Prefrontal circHomer1 regulates synaptic and behavioral adaptations induced by cocaine

Abstract

Substance use disorder (SUD) represents a substantial challenge in neuropsychiatric medicine, with the molecular mechanisms underlying its etiology remaining elusive. The molecular underpinnings of SUD suggest a pivotal role for circular RNAs (circRNAs) in its pathophysiology. Herein, we present a study on circHomer1, a circRNA enriched in neurons, which is abnormal expression upon cocaine exposure. Employing models of repeated cocaine exposure and conditioning place preference (CPP), alongside virus-mediated gene regulation techniques, we revealed the contribution of circHomer1 in cocaine-induced rewarding effects and synaptic adaptations. We found a notably downregulation of circHomer1 expression in the prelimbic cortex consequent to repeated cocaine exposure in both rat model and patients with cocaine use disorder. Elevation of circHomer1 levels resulted in a pronounced attenuation of cocaine-induced CPP, whereas suppression of circHomer1 expression enhanced the rewarding effects. These outcomes were specifically observed in excitatory neurons, implicating a cell type-specific function of circHomer1. Furthermore, the restoration of circHomer1 rescued the reduction of mushroom-type dendritic spines and rectified deficits in the frequency of spontaneous excitatory postsynaptic currents associated with prolonged cocaine exposure. Mechanistically, circHomer1 appears to modulate cocaine-induced behavioral responses through dopamine D1 receptor signaling pathways. Notably, circHomer1’s behavioral effects showed specificity for psychostimulants (cocaine and methamphetamine), without affecting food- or opioid-associated rewards. Our findings highlight the significant role of circHomer1 in regulating cocaine reward and identify a novel molecular regulator of the actions of cocaine on the brain’s reward circuitry, providing a new strategy for treating drug addiction.

Introduction

Substance use disorder (SUD) is a chronic, relapsing disease in which patients often have uncontrolled and reckless drug abuse [1]. With the increasing number of drug abusers, drug abuse not only affects the health of individuals but also endangers society and imposes a huge economic burden on the state [2]. Evidence has revealed that repeated addictive drug exposure leads to enduring cellular, circuits and neuroplasticity alterations in mesocorticolimbic reward system, which includes prefrontal cortex (PFC), nucleus accumbens (NAc) and ventral tegmental area (VTA) [3–8]. Such long-lasting structural and functional modifications are thought to increase sensitivity to the motivational effects of addictive drugs, culminating in a loss of control over intake [9, 10].

The medial prefrontal cortex (mPFC), a critical hub for integrating different brain networks involved in reward processing, salience attribution, and inhibitory control, is proposed to precipitate bingeing and relapse, perpetuating the addiction cycle [11–13]. Accumulating evidence demonstrates that complex drug-induced neuroadaptations in mPFC are mediated largely through dynamic regulation of gene expression [14–16]. Epigenetic mechanisms, such as DNA methylation, histone modifications, and certain types of noncoding RNAs like circRNAs, are known to modulate expression of gene networks in mPFC and other drug reward-associated brain regions, contributing to the drug-induced structural, synaptic, and behavioral plasticity [17–20]. However, the precise molecular mechanisms driving these changes in the mPFC remain unclear.

Circular RNAs (circRNAs), vastly conserved non-coding RNAs and naturally occurring in a covalently closed loop structure, are produced by back-splicing [21, 22]. Numerous neuronal circRNAs are derived from synaptic gene locus, with expression levels changing in parallel with the synapse formation and neuroplasticity [23]. Evidence shows that circRNAs, like circHomer1, may play a key role in various neuropsychiatric disorders [24–28]. CircHomer1, derived from Homer protein homolog 1 (Homer1), is a neuronal-enriched circRNA abundantly expressed in the frontal cortex [29]. CircHomer1 knockdown in the mouse orbitofrontal cortex leads to specific deficits in cognitive flexibility [29]. And its inhibition appears to ameliorate methamphetamine-induced neuronal injury through inhibiting Bbc3 expression in HT-22 cells [30]. Moreover, circHomer1 regulates the expression of synaptic-related proteins and is involved in synaptic plasticity, learning, and memory, which are abnormal symptoms of patients with SUD [28–31]. Nonetheless, the specific influence of circHomer1 on drug-induced rewarding effects is largely unknown.

In this study, we utilized the conditioned place preference (CPP) model, self-administration model, viral-mediated circHomer1 expression, fluorescence in situ hybridization, and patch clamp to investigate the cell type-specific influence of circHomer1 on drug-induced behavioral and synaptic plasticity. We demonstrated that repeated cocaine exposure notably downregulated the expression of circHomer1 in the prelimbic cortex (PrL). However, recovering the expression of circHomer1 in the PrL reduced cocaine preference and intake, and repaired the hypoactivity of PrL neuron induced by cocaine. This indicated that circHomer1 may be a key modulator for the cocaine responses.

Material and methods

Animals

Male Sprague-Dawley rats (260 – 280 g) used in this study were obtained from Charles River Co., LTD. (Beijing, China). The rats were housed four to a cage in the animal care facility at a temperature (22 °C ~ 24 °C)- and humidity (40% ~ 60%)-controlled facility with a reverse 12 h-light/12 h-dark cycle (8:00–20:00) with food and water available ad libitum. The behavioral experiments were conducted during the dark phase of the cycle. All of the procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experiments were approved by the Biomedical Ethics Committee for Animal Use and Protection of Peking University.

Our estimates of the number of animals that were required for behavioral testing (n  ≥  7 per group), Ex vivo electrophysiology (n  ≥  3 per group), immunofluorescence (n  ≥  3 per group), circHomer1 expression (n  ≥  5 per group), and spine analysis (n  ≥  3 per group) were based on our past experience in the laboratory and previous research. All rats were randomly assigned to the experimental and control groups. Experiments were not performed blindly in the research, but data collection and analyses were carried out blinded by using a computer-based recording system that was not influenced by human bias. Rats were excluded from the analysis based on the accuracy of injection site of viruses or guide cannulas location.

Drug treatment

Cocaine, methamphetamine, and morphine were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and were dissolved in 0.9% physiological saline. SCH23390 (SCH) (D054, Sigma, USA) and raclopride (RAC) (R121, Sigma, USA) were also dissolved in 0.9% physiological saline. Clozapine N-oxide (CNO) (C0832, Merck, USA) was dissolved in dimethylsulfoxide (DMSO) and then diluted with 0.9% physiological saline.

For chronic or acute cocaine exposure, cocaine (10 mg/kg) was administered via intraperitoneal (i.p.) injection respectively for repeated 14 days or one day. For the CPP test, cocaine (10 mg/kg or 5 mg/kg) were intraperitoneally injected, while methamphetamine (1 mg/kg) and morphine (10 mg/kg) were subcutaneously injected. SCH (2.0 μg/0.5 μl/side), RAC (5.0 μg/0.5 μl/side) and CNO (1.5 mg/kg, i.p.) were injected 30 min before cocaine injection.

Recombinant adeno-associated virus (rAAV)

To manipulate circHomer1 expression, recombinant adeno-associated virus serotype 9 (rAAV9) vectors expressing enhanced green fluorescent protein (eGFP) were employed. For ubiquitous overexpression, we used rAAV9 with a cytomegalovirus (CMV) promoter driving circHomer1 (rAAV-CMV-circHomer1-GFP, referred to as CMV-OE-circHomer1). For targeted knockdown, a vector expressing short hairpin RNA (shRNA) against circHomer1 under the U6 promoter was used (rAAV-U6-sh-circHomer1-GFP, referred to as sh-circHomer1). To achieve excitatory neuron-specific overexpression, circHomer1 was driven by the calcium/calmodulin-dependent protein kinase II alpha (CamKIIα) promoter (rAAV-CamKII-circHomer1-GFP, referred to as CamKII-OE-circHomer1). Corresponding control vectors included rAAV-CMV-GFP (referred to as CMV-GFP), rAAV-CamKII-GFP (referred to as CamKII-GFP), and rAAV-U6-sh-control-GFP (referred to as sh-control). All aforementioned circHomer1 manipulation and control vectors were obtained from Vigene Bioscience (Shandong, China).

For chemogenetic experiments, the rAAV9 virus that specifically activates D1 neurons [rAAV-D1-hM3D(Gq)-mCherry-WPREs] was purchased from BrainVTA, Wuhan, China.

