Pharmacological Interventions on The Dopamine Motivation System Effectively Disturbed Cue-Induced Memory Reconsolidation Cocaine-Seeking Behavior for Rats

Drug addiction is a disorder related to dysfunction in the neural reward memory circuits, and it is characterized by compulsive drug use despite terrible negative consequences. Memory reconsolidation, during which aroused memory is easy to strengthening, weakening or updating, plays an extremely important role in drug addiction. Effectively interfering with the drug memory reconsolidation process would be key in treating drug addiction, but this intervention currently remains impossible. The dopamine motivation system has been widely recognized as an important system for reward, but whether the dopamine motivation system participates in drug memory reconsolidation is unclear. We aimed to explore the role of the dopamine motivation system during the cue-induced cocaine memory reconsolidation process by examining the effect of different pharmacological interventions on the dopamine motivation system during cue-induced cocaine self-administration-related memory reconsolidation drug-seeking behavior. Using a combined behavioral and biological method, our results showed that high concentrations of SCH 23390 and raclopride, or VTA lesions, could effectively disturb subsequent cue-induced cocaine self-administration-related memory reconsolidation drug-seeking behavior in rats. However, low concentrations of SCH 23390 and raclopride could not block this behavior. In summary, only a high dose of dopamine D 1 and D 2 receptor antagonists, or VTA lesions, could effectively disturb subsequent cue-induced cocaine self-administration-related memory reconsolidation drug-seeking behavior. These ndings indicated that pharmacological interventions in the dopamine motivation system could effectively disturb subsequent cue-induced drug memory reconsolidation. Thus, pharmacological interventions on the dopamine motivation system might have therapeutic potential for drug addiction.


Introduction
Drug addiction is a disorder related to dysfunction in the neural reward memory loop that is characterized by compulsive drug use despite terrible negative consequences (Hyman, Malenka, & Nestler, 2006; Y. . Through associative learning, drug-paired environmental stimuli could have very powerful in uence on drug-seeking behavior; therefore, drug memory reconsolidation critically underlies persistent drug-seeking behavior. Drug-related memory could be reactivated after re-exposure to drug-paired environmental stimuli (Namely, cues) or the drug itself (Everitt, Giuliano, & Belin, 2018;Gauthier et al., 2017;Torregrossa & Taylor, 2016), and a relapse in drug-seeking behavior could occur (Childress et al., 1999). Further studies will need to be powerful enough to diminish drug-seeking behavior under drugpaired environmental stimuli. Therefore, extinguishing the impact of drug-paired environmental stimuli is an extremely important goal for the valid treatment of drug addiction (Gauthier et al., 2017).
Environmental stimuli can improve learning and memory and induce neuroplasticity in the brain ( (Chen et al., 2017). DA plays important roles in many aspects of cognition, including reward, punishment, attention, learning and memory (Smythies, 2005). Dysfunction in DA and DA neurons could lead to neurological and psychiatric diseases, such as drug addiction, Parkinson's disease, schizophrenia, hyperprolactinemia and Tourette's syndrome (Belliotti et al., 1999;Zhang et al., 2007). After chronic exposure to several kinds of addictive substances, many molecular and cellular adaptive changes that occur in the DA mesocorticolimbic system may contribute to drug addiction. Neuroadaptations in the dopamine motivation system could mediate enhanced motivation towards related reinforced predictors. Cocaine inhibited the clearance of dopamine from the synaptic cleft by blocking plasma membrane monoamine transporters.
The role of dopamine (DA) in motivated behaviors and cognitive processes has been well established. The DA motivation system mainly includes DA neurons in the ventral tegmental area (VTA) and the substantia nigra (SN). On the one hand, limbic structures, including the hippocampus and nucleus accumbens, which have traditionally been associated with motivation and reinforcement learning, receive dopaminergic innervation from the VTA, which is known to be a key dopaminergic center in the brain ( In this study, to test the hypothesis that interventions targeting on the dopamine motivation system could affect drug memory reconsolidation, we employed a cue-induced cocaine self-administration paradigm to explore the effect of pharmacological interventions on rebalancing the dopamine motivation system and on cue-induced cocaine SA-related memory reconsolidation drug-seeking behavior. These results might help us to elucidate the neuropharmacological mechanism underlying the function of the dopamine motivation system during drug memory reconsolidation.

