Prefrontal circHomer1 regulates synaptic and behavioral adaptations induced by psychostimulants

doi:10.21203/rs.3.rs-4338599/v1

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Prefrontal circHomer1 regulates synaptic and behavioral adaptations induced by psychostimulants

https://doi.org/10.21203/rs.3.rs-4338599/v1

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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 contributory function 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. The modulatory actions of circHomer1 on cocaine-induced behavioral and synaptic responses were mediated by the dopamine receptor D1. Intriguingly, the effects of circHomer1 were selective to psychostimulant drugs, with no influence on food or opioid reward. Our findings highlight the significant role of circHomer1 in regulating psychostimulants reward and identify a novel molecular regulator of the actions of psychostimulants on the brain’s reward circuitry, providing a new strategy for treating drug addiction.

drug addiction

circular RNA

circHomer1

prelimbic cortex

rewarding effects

synaptic plasticity

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 [24–27]. CircHomer1, derived from Homer protein homolog 1 (Homer1), is a neuronal-enriched circRNA abundantly expressed in the frontal cortex [28]. CircHomer1 knockdown in the mouse orbitofrontal cortex leads to specific deficits in cognitive flexibility [28]. And its inhibition appears to ameliorate methamphetamine-induced neuronal injury through inhibiting Bbc3 expression in HT-22 cells [29]. 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 [28–30]. 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 expression 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 is a key modulator for the cocaine responses.

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.

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)

EGFP-expressing rAAV9 with CMV promoter was used to overexpress circHomer1 (OE-circHomer1), while a corresponding inhibitor shRNA was used to knockdown circHomer1 (sh-circHomer1). Additionally, EGFP-expressing rAAV9 with CamKⅡ promoter was used to overexpress circHomer1 specifically in excitatory neurons (CamKⅡ-OE-circHomer1). The control vectors used were CMV-GFP, CamKⅡ-GFP, and sh-control. All vectors were provided by 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 prelimbic cortex (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 [31]. 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.) [31–33]. After each injection, the rats were placed in the corresponding conditioning chambers and then returned to their home cage 45 minutes 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 [34].

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; 0.2 µl, 16° angle. SCH/RAC/vehicle was delivered into the PrL 30 min prior to cocaine injection. Other procedures were consistent with those described above. For CNO pre-treatment, CNO (1.5mg/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 [31]. 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 h. 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. 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 [35].

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. The procedures for Basescope in situ hybridization were based on previous studies with minor modifications [36, 37]. 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 (Advanced Cell Diagnostics) for 2 h at 40°C in the HybEZ™ 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 with a confocal microscope (Sted) using a 40× oil-immersion 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 [37, 38]. The sections hybridized with the probe were incubated with the primary antibody in PBST (PBS containing 0.05% Tween-20) 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), and rabbit anti-Iba1 (1:500, ab178847, 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 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 confocal microscope (Sted). 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 spine counting on 4–6 secondary or tertiary dendrites. Mushroom spines are defined as having a head diameter larger than 0.5 µm and a head-to-neck diameter ratio greater than 1:1 [39].

Ex vivo electrophysiology

The experiments were performed as previously described [40]. 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 minutes. Subsequently, they were maintained at room temperature for a minimum of an additional 30 minutes 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 circRNA

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 [41]. 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 1 [42]. 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. Normally distributed data were tested using one-way or two-way ANOVA followed by a post hoc multiple comparison test or unpaired t test for two-group comparisons. Non-normally distributed data were tested using the Kruskal-Wallis test followed by a post hoc multiple comparison test or the Mann Whitney U test for two-group comparisons. The results are expressed as mean ± SEM. P < 0.05 were considered statistically significant.

1. 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 response to cocaine, we first examined the changes in the expression of circHomer1 in various brain regions associated with addiction following repeated cocaine exposure (Fig. 1a). We found that in rats subjected to chronic cocaine exposure, the expression of circHomer1 was substantially decreased in the PrL at 30 min and 24 h after the last cocaine injection (Fig. 1b). The expression level of circHomer1 in CeA and VTA also significantly diminished, but only at 30 min after the last cocaine injection, not at 24h (Fig. 1b). While circHomer1 in BLA and NAc core remain unchanged at both timepoint post-injection (Fig. 1b). These findings indicated that circHomer1 was not specific to the PrL, but was substantially changed in the PrL when rats were repeatedly exposed to cocaine. Additionally, the downregulated expression of circHomer1 in the PrL was confirmed by BaseScope in situ hybridization (Fig. 1c). Crucially, the expression of circHomer1 was significantly decreased in the dorsolateral prefrontal cortex (dlPFC) of patients with cocaine use disorder (Fig. 1d), which mirrors our findings in rat model following chronic cocaine exposure, suggesting that circHomer1 may serve as a pivotal regulatory element in cocaine use disorder across species.