Stereotaxic microinjection

The rats were anesthetized with isoflurane (4–5% for induction, 1–2% for maintenance) and placed in a stereotaxic frame (RWD, Shenzhen, China). The skull of the rat was then exposed and leveled. The rAAV was bilaterally delivered into the PrL at the following coordinates: anterior-posterior (AP): + 0.30 cm, medio-lateral (ML): ± 0.06 cm, dorso-ventral (DV): - 0.40 cm. The infusion rate was 0.06 μl/min for a total volume of 0.5 μl. The microinjector was left in the place for 10 min after microinjection to allow for the diffusion of the rAAV complexes. After surgery, penicillin sodium (0.02 mg/kg, i.p.) were injected to the rats once daily for five consecutive days to prevent infection. Rats were housed with free access to food and water and provided with standard care. Twenty-one days after surgery, rats that were microinjected with rAAV were used for behavioral tests.

Conditioned place preference (CPP)

The procedures for CPP training were based on previous studies with minor modifications [32]. The apparatus for CPP conditioning and testing consisted of three polyvinyl chloride boxes, which were identical except for their floors. The boxes had two large side chambers (27.9 cm long × 21.0 cm wide × 20.9 cm high) with a different type of floor (bar or grid), separated by a smaller chamber (12.1 cm long × 21.0 cm wide × 20.9 cm high) with a smooth polyvinyl chloride floor. In each box, the three chambers were separated by manual guillotine doors.

Before the CPP training, the rats were handled for seven days to acclimate to the experimenter’s touch. To determine baseline preference, rats were placed in the middle chamber and given 15 min to freely explore the three compartments with the door open. A computer recorded the time that each rat spent in each compartment to determine the baseline preference. Rats must spend approximately one-third of their time in each chamber; otherwise they will be excluded. Each rat was trained for 8 consecutive days with alternating injections of drug (cocaine, 10 mg/kg or 5 mg/kg, i.p.; methamphetamine, 1 mg/kg, s.c.; morphine, 10 mg/kg, s.c.) and saline (1 ml/kg, i.p. or s.c.) [32–34]. After each injection, the rats were placed in the corresponding conditioning chambers and then returned to their home cage 45 min later. The day after the last conditioning session, all rats were allowed to explore the three compartments freely for 15 min under conditions identical to those described in the baseline test. The CPP score was calculated as the time that each rat spent in the drug-paired chamber minus the time spent in the saline-paired chamber [35].

For SCH and RAC pre-treatment, guide cannulas (24 gauge; RWD, Shenzhen, China) were bilaterally implanted 1 mm above the PrL. The coordinates for PrL were the following: AP: +0.30 cm, ML: ± 0.06 cm, DV: −0.28 cm; 0.5 μl, 16° angle. SCH/RAC/vehicle was separately delivered into the PrL 30 min prior to cocaine injection. Other procedures were consistent with those described above. For CNO pre-treatment, CNO (1.5 mg/kg) was intraperitoneally injected 30 min before cocaine injection. All other procedures were identical to those described above.

Cocaine self-administration procedures

The procedures for cocaine self-administration (SA) training were based on previous studies with minor modifications [32]. Rats were anesthetized with isoflurane (4–5% for induction, 1–2% for maintenance). Catheters were inserted into the right jugular vein with the tip terminating at the opening of the right atrium as previously described. All rats were allowed to recover for 4–5 days after surgery. The operant chambers used (AniLab Software and Instruments) were equipped with two nose poke operandi (ENV-114M; Med Associates) that were 5 cm above the floor of the chambers. Nose pokes in the active operandum led to cocaine infusions that were accompanied by a 5-s tone-light cue. Nose pokes in the inactive operandum were also recorded but had no programmed consequences. The rats were trained to self-administer intravenous cocaine hydrochloride (0.75 mg/kg/infusion) during three 1-h daily sessions separated by 5 min over 7 days. A fixed-ratio one (FR1) reinforcement schedule was used. Each injection was accompanied by the illumination of a cue light above the active nose poke, followed by an additional 20 s timeout period when the cue and house lights were extinguished and additional nose poke responses had no programmed consequence. The number of drug infusions was limited to 20 per hour. At the end of the training phase, all rats underwent extinction. The conditions were the same as during training, except that drug was no longer available. The rats were given extinction training until responding on the active nose poke operandum decreased to less than 20% of the mean responding during the last three days of cocaine self-administration for at least two consecutive days.

Cocaine induced reinstatement test

Before the SA reinstatement test, the rats received a saline (1 ml/kg) or cocaine (10 mg/kg) injection. Conditions during the reinstatement test were the same as those during cocaine self-administration training.

RNA extraction, cDNA synthesis, and quantitative real-time PCR (qPCR)

Sample tissues were isolated, and total RNAs were extracted using TRIzol reagent (Invitrogen, Catalog no. 15596–026). RNA concentration and quality were determined using a NanoDrop spectrophotometer (Thermo Scientific, USA). 500 ng of total RNA from each sample was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech, Catalog no. R212-01). The reaction parameters were 25 °C for 5 min, 50 °C for 50 min and 85 °C for 15 s. All cDNA samples were stored at −20 °C for further use. The qPCR was performed with SYBR Master Mix (QPK201, TOYOBO, Japan) in a QuantStudio 5 Real-Time PCR System (Applied Biosystems) under the following conditions: 95 °C for 2 min and 40 cycles of 95 °C for 30 s, 60 °C for 30 s. Relative expression levels were determined using the 2^−ΔΔCt method [36]. GAPDH mRNA was used as reference genes to analyze Homer1a, Homer1b/c and circHomer1. Specific divergent primers designed at the backsplice junction of circHomer1 were used to detect its expression. All primers were obtained from previous study and listed in Supplementary Table 1 [23].

Basescope in situ hybridization

To detect the distribution and quantification of circHomer1 in different cells, we customized an RNA probe set that specifically targets the junction sites of circHomer1 (Cat No. 1055641-C1, Advanced Cell Diagnostics, USA). The procedures for Basescope in situ hybridization were based on previous studies with minor modifications [37, 38]. Twenty-four hours after the last cocaine injection, the rats were perfused with with 0.1 mol/L phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA; pH 7.4). The brains were then removed and post-fixed in 4% paraformaldehyde for 24 h. Afterward, the brains were then dehydrated in 30% sucrose (w/v) dissolved in 0.1 mol/L phosphate buffer. The brains were coronally sectioned at 12 μm using a sliding microtome, and five to six sections spanning the rostrocaudal axis of the PrL were collected and stained, with each section being taken from different rats. All sections were washed three times for 5 min each with phosphate buffered saline (0.1 mol/L PBS), and then treated with protease for 10 min. After washing off the protease, we incubated brain sections with the probe sets specifically targeting circHomer1 for 2 h at 40 °C in the HybEZ^TM oven. Following probe incubation, sections underwent a series of incubations with preamplifier probes, amplifier probes, and fluorescently labeled probes at 40 °C. We acquired fluorescent images using the fluorescence microscope (Olympus, Tokyo, Japan) with a 20× objective lens. Fluorescent dot numbers were obtained for each specific probe set (red dots defined as those for circHomer1).

Immunofluorescence

To detect the co-localization of circHomer1 and cell marker, the immunofluorescence based on previous studies with minor modifications was used [38, 39]. The sections hybridized with the probe were incubated with the primary antibody in PBST (PBS containing 0.05% triton-100) with 1% normal goat serum overnight at 4 °C. The following primary antibodies were used in our experiments: mouse anti-CamKⅡ (1:100, sc-13141, Santa Cruz Biotechnology), mouse anti-GAD67 (1:100, ab26116, Abcam), mouse anti-NeuN (1:100, MAB377, Millipore), rabbit anti-GFAP (1:500, ab23922, Abcam), rabbit anti-Iba1 (1:500, ab178847, Abcam), and rabbit anti-Dopamine Receptor D1 (D1R) (1: 100, ab279712, Abcam). The sections were then washed three times in PBST and incubated with the indicated secondary antibodies for 3 h at room temperature. The following secondary antibodies were used in our experiments: Alexa Fluor 488 goat anti-rabbit IgG (for GFAP, D1R and Iba1, 1:500; ab150077, Abcam), and Alexa Fluor 488 goat anti-mouse IgG (for CamKⅡ, GAD67, and NeuN, 1:500; ab150113, Abcam). In order to determine the specificity of CamKⅡ, we utilized Alexa Fluor 594 goat anti-mouse IgG (1:1000; ab150116, Abcam) as the secondary antibody. Finally, after three additional washes with PBST, the sections were mounted on Antifade Mounting Medium (S2110, Solarbio) and imaged using the fluorescence microscope (Olympus, Tokyo, Japan). Four to six images were randomly selected from individual animals for counting the co-localization of circHomer1 and cell markers. The co-localization of cells is defined as GFP cell markers surrounded by red circHomer1 dots.

