.

Mesolimbic circuitry has long been identified as critical for reward learning, motivated behavior, and drug reinforcement [66–68], and all drugs commonly abused by humans have been shown to increase synaptic DA concentrations in mesolimbic dopamine systems [15]. To further probe the role of the mesolimbic pathway in the observed shifts in reward sensitivity during adolescent morphine withdrawal, we sought to determine if chronic adolescent morphine administration drives neurobiological changes in DA neurons in the VTA. As VTA_DA neurons are more excitable during adolescence relative to adulthood [18], we hypothesized that the reduction in reward sensitivity observed following the cessation of adolescent morphine administration may be driven by a decrease in the excitability of DA neurons. DA neurons recorded from morphine-withdrawing adolescent rats exhibited reductions in excitability measures (i.e., RMP, threshold) and reduced ability to generate action potentials given depolarizing current steps, demonstrating a physiological correlation of DA neuron behavior with reduced reward seeking. These data suggest that during adolescent morphine withdrawal, DA neurons exhibit reduced excitability which may mediate the reduced reward sensitivity observed in the aRF task.

The acute engagement of MORs on presynaptic GABAergic neurons likely mediates immediate effects on VTA_DA excitability and therefore reward-related behaviors through disinhibition [69–71]; however, the mechanism(s) by which DA release is reduced during protracted withdrawal are less clear. Our results indicate that morphine withdrawal hyperpolarizes RMPs, reduces AP thresholds, and reduces spike generation on current steps of VTA_DA neurons, which likely results in reduced DA release and reward sensitivity. During withdrawal, presynaptic GABAergic neurons exhibit cAMP-mediated changes resulting in increased GABA release which may maintain DA neurons in a state of lower excitability [72]. Future work should examine the role of VTA GABA receptors during morphine withdrawal and determine if sustained changes in GABA input to VTA_DA neurons explain our observed phenotype.

Drugs with abuse liability in humans have been shown to facilitate ICSS reward sensitivity in adult rats [33]. Across both sexes, similar chronic administration protocols (7-day) of low (3.2mg/kg) and high doses (18mg/kg) of morphine during adulthood have consistently resulted significant facilitatory effects on sensitivity to ICSS [48,73], with withdrawal producing the opposite effect [48–51]. In vivo recordings of DA release in adult rats have supported a direct role of mesolimbic dopamine circuits in these behavioral shifts. Measurements with both microdialysis and fast scan cyclic voltammetry have shown that initial opioid administration results in a significant and prolonged increase in DA release in the NAc [74]. Repeated opioid use decreases the responsivity of DA not only to opioid administration but also to natural reinforcers, resulting in a motivational deficit that begins the shift from positive to negative reinforcement of drug use [3]. Opioid withdrawal results in a net decrease in DA release [7,75]. The severity of withdrawal symptoms is correlated with the degree to which DA release is decreased, and decreases in DA release persist beyond the somatic symptoms of withdrawal [76,77]. Together, such adaptations in mesolimbic pathways may play a critical role in both initial and long-term OUD.

While limited research has been conducted to determine the effects of adolescent opioid exposure on the mesolimbic pathway, our findings are consistent with those that show of adolescents displaying heighted drug associative learning in conditioned place preference paradigms [78,79], increased sensitivity to the acutely rewarding effects of morphine in self-administration tasks [80], and increased DA release in the NAc to morphine administration [81] relative to adults. In addition, repeated chronic use can result in decreased DA release to both opioid administration and natural reinforcers [7,82]. Here, we find that the positive effect of opioid use on ICSS sensitivity is comparable in adolescents and adults treated with morphine for the first time, but the negative effect during withdrawal is greater and more prolonged in adolescents. This difference may contribute to the enhanced responsiveness to ICSS in adults re-exposed to morphine. The current study did not address whether the effects of re-exposure to morphine depend on the age of pre-exposure, only that adolescent exposure itself produces lasting effects. It is well established that drugs induce long-term sensitization that contributes to reinstatement of drug-taking [5]. Future work will examine the effect of pre-exposure timing by comparing groups first treated with morphine either during adolescence or in early adulthood, after the closing of critical-period development.

The allostatic model of addiction proposes that long term OUD is facilitated by neurobiological shifts in both the initial positive reward of opioid use and the negative reinforcement aspects of withdrawal [1–5]. During adolescence, reward systems are particularly sensitive and vulnerable to alterations in developmental trajectories in response to experiences. Our results demonstrate that the use of opioids during this critical period drives changes in reward sensitivity and reward system excitability that last into adulthood. Chronically enhancing the sensitivity of reward systems during adolescence may contribute to the long-term vulnerability to OUD observed in epidemiological studies, both by increasing the rewarding effects of opioid use during adulthood and exacerbating the negatively reinforcing aspects of opioid withdrawal. Future research should explore additional mechanisms of observed shifts in reward sensitivity, as well as how alterations in reward pathways manifest in maladaptive decision-making.

Supplementary Material

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.physbeh.2025.114987.

Data availability

https://zenodo.org/records/15564482 (All data and complete reporting of all statistical analyses, including overall fit of LMMs, are available at:)