To determine the relative Homer1 mRNA changes after chronic cocaine exposure, we measured the expression of total Homer1 mRNA in linear RNA-enriched cDNA samples with specific primers designed to avoid circRNA detection, we found the expression of total Homer1 mRNA was no changes neither in the PrL at 30 min or 24 h after the last cocaine injection (Supplementary Fig. 2). We thus conclude that circHomer1, but not linear Homer1 mRNA, is notably reduced in the PrL after chronic 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. The expression of rAAV-mediated GFP was restricted to the PrL (Fig. 1e, f). The expression of circHomer1 was markedly increased in the PrL of rats in CMV-OE-circHomer1 group (Fig. 1g), 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. 1h). 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. 1h). This 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. 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. 1i), indicating the effectiveness of shRNA-mediated suppression. The rAAV microinjection did not change 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. 1j). However, the preference for the cocaine-paired compartment was developed in the sh-circHomer1 group rats (Fig. 1j). 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.

2. 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. As shown in Supplementary Fig. 4a and 4b, circHomer1 predominantly co-localized with NeuN, a neuronal marker, but not with glial fibrillary acidic protein (GFAP, an astrocyte marker) and Iba-1 (a microglia marker) in the PrL, indicating that circHomer1 is primarily located in PrL neurons. 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-2e), a marker of excitatory neurons, but not GAD67 (a marker of inhibitory neurons) (Fig. 2f-2h) in the PrL. This results suggest that circHomer1 may modulate the rewarding effects of cocaine by acting on excitatory neurons in the PrL region.

3. CircHomer1 in PrL excitatory neurons mediated cocaine-induced rewarding effects

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. 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. 3a-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 significantly lower cocaine-induced CPP score compared to CamKⅡ-GFP rats (Fig. 3e).

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 rewarding effects, highlighting its significance in the development of cocaine addiction.

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

Previous studies have indicated that in vivo knockdown of circHomer1 results in significant changes in the abundance of alternative isoforms, which are associated with synaptic plasticity [28]. In an attempt to better understand whether circHomer1 rescued cocaine-induced behavioral adaptation through regulating synaptic plasticity, we assessed both the structural and functional plasticity of PrL excitatory neurons. As shown in Fig. 4a-4d, repeated cocaine exposure significantly decreased the density of mushroom dendritic spines in the PrL but not the total dendritic spines. However, the overexpression of circHomer1 rescued this reduction induced by cocaine (Fig. 4b and 4d). This suggests that circHomer1 may play a key role in regulating the structural plasticity of excitatory neurons and enhancing the maturation of dendritic spines. Further, we explored functional plasticity using patch-clamp recording. We found that repeated cocaine exposure significantly decreased the frequency of spontaneous excitatory postsynaptic current (sEPSC) without affecting their amplitude. However, overexpression of circHomer1 reversed the decrease in sEPSC frequency (Fig. 4e-4i).

Altogether, our results indicate that repeated cocaine exposure damaged the synaptic plasticity and caused the hypoactivity of PrL neuron while circHomer1 negatively regulates the synaptic plasticity. To a certain extent, circHomer1 rectified deficits in synaptic plasticity induced by chronic cocaine exposure to prevent cocaine-induced rewarding effects.

5. Cocaine alters circHomer1 expression via activating dopamine receptor D1 (D1R)

Accumulating evidence indicates that D1R and dopamine receptor D2 (D2R) are both essential for the rewarding effects of psychoactive drugs [43]. 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). These results indicate that cocaine may reduce circHomer1 expression through activating D1R signaling in the PrL.

To discern whether D1R signaling could modulate circHomer1 associated cocaine-induced rewarding effects, we bilaterally expressed hM3Dq, a designer receptor exclusively activated by designer drugs, via D1R promoter-driven rAAV infection, coupled with CamKⅡ-OE-circHomer1 or CamKⅡ-GFP in the PrL (Fig. 5d and 5e). The clozapine N-oxide (CNO, an hM3Dq agonist) was applied to increase PrL D1R neuronal activity via intraperitoneal injection. Activation of PrL D1R neurons with CNO injection in rats subjected to cocaine CPP training intensified cocaine CPP compared with vehicle injection (Fig. 5f). Additionally, circHomer1 overexpression reversed the increase of cocaine CPP induced by CNO-activated PrL D1R neurons (Fig. 5f).

Collectively, these data indicate that D1R signaling directly or indirectly downregulated the expression of circHomer1 in the PrL after cocaine exposure. Overexpressed circHomer1 disrupts the cocaine rewarding effects induced by activating the PrL D1 neuron. In a way to show, circHomer1 is a key downstream of D1R signaling pathway in cocaine-induced rewarding effects.

6. 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.

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 circHomer1 is 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 diminishing circHomer1 expression in the PrL intensified its rewarding effects. Moreover, we found cocaine caused the hypoactivity in the PrL by decreasing the maturation of mushroom dendritic spines and the frequency of sEPSC, and elevated circHomer1 level rescued the activity of PrL excitatory neuron. The regulatory effects of circHomer1 on cocaine-driven behavioral and synaptic alterations were facilitated through the D1R signaling. Notably, the influence of circHomer1 appeared to be selective to psychostimulants, without affecting rewards associated with food or opioids.