Dendritic spine counting

Coronal sections (60 μm thick) were processed for imaging. All images were acquired at a resolution of 1,024 × 1,024. High-resolution z-stacks of GFP-positive cells were acquired using a 60× oil-immersion lens from each PrL section per rat, with a step size of 1 μm. High-resolution z-stacks of randomly selected secondary or tertiary dendritic branch segments from individual cells were acquired for spine counting. The acquisition was done with a 5× optical zoom for the dendritic branch segments. Four to six pyramidal neurons were randomly selected from each individual animal, and each neuron was scanned for spines counting on 4-6 secondary or tertiary dendrites. The dendritic spines are classified into four categories [40]: mushroom spines, long-thin spines, stubby spines and filopodia spines. Mushroom spine is defined as having a head diameter larger than 0.5 μm and a head-to-neck diameter ratio greater than 1:1 [40, 41]. The length of the spine is more than twice the average neck width, and the average neck width is less than or equal to the maximum width of the head, which are classified into long-thin spine [40]. The spine with the maximum width of the head is less than or equal to the average neck width is classified as filopodia and with a total length of less than 1 μm is classified as stubby [40].

Ex vivo electrophysiology

The experiments were performed as previously described [42]. The rats were deeply anesthetized with chloral hydrate and then decapitated. The brains were swiftly extracted and immersed in a frigid cutting solution consisting of the following composition: 87 mM NaCl, 26 mM NaHCO₃, 3.0 mM KCl, 1.0 mM NaH₂PO₄, 1.3 mM MgSO₄, 1.5 mM CaCl₂, 20 mM D-glucose, and 75 mM sucrose saturated with a mixture of 95% O₂ and 5% CO₂. Coronal sections that encompassed the PrL region were delicately sliced at a thickness of 230 μm using a vibratome (Leica VT1200), while being immersed in the same ice-cold cutting solution enriched with 95% O₂ and 5% CO₂. The slices were subsequently transferred to a specialized incubation chamber, in which they were submerged in artificial cerebrospinal fluid (aCSF) comprising the following constituents: 124 mM NaCl, 26 mM NaHCO₃, 3.0 mM KCl, 1.0 mM NaH₂PO₄, 1.3 mM MgCl₂, 2 mM CaCl₂, and 20 mM D-glucose. This aCSF solution was meticulously saturated with a blend of 95% O₂ and 5% CO₂, simulating the optimal physiological conditions. Initially, the slices were incubated at a precise temperature of 34 °C for a duration of 30 min. Subsequently, they were maintained at room temperature for a minimum of an additional 30 min before being employed for recording purposes.

Each individual slice was carefully transferred to a submerged chamber, where it experienced continual exposure to aCSF that had been thoroughly saturated with a precise blend of 95% O₂ and 5% CO₂, flowing at a controlled rate of 2 ml/min, regulated by a meticulous flowmeter. Initially, the slice was examined using a 4× objective, facilitating the precise localization of the PrL region, which was identified by its proximity to the forceps minor corpus callosum and the midline. Subsequently, under the illumination of near-infrared light, layer V of the PrL was observed using a high-resolution 40× water-immersion objective. The identification of layer V pyramidal cells was meticulously conducted based on their cellular morphology, size, and distinctive electrophysiological properties.

All experimental procedures were meticulously carried out under a controlled temperature of 32 °C. For the preparation of electrodes, thick-wall borosilicate glass was skillfully manipulated using a horizontal puller, resulting in electrodes with resistances ranging from 2.5 to 3.5 MΩ. Ensuring optimal electrical connectivity, the seal resistance exceeded the threshold of 1 GΩ. To capture cellular activity at the soma, whole-cell recordings were conducted using the advanced MultiClamp 700B amplifier.

For the current-clamp recordings, the finely crafted pipette solution consisted of a precise composition: 120 mM potassium gluconate, 10 mM KCl, 4 mM ATP-Mg, 0.3 mM GTP, 10 mM HEPES, 5 mM Na₂-phosphocreatine, and 2 mM EGTA (with a pH value of 7.2 and an osmolarity of 270–280 mOsm, achieved through the addition of sucrose). To maintain neuronal membrane potentials around −60 mV, a gradual current was carefully applied. Series resistance was impeccably compensated for utilizing the bridge circuit integrated within the esteemed MultiClamp 700B amplifier. To evaluate the spike rate, the frequency of spikes was meticulously recorded in discreet 500 ms intervals. These results were then visually represented in relation to the intensity of the applied current. Within 20 seconds of establishing whole-cell configuration, the resting potentials of the neurons were accurately measured. Input resistance was methodically ascertained by administering hyperpolarizing current pulses of either −50 pA or −100 pA, inducing voltage shifts ranging from 5 to 15 mV below the resting membrane potential. The threshold, a key measurement, was determined as the precise moment when the slope of the rising membrane potential exceeded an impressive rate of 50 mV/ms. Additional measurements such as after-hyperpolarization, half-width, and overshoot were carefully estimated from all action potentials observed during the 200-pA current injection step. To conduct these experiments, the esteemed Clampex program (Molecular Devices) was employed. All data points were keenly digitized at an impressive rate of 20 kHz. For meticulous analysis, the esteemed Clampfit 10.7 software (Molecular Devices) was utilized.

Data collection and analysis of human brain circRNAs

An RNA sequencing raw data involving the dorsolateral prefrontal cortex (dlPFC) of patients with cocaine use disorder was obtained from GSE99349 in GEO database (http://www.ncbi.nlm.nih.gov/geo/). The procedures for identifying the expression of circHomer1 were based on our previous study [43]. In our analysis, we excluded participants with the age over 50 years old to align the human data with the age range of the rats used in our experiments. All including samples were listed in Supplementary Table 2 [44]. To shed light on the statistical significance of circHomer1 expression, we employed the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) in each participant as the basis of our rigorous analysis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8. Unless otherwise stated, normally distributed data was tested using one-way ANOVA followed by Tukey’s post hoc; repeated two-way ANOVA followed by Bonferroni’s post hoc or Holm-Sidak’s post hoc; two-way ANOVA followed by Tukey’s post hoc; three-way ANOVA followed by Tukey’s post hoc; and two-tailed, unpaired t test. Non-normally distributed data were tested using the Kruskal-Wallis test with Dunn’s post hoc and the two-tailed, unpaired Mann Whitney U test. The results are expressed as mean ± SEM. P  <  0.05 were considered statistically significant. The detailed statistical analyses are showed in Supplementary Table 3.

Results

CircHomer1 in PrL is required for cocaine-induced rewarding effect

CircHomer1, an extensively preserved circRNA, originates from the exons 2 to 5 of Homer1 through the intricacies of backsplicing (Supplementary Fig. 1). To investigate the role of circHomer1 in cocaine response, we analyzed its expression dynamics across addiction-related brain regions following repeated cocaine exposure (Fig. 1a). Chronic cocaine administration induced region- and time-dependent alterations in circHomer1 expression. The prelimbic cortex (PrL) exhibited sustained circHomer1 reduction across all examined time points (30 min, 1 h, 6 h, 24 h, and 48 h) post-final cocaine injection (Fig. 1b). Other regions showed temporally restricted responses: significant downregulation occurred at 30 min in the central amygdala (CeA) and ventral tegmental area (VTA), at 1 h in the infralimbic cortex (IL) and basolateral amygdala (BLA), and at 6 h in the BLA (Fig. 1b). Notably, nucleus accumbens (NAc) core circHomer1 levels remained unchanged across all post-injection intervals (Fig. 1b). These region-specific temporal dynamics were further confirmed by BaseScope in situ hybridization, which validated the persistent circHomer1 reduction in PrL (Fig. 1c and d). Significantly, analysis of dorsolateral prefrontal cortex (dlPFC) samples from patients with cocaine use disorder revealed marked circHomer1 reduction (Fig. 1e), paralleling our observations in chronically cocaine-exposed rats. This cross-species conservation of circHomer1 dysregulation suggests its potential role as a therapeutic target in cocaine use disorder.

Fig. 1. CircHomer1 diminished after repeated cocaine exposure and was essential for conditioned place preference induced by cocaine in the prelimbic cortex.

a Timeline of the experiment. The rats were injected intraperitoneally with cocaine for 14 days and were decapitated to detect the expression of circHomer1 at 30 min, 1 h, 6 h, 24 h and 48 h after the last cocaine injection. b Detection of the expression of circHomer1 using qPCR in addiction-related brain regions following chronic cocaine exposure. First row (30 min): Unpaired t test, PrL: t₉ = 2.94, P = 0.016; CeA: t₉ = 3.34, P = 0.009; VTA: t₉ = 2.44, P = 0.037. saline: n = 6/group, cocaine: n = 5/group. Second row (1 h): Unpaired t test, PrL: t₁₄ = 2.54, P = 0.024; IL: t₁₄ = 2.46, P = 0.028; BLA: t₁₄ = 2.90, P = 0.012. PrL/IL/BLA/CeA/NAc core: n = 8/group; VTA: saline n = 7, cocaine n = 8. Third row (6 h): Unpaired t test, PrL: t₁₃ = 2.87, P = 0.013; BLA: t₁₄ = 2.83, P = 0.013. PrL/IL/VTA: saline n = 8, cocaine n = 7; BLA/CeA/NAc core: n = 8/group. Fourth row (24 h): Unpaired t test, PrL: t₁₀ = 3.05, P = 0.012. n = 6/group. Fifth row (48 h): Unpaired t test, PrL: t₁₂ = 2.21, P = 0.047. n = 7/group. c Visualization of the expression of circHomer1 in the PrL at 30 min, 1 h, 6 h, 24 h and 48 h after the last cocaine injection. Red dots indicate expressed circHomer1, while blue dots indicate the location of the cell nucleus. Scale bars: 50 μm. d Quantification of the circHomer1 expression using in situ hybridization in the PrL at 30 min, 1 h, 6 h, 24 h and 48 h after the last cocaine injection. Unpaired t test, 30 min: t₁₀ = 2.66, P = 0.024; 1 h: t₁₀ = 2.48, P = 0.033; 6 h: t₁₀ = 4.73, P = 0.0008; 24 h: t₁₀ = 7.71, P < 0.0001; 48 h: t₁₀ = 3.36, P = 0.007. n = 6/group. e Decreased expression of circHomer1 in the dlPFC of patients with cocaine use disorder. Unpaired t test: t₂₆ = 2.21, P = 0.037. control: n = 12; CUD: n = 16. f Timeline of the CPP. Before cocaine CPP training, the rats were bilaterally microinjected in the PrL with CMV-OE-circHomer1 to overexpress circHomer1 or sh-circHomer1 to knockdown circHomer1. Corresponding controls received CMV-GFP or sh-control. Following the rAAV expression, cocaine at dose of 10 mg/kg or 5 mg/kg was used to explore whether overexpression of circHomer1 could suppress cocaine CPP or knockdown of circHomer1 could enhance cocaine CPP, respectively. g GFP fluorescence indicates local expression of rAAV in the PrL of rats, as visualized under fluorescence microscopy. The injection site of rAAV (left), Scale bars: 2 mm. The expression of rAAV (right), Scale bars: 100 μm. h Elevated expression of circHomer1 in the PrL of rats injected with CMV-OE-circHomer1. Unpaired t test: t₁₆ = 6.38, P < 0.0001. n = 9/group. i CircHomer1 overexpression resulted in a reduction in cocaine CPP. Repeated two-way ANOVA: the interaction of CPP training × the viral vector (F_{1, 18} = 5.54, P = 0.030). n = 10/group. j The expression of circHomer1 significantly decreased in the PrL of rats injected with sh-circHomer1. Unpaired t test: t₁₆ = 9.49, P < 0.0001. n = 9/group. k Knockdown of circHomer1 enhanced cocaine CPP. Repeated two-way ANOVA: the interaction of CPP training × the viral vector (F_{1, 14} = 6.71, P = 0.021). n = 8/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ##P < 0.01. The data are expressed as mean ± SEM. CPP: conditioned place preference. CUD: cocaine use disorder. FPKM: Fragments Per Kilobase of exon model per Million mapped fragments.

Then, we examined expression changes in Homer1 linear transcripts following chronic cocaine exposure, focusing on three predominant isoforms: Homer1a and Homer1b/c. Homer1b and Homer1c were analyzed collectively due to their high structural and functional similarity, differing by only 36 base pairs [45, 46]. While Homer1a expression in PrL remained unchanged at both 30 min and 24 h post-cocaine injection, Homer1b/c showed significant upregulation at 24 h, but not at 30 min, following final cocaine administration (Supplementary Fig. 2). Given previous findings demonstrating circHomer1 binding to Homer1b in orbitofrontal cortex and its role in cognitive flexibility regulation [26], we hypothesized that circHomer1 might function through competitive sequestration of Homer1b/c, potentially explaining the transient Homer1b/c upregulation observed following repeated cocaine exposure.

Additionally, we detected the expression of circHomer1 after acute cocaine exposure, we found single cocaine exposure did not change the expression of circHomer1 (Supplementary Fig. 3). The reduction in circHomer1 levels induced by cocaine does not represent a mere pharmacological response, but rather signifies a lasting adaptive alteration within the PrL brought about by prolonged exposure to cocaine. This indicates that the enduring aberrant expression of circHomer1 may be the etiology and pathogenesis of cocaine addiction.

Next, we assessed whether manipulating circHomer1 expression in the PrL would impact cocaine-induced CPP. We first employed a recombinant adeno-associated virus (rAAV) with the CMV promoter to overexpress circHomer1 (CMV-OE-circHomer1), while using an empty vector (CMV-GFP) as a control (Fig. 1f). The expression of rAAV-mediated GFP was restricted to the PrL (Fig. 1g). The expression of circHomer1 was markedly increased in the PrL of rats in CMV-OE-circHomer1 group (Fig. 1h), which demonstrated that CMV-OE-circHomer1 induced significant overexpression of circHomer1. Before cocaine conditioning (baseline), both the CMV-OE-circHomer1 and CMV-GFP injected groups displayed comparable CPP scores, indicating no inherent preference (Fig. 1i). However, rats in CMV-OE-circHomer1 group showed a significantly decreased cocaine-induced CPP score compared to that of CMV-GFP rats after cocaine conditioning (10 mg/kg) (Fig. 1i). These findings suggest that overexpression of circHomer1 can mitigate the rewarding effect of cocaine.

We then evaluated whether inhibiting circHomer1 expression in the PrL could enhance cocaine-induced CPP. We utilized a rAAV vector with a U6 promoter to express a specific shRNA (sh-circHomer1) for targeted knockdown of circHomer1 expression in vivo (Fig. 1f). A scrambled sequence (sh-control) was used as a control. The expression of circHomer1 was notably decreased in the PrL of sh-circHomer1 group rats (Fig. 1j), indicating the effectiveness of shRNA-mediated suppression. The rAAV microinjection did not influence the preference of the rats, as both groups exhibited similar CPP score in the baseline. Post-cocaine conditioning (5 mg/kg), the sh-control group did not exhibit a place preference for the cocaine-paired compartment (Fig. 1k). However, the preference for the cocaine-paired compartment was developed in the sh-circHomer1 group rats (Fig. 1k). These results underscore the importance of circHomer1 in the PrL for cocaine-induced rewarding effects.

Collectively, these findings demonstrate that lasting adaptive alteration of circHomer1 expression within the PrL is essential for cocaine-induced rewarding effects.