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, 44]. The implications of circRNAs in the pathogenesis of neuropsychiatric diseases have been documented [37, 45–47], but their involvement in SUD remains poorly understood [20, 48–50]. CircHomer1, derived from exons 2–5 of the Homer1 longest transcript [28], 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 [51], neuronal plasticity [23], cognitive flexibility and regulating synaptic gene expression [28]. 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, 28, 52]. 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. These evidences suggest that circHomer1 may be a key regulator in cocaine addiction, offering potential avenues for clinical applications. Notably, our data imply that the influence of circHomer1 extends beyond cocaine; it also modulates methamphetamine-induced hyper-reward, indicating its broad relevance in the dysfunction induced by various psychostimulants. Future studies are needed to investigate circHomer1-psychostimulants interactions and identify the potential protein partners of circHomer1 to better understand its gene-targeting mechanisms.

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 [53, 54]. CircHomer1, which is highly abundant in the mammalian brain and enriched in synaptoneurosomes compared with cytoplasm [55], 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 [28, 56], raising the possibility that circHomer1 in PrL might modulate behaviors via regulating synaptic plasticity. This study showed that the densities of mushroom dendritic spines were significantly reduced after cocaine treatment in the PrL of rats, suggesting an attenuated synaptic transmission in the PrL by cocaine treatment. Most importantly, local overexpression of PrL circHomer1 in vivo restored the decreased densities of mushroom dendritic spines. Moreover, overexpressed circHomer1 in PrL excitatory neurons rescued the decreased frequency of sEPSC induced by repeated cocaine exposure. Cocaine administration is known to decrease the neuronal activity in the mPFC, suggesting that cocaine-induced hypofunction of the mPFC may be critically involved in drug-taking behavior [57, 58]. Consistent with previous findings, our research indicates that cocaine exposure acts as a catalyst for inducing hypofrontality of PrL (a subregion of mPFC), characterized by a reduced number of mature dendritic spines and decreased frequency of sEPSC. Crucially, restoring circHomer1 levels revives the cocaine-induced hypofunction in the PrL.

It is well-documented that D1R and D2R are both essential for the rewarding effects of psychoactive drugs [59, 60]. 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 [61, 62]. 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[63, 64], but the D2R in the mPFC is not as strongly linked to the rewarding effects of abused drugs [63, 65]. Additionally, an intra-mPFC injection of a D1R antagonist, but not a D2R antagonist, before the posttest significantly attenuated CPP expression [66, 67]. 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 [68–70]. 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 [69]. Given the report that D1R signaling modulates dendritic arborization and dendritic spine density in mPFC excitatory neurons [68, 71, 72], we consider that cocaine reduces the density of mature dendritic spines and the frequency of sEPSC in the PrL possibly through activation of D1R. Furthermore, we propose that the D1R downstream signaling pathway may be involved in regulating this reduction of circHomer1.

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 [73–76]. 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 [75, 77]. In addition, some agonists of DA receptors may possess addictive properties, while completely blocking DA receptors can lead to emotional and physiological motor abnormalities [75, 77]. In the present study, we found that circHomer1 may act as a regulator in the modulation of cocaine- and methamphetamine-induced rewarding effects and synaptic plasticity though D1R signaling, while not affecting natural rewarding processes. It is implied that circHomer1 may be a therapeutic strategy for combating psychostimulants’ addiction. Recent studies have shown that circHomer1 directly bonded to Homer1b mRNA (A transcript of Homer1) and inhibited each other’s synaptic localization [26]. The constitutively expressed Homer1b/c maintains dendritic spine structure and synaptic transmission [78]. It is tempting to hypothesize that circHomer1 competes with Homer1b regulating cocaine-induced behavior and synaptic adaptation. But, our current study showed that chronic cocaine treatment did not change the total Homer1 mRNA expression. Future work is needed to elucidate the exact molecular mechanisms that could underlie circHomer1-mediated effects in cocaine-induced rewarding and synaptic plasticity.

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.

Acknowledgments

This work was supported by several grants from STI2030-Major Projects (2021ZD0202100 and 2021ZD0200800), as well as the National Natural Science Foundation of China (82130040, 82288101, and 32161143022).

Conflict of interest

The author declares no competing interests.

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.

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Prefrontal circHomer1 regulates synaptic and behavioral adaptations induced by psychostimulants

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Abstract

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Introduction

Material and methods

Animals

Drug treatment

Recombinant adeno-associated virus (rAAV)

Stereotaxic microinjection

Conditioned place preference (CPP)

Cocaine self-administration procedures

Cocaine induced reinstatement test

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

Basescope in situ hybridization

Immunofluorescence

Dendritic Spine Counting

Ex vivo electrophysiology

Data collection and analysis of human brain circRNA

Statistical Analysis

Results

Discussion

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References

Additional Declarations

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