Cocaine modulates circHomer1 expression in excitatory neurons of the PrL

To assess the predominant cell types expressing circHomer1 in the PrL, we used the BaseScope technique in conjunction with immunostaining of specific cell markers in naive rats. Analysis revealed that circHomer1 transcripts exhibited robust co-localization with the pan-neuronal marker NeuN (Supplementary Fig. 4a, 4b). In contrast, significantly less co-localization was observed with the astrocyte marker glial fibrillary acidic protein (GFAP) or the microglia marker ionized calcium-binding adapter molecule 1 (Iba-1), indicating neuronal enrichment of circHomer1 within the PrL. Then, we identified the specific types of neurons in which circHomer1 functioned after repeated cocaine exposure using the BaseScope method in conjunction with immunostaining of specific neuronal markers. As shown in Fig. 2, compared with saline-treated group, repeated cocaine exposure significantly decreased the proportion of circHomer1 co-labeled with CamKII (Fig. 2c–e), a marker of excitatory neurons, but not GAD67 (a marker of inhibitory neurons) (Fig. 2f–h) in the PrL. This results suggest that circHomer1 may modulate the rewarding effects of cocaine by acting on excitatory neurons in the PrL region.

Fig. 2. Repeated cocaine exposure selectively reduced circHomer1 expression in excitatory neurons of the PrL.

a Timeline of the experiment. The rats were injected intraperitoneally with cocaine for 14 days and were decapitated to detect the co-localization of circHomer1 with cell markers at 24 h after the last cocaine injection. b The schematic of PrL for detecting the co-localization of circHomer1 with cell markers. Scale bars: 1 mm (left); 100 μm (right). c The co-localization of circHomer1 with CamKII (a marker of excitatory neurons) in the PrL of rats treated with saline or cocaine. White arrows indicate positive staining of biomarkers for excitatory neurons (green), while white triangles indicate colocalization with circHomer1 (red) and biomarkers (green). Scale bars: 50 μm. d and e, Repeated cocaine treatment increased the ratio of circHomer1 and CamKII double-positive cells among circHomer1-positive cells. Unpaired t test: t₁₄ = 4.56, P = 0.0004. n = 8/group. f The co-localization of circHomer1 with GAD67 (a marker of inhibitory neurons) in the PrL of rats treated with saline or cocaine. White arrows indicate positive staining of biomarkers for inhibitory neurons (green), while white triangles indicate co-localization with circHomer1 (red) and biomarkers (green). Scale bars: 50 μm. g and h, The ratio of circHomer1 and GAD67 double-positive cells among circHomer1-positive cells was unchanged. Unpaired t test: t₁₄ = 0.90, P = 0.345. n = 8/group. The data are expressed as mean ± SEM. *** P < 0.001.

CircHomer1 in PrL excitatory neurons regulated cocaine-induced behavioral response

To confirm whether circHomer1 in excitatory neurons indeed regulates cocaine-induced conditioning behavior, we utilized a rAAV with CamKⅡ promoter to selectively overexpress circHomer1 in excitatory neurons (CamKⅡ-OE-circHomer1), using an empty vector (CamKⅡ-GFP) as a control (Fig. 3a). To evaluate the transfection efficiency of rAAV with the CamKⅡ promoter in the PrL, we quantified the co-localization of GFP with the marker of excitatory neurons, CamKⅡ. As shown in Fig. 3b and Fig. 3c, the rAAV mainly targeted the PrL region, with approximately 98.56% of circHomer1 overexpressed rAAV transfected into excitatory neurons, as indicated by the co-localization of GFP with CamKⅡ. In addition, the expression of circHomer1 was significantly increased in the PrL of CamKⅡ-OE-circHomer1 group rats (Fig. 3d). These results demonstrated that the CamKⅡ-OE-circHomer1 group successfully induced the overexpression of circHomer1 in PrL excitatory neurons. Before cocaine conditioning, the rats in the CamKⅡ-OE-circHomer1 group and the CamKⅡ-GFP group displayed similar CPP scores in the CPP baseline (Fig. 3e). After cocaine conditioning, all rats exhibited cocaine-induced CPP, but rats injected with CamKⅡ-OE-circHomer1 showed a significant lower cocaine-induced CPP score compared to CamKⅡ-GFP rats (Fig. 3e).

Fig. 3. CircHomer1 in the PrL excitatory neurons was necessary for cocaine-induced rewarding effects.

a Experimental timeline of the CPP procedure. Before CPP training, the rats were bilaterally microinjected in the PrL with CamKⅡ-OE-circHomer1 to overexpress circHomer1, and corresponding CamKⅡ-GFP was microinjected as a control. After the rAAV was expressed, a dose of 10 mg/kg of cocaine was used to explore whether the specific overexpression of circHomer1 in the excitatory neurons could suppressed cocaine CPP expression. b Illustration of CamKⅡ-OE-circHomer1 transfected cell types in the PrL. The GFP shown that the expression of rAAV was restricted in PrL. Left: the injection site of rAAV, Scale bars: 1 mm. Right: the co-localization of GFP and CamKⅡ (the marker of excitatory neurons) in the PrL. White arrows indicate positive staining of biomarkers for excitatory neurons, Scale bars: 50 μm. Red indicates CamKⅡ-positive neuron, green indicates rAAV-infected cell, and blue indicated DAPI-stained nuclei. c Ratio of GFP and CamKⅡ double-positive cells among GFP-positive cells. n = 6. d The expression of circHomer1 significantly increased in excitatory neurons of the PrL in the group of CamKⅡ-OE-circHomer1. Mann Whitney U test, P < 0.0001. CamKⅡ-GFP: n = 9; CamKⅡ-OE-circHomer1: n = 8. e Overexpression of circHomer1 in the PrL excitatory neurons decreased cocaine CPP. Repeated two-way ANOVA: the interaction of CPP training × the viral vector (F_{1, 16} = 6.25, P = 0.024). n = 9/group. f Experimental timeline of the cocaine self-administration procedure. Three weeks after the injection of CamKⅡ-OE-circHomer1 or CamKⅡ-GFP in the PrL, the rats were trained to self-administer intravenous injections of cocaine under an FR1 schedule in daily 3-h sessions until cocaine intake stabilized. Then, the rats were given extinction training until responses of the active nose poke decreased to less than 20% of the mean responding during the last 3 days of cocaine self-administration for at least 2 consecutive days. Last, these rats underwent a saline (1 ml/kg, i.p.) or cocaine (10 mg/kg, i.p.)-induced reinstatement. n = 7/group. g The circHomer1 overexpression decreased the infusions during daily 3-h self-administration sessions. Repeated two-way ANOVA: the main effect of training days (F_{6, 72} = 2.48, P = 0.031) and the main effect of the viral vector (F_{1, 12} = 23.08, P = 0.0004). h Active nose pokes responses on the active operandum during the extinction session. Repeated two-way ANOVA: the interaction of extinction time × the viral vector (F_{11, 132} = 1.29, P = 0.240). i, Rats in the CamKⅡ-OE-circHomer1 group showed significantly fewer active nose pokes during cocaine priming. Repeated two-way ANOVA: the interaction of priming × the viral vector (F_{1, 12} = 4.28, P = 0.061). The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P < 0.05, ##P < 0.01.

To further assess the role of circHomer1 in excitatory neurons in cocaine addiction-like behavior, we injected CamKⅡ-OE-circHomer1 or CamKⅡ-GFP into PrL and examined the behavioral effects using an animal model of cocaine SA. Figure 3f shows the timeline and schematic of the entire cocaine SA procedure. During training cocaine SA, both CamKⅡ-OE-circHomer1 and CamKⅡ-GFP mice reliably self-administered cocaine at a dose of 0.75 mg/kg/infusion under fixed ratio 1 (Fig. 3g). However, circHomer1 overexpression significantly decreased the cocaine infusions each day during the training phase (Fig. 3g). Although the number of active responses was similar overall between groups across the entire extinction phase (Fig. 3h), CamKⅡ-OE-circHomer1 group significantly diminished the cocaine-priming-induced cocaine seeking during the reinstatement test but not saline-priming-induced cocaine seeking (Fig. 3i).

Collectively, these findings demonstrate that circHomer1 in the PrL excitatory neurons is necessary for cocaine-induced behavior responses, highlighting its significance in the development of cocaine addiction.

CircHomer1 regulated cocaine-induced synaptic plasticity in excitatory neurons

Building on previous evidence that circHomer1 knockdown affects alternative isoform expression and synaptic plasticity [29], we investigated whether circHomer1 modulates cocaine-induced behavioral adaptations through synaptic mechanisms. Following stereotactic delivery of CamKII-OE-circHomer1 or CamKII-GFP into PrL, we analyzed structural and functional plasticity in PrL excitatory neurons after 14-day cocaine exposure (Fig. 4a). While total dendritic spine density remained unchanged following chronic cocaine administration (Fig. 4b, c), morphological analysis revealed specific cocaine-induced synaptic alterations: increased stubby spine density (immature phenotype) with concurrent reduction in mushroom spines (mature phenotype), while filopodia and long-thin spine densities remained stable (Fig. 4d–g). Notably, circHomer1 overexpression normalized this cocaine-induced spine subtype redistribution (Fig. 4d–g). Functional analysis using patch-clamp recordings revealed cocaine-induced reductions in spontaneous excitatory postsynaptic current (sEPSC) frequency but not amplitude, an effect reversed by circHomer1 overexpression (Fig. 4h–l). These findings align with recent studies showing circHomer1 depletion decreases mushroom spine density and the frequency of sEPSC [28, 47]. Together, these structural and functional analyses establish circHomer1 as a key regulator of synaptic plasticity in PrL excitatory neurons, particularly in maintaining spine maturation and synaptic strength.

Fig. 4. CircHomer1 regulated cocaine-induced synaptic plasticity in excitatory neurons.

a Timeline of the experiment. The rats underwent stereotactic surgery to infuse CamKⅡ-OE-circHomer1 or CamKⅡ-GFP into PrL. After three weeks, cocaine (10 mg/kg) or saline (1 ml/kg) i.p. injections commenced, and tissue was collected 24 h after the final injection for analysis of dendritic spines and patch-clamp recording. b Representative images of dendritic spines in the PrL for each group. Solid arrow indicate mushroom spines, hollow arrow indicate long-thin spines, solid triangle indicated stubby spines, and hollow triangle indicate filopodia spines. CamKⅡ-GFP-saline: n = 50 dendritic segments from n = 3 rats; CamKⅡ-GFP-cocaine: n = 55 dendritic segments from n = 3 rats; CamKⅡ-OE-circHomer1-cocaine: n = 57 dendritic segments from n = 3 rats. c Quantification of the total spines in the PrL. Cocaine did not change the density of the total spines. But circHomer1 overexpression increased the density of the total spines. One-way ANOVA: F_{2, 159} = 13.61, P < 0.0001. d Quantification of the mushroom spines in the PrL. Left: Cocaine decreased the numbers of mushroom spines, and the overexpression of circHomer1 in PrL excitatory neurons reversed the cocaine-induced reduction. One-way ANOVA: F_{2, 159} = 57.07, P < 0.0001. Right: Cocaine decreased the ratio of mushroom spines, and the overexpression of circHomer1 in PrL excitatory neurons reversed the cocaine-induced reduction. One-way ANOVA: F_{2, 159} = 35.16, P < 0.0001. e Quantification of the stubby spines in the PrL. Left: Cocaine increased the numbers of stubby spines but cirHomer1 did not reversed the cocaine-induced increase. One-way ANOVA: F_{2, 159} = 15.83, P < 0.0001. Right: Cocaine increased the ratio of stubby spines, and the overexpression of circHomer1 in PrL excitatory neurons reversed the cocaine-induced increase. One-way ANOVA: F_{2, 159} = 15.65, P < 0.0001. f Quantification of the long-thin spines in the PrL. Left: Cocaine did not change the numbers of the long-thin spines. One-way ANOVA: F_{2, 159} = 0.10, P = 0.903. Right: Cocaine did not change the ratio of the long-thin spines. But circHomer1 overexpression decreased the ratio of the long-thin spines. Kruskal-Wallis test: P < 0.0001. g Quantification of the filopodia spines in the PrL. Left: CircHomer1 overexpression increases the numbers of filopodia spines. One-way ANOVA: F_{2, 159} = 3.15, P = 0.045. Right: Cocaine did not influence the ratio of filopodia spines. One-way ANOVA: F_{2, 159} = 0.84, P = 0.434. h Representative raw traces of sEPSC from PrL excitatory neurons. CamKⅡ-GFP-saline: n = 15 neurons from n = 3 rats; CamKⅡ-GFP-cocaine: n = 14 neurons from n = 3 rats; CamKⅡ-OE-circHomer1-cocaine: n = 15 neurons from n = 3 rats. i and k Cumulative frequency plots of inter-event intervals and amplitude. j and l, The frequency and amplitude of sEPSC in PrL excitatory neurons. The scattergram shows a significant decrease in sEPSC frequency but not amplitude in the CamKⅡ-GFP-cocaine group. However, the overexpression of circHomer1 in the PrL excitatory neurons reversed the decrease caused by cocaine. Kruskal-Wallis test: P < 0.0001 for the frequency, P = 0.0003 for the amplitude. The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The expression of circHomer1 may be altered by cocaine via the dopamine D1 receptor (D1R) pathway

Accumulating evidence indicates that D1R and dopamine D2 receptor (D2R) are both essential for the rewarding effects of psychoactive drugs [48]. We hypothesized that the D1R and D2R may mediate the adaptive changes in circHomer1 expression after cocaine exposure. To test this, we microinjected SCH (a D1R antagonist), RAC (a D2R antagonist), or a vehicle into the PrL prior to the cocaine-context pairing during training (Fig. 5a). The results revealed that the administration of SCH, but not RAC, resulted in the suppression of cocaine-induced CPP expression in the PrL when compared to the vehicle (Fig. 5b). To further explore whether dopamine receptors regulate the expression of circHomer1, we next examined the circHomer1 expression in the PrL after injection of SCH or RAC. We observed that pre-treatment with SCH reversed the downregulation of circHomer1 expression induced by cocaine (Fig. 5c). However, RAC did not have a significant effect on the altered expression of circHomer1 caused by cocaine (Fig. 5c). Importantly, we found approximately 82.08% of the D1-positive neurons exhibited the expression of circHomer1 (Fig. 5d).These results indicate that cocaine may reduce circHomer1 expression through activating D1R signaling in the PrL.

Fig. 5. The cocaine-induced decrease in circHomer1 expression in the PrL was mediated by D1R signaling.

a Timeline of SCH and RAC pre-treatment prior to the cocaine-induced CPP. Vehicle, SCH or RAC was given 30 min prior to saline or cocaine injection on the drug training day. PrL samples for qPCR were collected 24 h after the CPP test. b SCH but not RAC pre-treatment significantly attenuated the CPP expression induced by cocaine. Repeated three-way ANOVA: the interaction of CPP training × antagonist × cocaine (F_{2, 37} = 2.92, P = 0.066). saline-vehicle, n = 8; cocaine-vehicle, n = 7; saline-SCH, n = 7; cocaine-SCH, n = 8; saline-RAC, n = 5; cocaine-RAC, n = 8. c SCH rescued the cocaine-induced decrease in the expression of circHomer1 in the PrL of rats. Two-way ANOVA: the main effect of antagonist (F_{2, 36} = 3.99, P = 0.027) and the main effect of cocaine (F_{1, 36} = 5.85, P = 0.021). saline-vehicle, n = 8; cocaine-vehicle, n = 7; saline-SCH, n = 7; cocaine-SCH, n = 7; saline-RAC, n = 5; cocaine-RAC, n = 8. d Approximately 82.08% of the D1-positive neurons exhibited the expression of circHomer1. Left: the schematic of the co-localization of circHomer1 with D1R; right: ratio of circHomer1 and D1R double-positive cells among D1R-positive cells. Scale bar: 50 μm. n = 6. e Timeline of D1 neurons activation and circHomer1 overexpression prior to the cocaine-induced CPP. The rats underwent stereotactic surgery to infuse rAAV-D1-hM3D(Gq)-mCherry mixed with CamKⅡ-OE-circHomer1 or CamKⅡ-GFP into the PrL in order to overexpress circHomer1 and activate D1 neurons at the same time. After three weeks, cocaine (5 mg/kg) CPP commenced and CNO was administered 30 min prior to cocaine injection on the drug training day to activate D1 neurons. PrL samples for detecting rAAV expression were collected 24 h after the CPP test. n = 10/ group. f The schematic of stereotaxic viral injection into the rat’s PrL. The left fluorescent image demonstrates the spread of viral-mediated transgene expression in the PrL. Scale bar: 2 mm. Right fluorescent image is shown to demonstrate the co-expression of rAAV-D1-hM3D(Gq)-mCherry with CamKⅡ-OE-circHomer1 or CamKⅡ-GFP in PrL excitatory neurons. White arrows indicate positive co-expressed neurons of mCherry and GFP merged. Scale bar: 100 μm. g Overexpression of circHomer1 reversed the enhancing effect of activating D1 neurons on cocaine-induced CPP. Repeated three-way ANOVA: the interaction of CPP training × virus vector × CNO (F_{1, 36} = 5.52, P = 0.025). The data are expressed as mean ± SEM. *P < 0.05, ****P < 0.0001, ##P < 0.01, ###P < 0.001. SCH: SCH23390, the D1R antagonist; RAC: raclopride, the D2R antagonist; CNO: clozapine-n-oxide, 1.5 mg/kg.

To investigate whether D1R signaling modulates the contribution of circHomer1 to cocaine reward, we employed a chemogenetic strategy targeting D1R-expressing neurons in the PrL. Rats received bilateral PrL injections of rAAV vectors driving expression of the excitatory designer receptor hM3Dq under the control of a D1R promoter. These were co-injected with CamKII-OE-circHomer1 or a control CamKII-GFP to restrict circHomer1 overexpression to excitatory neurons (Fig. 5e, f). Following cocaine-induced CPP training, clozapine N-oxide (CNO), an hM3Dq agonist, or vehicle was administered systemically to activate PrL D1R neurons. Chemogenetic activation of PrL D1R neurons via CNO significantly potentiated the cocaine CPP score compared to vehicle injection (Fig. 5g). Crucially, this potentiation of cocaine reward by D1R neuron activation was completely reversed by concurrent circHomer1 overexpression (Fig. 5g).

Collectively, these findings demonstrate that D1R signaling modulates cocaine-induced suppression of circHomer1 expression in the PrL, potentially through direct transcriptional regulation or indirect pathway crosstalk. Critically, our functional rescue experiments establish that circHomer1 overexpression reverses cocaine reward phenotypes by counteracting the effects of D1R neuronal activation in the PrL. These results may position circHomer1 as a downstream effector of D1R-mediated signaling in the neural circuitry underlying cocaine-induced behavioral response.

CircHomer1 modulates the rewarding effects induced by psychostimulants but not food or opioid drugs

The above findings figured that circHomer1 in PrL is a regulator of cocaine reward, but its impact on the rewarding effects of natural rewards and other addictive substances remains unclear. To this end, we microinjected CamKⅡ-OE-circHomer1 into the PrL to selectively increase the expression of circHomer1 in PrL excitatory neurons prior to CPP training with methamphetamine, morphine, or food (Fig. 6a–c). We found that the overexpression of circHomer1 markedly attenuated the rewarding effect induced by methamphetamine (Fig. 6a). However, overexpressed circHomer1 did not alter the rewarding effect of morphine (Fig. 6b) and food (Fig. 6c). These results indicate that circHomer1 in PrL excitatory neurons selectively regulates reward induced by psychostimulants, such as cocaine and methamphetamine, without affecting response to sedatives like morphine or natural rewards.

Fig. 6. The influence of circHomer1 is selective for psychostimulants.

a Overexpression of circHomer1 in the PrL excitatory neurons decreased methamphetamine CPP. Repeated two-way ANOVA: the interaction of CPP training × virus vector (F_{1, 16} = 10.35, P = 0.006). CamKⅡ-GFP: n = 8; CamKⅡ-OE-circHomer1: n = 10. b CircHomer1 overexpression in the PrL excitatory neurons did not suppress morphine-induced rewarding effects. Repeated two-way ANOVA: the interaction of CPP training × virus vector (F_{1, 15} = 2.56, P = 0.130). CamKⅡ-GFP: n = 8; CamKⅡ-OE-circHomer1: n = 9. c Overexpression of circHomer1 in the PrL excitatory neurons did not suppress food-induced natural rewarding effects. Repeated two-way ANOVA: the interaction of CPP training × virus vector (F_{1, 13} = 0.32, P = 0.584). CamKⅡ-GFP: n = 7; CamKⅡ-OE-circHomer1: n = 8. The data are expressed as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, ##P < 0.01.

Discussion

Our study demonstrated the pivotal role of circHomer1 in modulating behavioral and synaptic responses to drug abuse (Fig. 7). Here, we provide novel evidence that the level of circHomer1 is substantially decreased in the PrL following repeated cocaine administration. Furthermore, we show that circHomer1 expression levels are also reduced in the dlPFC of patients with cocaine use disorder (CUD). Using rAAV-regulating the expression of circHomer1 in vivo, we demonstrated that circHomer1 overexpression in the PrL markedly mitigated the cocaine-induced rewarding effects, while inhibiting circHomer1 expression in the PrL intensified its rewarding effects. Moreover, cocaine impairs functional synapse formation and synaptic transmission in PrL, effects that are normalized by circHomer1 elevation. These circHomer1-mediated effects on cocaine-induced behavioral and synaptic adaptations operate through D1R signaling pathways. Notably, circHomer1’s modulatory effects on behavioral response show specificity for psychostimulants (cocaine and methamphetamine), without influencing opioid or natural reward processing.

Fig. 7. Schematic illustration depicting the involvement of circHomer1 in the modulation of cocaine-induced behavioral and synaptic responses in the PrL.

The circHomer1 is found to be widely distributed in neurons under physiological conditions (left). However, repeated cocaine exposure has been found to significantly impact the expression of circHomer1 by activating the D1R pathway, which results in an intensification of hyper-rewarding behavior, as well as a reduction in mushroom spines and excitatory synaptic transmission in the PrL (middle). Importantly, the overexpression of circHomer1 levels suppressed the rewarding effects and the hypoactivity of excitatory neurons induced by cocaine in the PrL (right).

CircRNAs are emerging as crucial players in gene regulation, involved in numerous cellular processes, including regulating transcription of their host genes, sponging miRNAs and RNA binding proteins (RBPs), as well as influencing translation [22, 49]. The implications of circRNAs in the pathogenesis of neuropsychiatric diseases have been documented [38, 50–52], but their involvement in SUD remains poorly understood [20, 53–55]. CircHomer1, derived from exons 2-5 of the Homer1 longest transcript [29], is a neuronal-enriched circRNA that has been reported to play a role in a myriad of biological and behavioral functions, including methamphetamine-induced CPP preference [56], neuronal plasticity [23], cognitive flexibility and regulating synaptic gene expression [29]. Previous studies have found that circHomer1 is highly homologous to hsa_circ_0006916 in the human brain, with a conservation rate of 94%. It is also highly homologous to mmu_circ_0000491 in mice, with a conservation rate of 97% [23, 29, 57]. Furthermore, our study revealed a consistent reduction in circHomer1 expression in both the PrL of rats subjected to repeated cocaine administration and the dlPFC of patients with cocaine use disorder. Given that drugs of abuse induce persistent behavioral alterations, identifying molecular mediators of stable gene expression and behavioral changes is crucial. Recent evidence demonstrates that activity-dependent circHomer1 modifications persist for at least 48 h [47]. Consistent with our findings, we found that the decrease of circHomer1 expression caused by cocaine was maintained for at least 48 h. These evidences suggest its role as a key regulator of cocaine-induced long-term behavioral adaptations. Notably, our data indicated that the influence of circHomer1 extends beyond cocaine; it also modulates methamphetamine-induced hyper-reward, implying its broad relevance in the dysfunction induced by various psychostimulants.

Previous studies have found that drug-induced synaptic plasticity may underlie behavioral responses to drugs of abuse in the mPFC and the development of addiction. For example, Huang and his colleagues found that repeated cocaine administration in vivo promoted the induction of long-term potentiation and long-term depression in layer V pyramidal neurons of the mPFC [58, 59]. CircHomer1, which is highly abundant in the mammalian brain and enriched in synaptoneurosomes compared with cytoplasm [60], is significantly up-regulated in both the neuronal somata and dendrites following a homeostatic downscaling of neuronal activity [23]. This indicates its critical role in regulating synaptic function. Additionally, circHomer1 has been reported to play important roles in synaptic transmission and synaptic plasticity [29, 61], raising the possibility that circHomer1 in PrL might modulate behaviors via regulating synaptic plasticity. Recent work by Jenks and colleagues demonstrated that circHomer1 disruption impairs experience-dependent plasticity [47]. Consistent with this, we found cocaine exposure induces specific alterations in PrL dendritic spine morphology: decreased mushroom spine density with concurrent increases in stubby spines. These structural changes parallel functional deficits, as mushroom spines, with their enlarged heads and robust synaptic connectivity, are essential for stable synaptic transmission, while stubby spines show limited synaptic plasticity [62, 63]. The observed morphological changes correlate with reduced spontaneous excitatory postsynaptic current (sEPSC) frequency, indicating compromised synaptic function. This cocaine-induced suppression of dendritic spine dynamics and synaptic transmission leads to cortical hypoactivity, a key contributor to compulsive drug-taking behavior [64, 65]. Notably, PrL circHomer1 overexpression restored both structural and functional synaptic properties. The observed effects on filopodia, which serve as cellular antennae and spine precursors [66], further support circHomer1’s role in synaptic development. CircHomer1 overexpression significantly increased total spine density, particularly mushroom and filopodia spines (Fig. 4d, g), suggesting its dual role in spine formation and maturation. While circHomer1 overexpression did not reduce cocaine-induced stubby spine numbers, possibly due to enhanced spine formation and subsequent maturation, it normalized the proportional distribution of spine types disrupted by cocaine (Fig. 4e).The mechanism underlying circHomer1’s enhancement of synaptic plasticity may parallel that of Homer1a, the activity-dependent short isoform that acts as a dominant-negative regulator of constitutive Homer1b/c. Homer1a protein modulates mGluR clustering and mGluR-dependent NMDA/AMPA receptor currents through this interaction [67–69]. Similarly, circHomer1 appears to exert dominant-negative effects through direct binding to dendritic Homer1b mRNA [46]. Given our observation of cocaine-induced Homer1b/c upregulation (Supplementary Fig. 2), consistent with previous findings, circHomer1 likely regulates cortical plasticity through Homer1b/c interactions.

It is well-documented that D1R and D2R are both essential for the rewarding effects of psychoactive drugs [70, 71]. However, evidence shows that activation of D1R in the mPFC is particularly significant in the reinstatement of cocaine seeking and the development of reward-related behavior induced by cocaine, while D2R activation does not have the same effect [72, 73]. Subtypes of dopamine receptors exhibit distinct relationships with drug addiction. D1R stimulation in the mPFC is essential for cocaine addiction and is directly associated with the rewarding effects of cocaine[74, 75], but the D2R in the mPFC is not as strongly linked to the rewarding effects of abused drugs [74, 76]. Additionally, an intra-mPFC injection of a D1R antagonist, but not a D2R antagonist, before the test significantly attenuated CPP expression [77, 78]. Consistent with these findings, we observed that the administration of D1R antagonists in the PrL reduced cocaine-induced CPP, whereas this effect was not observed with D2R antagonists. Furthermore, we found that the suppression of circHomer1 expression by cocaine could be reversed by D1R antagonists. CircHomer1 overexpression reduced the cocaine CPP induced by chemicogenetic activation of D1R neurons. These evidences suggest that cocaine reduces the expression of circHomer1 in the PrL, possibly through the activation of D1R. Intriguingly, we found that the inactivation of the D2R subtype could also increase the proportion of circHomer1 expression after cocaine exposure (Fig. 5c). We speculated that beyond the influence of D1R, the D1R-D2R hetero-oligomer may also play a role in regulating circHomer1 expression. In mPFC excitatory neurons, dopamine receptor D1 and D2 subtypes have partially overlapped expressions, indicating that the stably co-expressed D1R and D2R could form a hetero-oligomeric unit [79–81]. This function of the D1R-D2R hetero-oligomer could be blocked by antagonists to either the D1R or D2R, but the signaling pathway could not be activated solely by D1R signaling or D2R signaling [80]. Given the report that D1R signaling modulates dendritic arborization and dendritic spine density in mPFC excitatory neurons [79, 82, 83], we consider that cocaine reduces the density of mature dendritic spines and the frequency of sEPSC in the PrL possibly through activation of D1R. Importantly, Recent research reported that the biogenesis of circHomer1 was the downstream of certain neurotransmitter receptors, and its expression is increased by a D2R antagonist [84]. In our study, however, we found administration of a D1R antagonist, but not D2R antagonist, in the PrL significantly influenced cocaine-induced reduction in circHomer1 expression, suggesting that D1R rather than D2R downstream signaling pathway may be involved in regulating this reduction of circHomer1 in the PrL. Cocaine is known to increase dopamine concentration in the NAc synaptic cleft by inhibiting dopamine transporters [85, 86]. While our results shown that a single dose of cocaine did not affect circHomer1 expression, and circHomer1 overexpression was able to restore synaptic plasticity changes caused by repeated cocaine exposure. This may indicate that circHomer1 does not affect the immediate reward effects of cocaine, but instead involved in long-term plasticity changes caused by repeated cocaine exposure. Moreover, by detecting the expression of circHomer1 in NAc after repeated cocaine exposure, we found that cocaine did not affect the expression of circHomer1 in this region, implying that circHomer1 may not directly involved in NAc functioning. However, we found a decrease in circHomer1 expression in the VTA at 30 min after repeated cocaine exposure, implying that circHomer1 may play a role in the VTA. Previous study reported that the VTA control the release of dopamine in the NAc to regulate reward-related behaviors [3]. Whether circHomer1 regulates dopamine concentrations in NAc through the projection from VTA to NAc or from PrL to NAc is an interesting question could be further explored in the future.

The rewarding mechanism of dopaminergic system and its receptors are fundamental to the development and maintenance of drug addiction, relapse after long-time abstinence and compulsive drug-seeking behavior [87–90]. Targeting dopaminergic system to find effective drugs to treat addiction is currently a research focal point. However, neither the agonists nor the antagonists of dopamine (DA) receptors can completely prevent the occurrence of abnormal behavior induced by substance abuse [89, 91]. In addition, some agonists of DA receptors may possess addictive properties, while completely blocking DA receptors can lead to emotional and physiological motor abnormalities [89, 91]. In the present study, we found that circHomer1 may act as a regulator in the modulation of cocaine-induced rewarding effects though D1R signaling, while not affecting natural rewarding processes. It is implied that circHomer1 may be a therapeutic strategy for combating psychostimulants’ addiction. However, we still don’t know the molecular mechanism by which circHomer1 works. Recently, Mellios and colleague identify cyclic AMP response element binding protein (CREB) as a key regulator of circHomer1 biogenesis, operating through multiple kinase pathways (ERK, PKA, MEK1/2, and Rap1) [84]. Given CREB’s established role in cocaine reward mechanisms [92, 93] and its regulation by various neurotransmitter receptors (NMDA, D2R, mGLuR5, 5-HT2A) in circHomer1 biogenesis, D1R may similarly modulate circHomer1 expression through CREB-dependent pathways. These findings suggest a model where D1R influences CREB-dependent circHomer1 biogenesis, with circHomer1 subsequently modulating cocaine-induced behavioral and synaptic adaptations through Homer1b/c-dependent mechanisms. However, the precise molecular interface between D1R signaling and CREB-mediated circHomer1 regulation requires further investigation.

In summary, our study identifies circHomer1 as a key regulator in the neuronal response to cocaine, with its levels inversely affecting cocaine’s rewarding effects and synaptic changes in the prefrontal cortex of rat model and patients with SUD. Specifically, increasing circHomer1 level mitigates psychostimulants-induced behavioral adaptations and restores synaptic integrity in excitatory neurons but not influences the rewarding effects induced by food and opioid. These insights reveal the potentiality of circHomer1 as a novel target for developing addiction treatments, given its selective modulation of psychostimulants rewards without affecting natural rewards.

Author contributions

JS, Y-XX and YC conceived and designed the study. YC, ZZ, XL, WC, SH and GZ performed the experiments. YC, ZZ, YZ, LZ, JS and Y-XX analyzed and interpreted the data. YC, JS and Y-XX wrote and revised the manuscript. All authors contributed to the article and approved the final version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author (Jie Shi and Yan-Xue Xue) upon reasonable request.

Competing interests

The author declares no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41380-025-03301-9.