Animals
All experimental procedures in our study were consistent with the guidelines of the Committee for Animal Care and Use (No. TDLL2018-03-180, Tangdu Hospital, the Fourth Military Medical University, Xi'an, Shaanxi, China). And the experimental protocols were approved by the Committee for Animal Care and Use of Tangdu Hospital, the Fourth Military Medical University. Sprague-Dawley rats (Male; 300 ~ 350 g) were individually housed in our animal center under controlled temperature (21 ± 2°C) and humidity (40%~60%) on a 12-hour light-dark cycle (lights on at 7:00 a.m.), and they were provided with food and water ad libitum.

Drug preparation
Cocaine hydrochloride (Qinghai Pharmaceutical Co. Ltd., Xining, Qinghai, China), which was dissolved in saline to a nal concentration of 8 mg/mL, was stored at room temperature away from light. Surgical procedure Jugular vein catheterization surgery Rats were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.) and xed in the prone position. A skin opening approximately 0.8 cm in length was cut longitudinally in the right neck to fully expose the right jugular vein. A sterile special silicone catheter was inserted into the right jugular vein. Following surgery, the jugular vein catheter was rinsed daily with saline containing heparin (10 U/mL) and penicillin (200,000 IU/rat) to prevent catheter blockage and infection, respectively.

VTA lesion surgery
After anesthetization, the rats in the VTA-lesioned group were xed in a stereotaxic apparatus (68025, RWD Life Science Co., Ltd, Shenzhen, Guangdong, China) in the prone position(Y. . 6-OHDA (10 µg/µL, 0.8 µL) was intracranially infused into the VTA (according to the rat stereotaxic atlas (Paxinos and Watson, 2005): -5.20 mm anterior to bregma, 0.80 mm lateral to the midline (left side), and 8.80 mm ventral to the brain surface) using 1 µL Hamilton syringes (Plastics One) at a rate of 0.1 µL/min for every rat. The injection needle was held in place for an additional 15 min so that 6-OHDA could completely diffuse from the needle tip.
Drug self-administration apparatus The self-administration (SA) apparatus (40 cm × 40 cm × 50 cm; AniLab Co., Ltd., Ningbo, Zhejiang, China) received an input signal once the rat made a valid nose poke into the port, and then, it could produce as many output signals as we required (for example, for the pump, signal lights, nose poke light, or sound). According to the experimental requirements, we rst needed to edit an appropriate experimental method ( xed ratio, FR = 1). Speci cally, when a rat nished a valid nose poke, the system would pump one bolus of drug (T = 1.25 s, ν = 1.60 mL/min), accompanied by several changes in environmental cues such as the signal lights (green light is off, and red light is on for 20 s), valid nose poke light (T = 20 s), and sound (buzzer, T = 1.25 s). Every time a valid nose poke was completed, the system would immediately enter into a 20-s refractory period. Then, the system would enter into the next cycle (green light would remain on until the next valid nose poke).
Drug self-administration training procedure and cue-induced drug addictive memory reconsolidation procedure After 3 d of recovery following jugular vein catheterization surgery, all rats underwent a drug selfadministration training procedure on a 14-day training schedule (Fig. 1). Training lasted 2 h per day, and the number of nose pokes (valid nose poke and invalid nose poke) and the number of pumps (with drug) were recorded.
In our study, when the number of valid nose pokes increased signi cantly and reached a relatively stable level at the end of the training period (varying less than 3 times or 15% for three consecutive days), the drug SA model for rats was considered basically successful.
After the successful establishment of the SA model, the rats began the memory reconsolidation procedure. Experimental rats were again placed into the SA apparatus for 2 h. When rats nished a "valid" nose poke, the system would only provide the related environmental cues without pumping cocaine.

Tissue preparation
After deep anesthetization, rats were transcardially perfused with 200 mL of ice-cold 0.1 M sodium phosphate-buffered saline (PBS; pH 7.4), followed by 400 mL of 4% (w/v) paraformaldehyde in 0.1 M PB. The brains were removed and post xed at 4°C overnight in 4% (w/v) paraformaldehyde in 0.1 M PB. After cryoprotection with 30% (w/v) sucrose in 0.1 M PB, the brain was sliced into 30 µm-thick transverse sections using a cryostat (-20°C). The brain sections including the VTA were stored in freeze protection solution (Ethylene glycol: Glycerol: PBS = 3:3:4).

Immunohistochemical staining
The sections were rst incubated at room temperature in 0.3% Triton X-100 for 20 min, 3% hydrogen peroxide for 15 min and normal goat serum for 30 min. Then, the sections were incubated sequentially at room temperature as follows: Firstly, anti-tyrosine hydroxylase (TH) mouse Ig G (1 µg/mL, ab129991, Abcam, Cambridge, UK) for 24 h at 4°C. Secondly, a biotinylated goat antibody against mouse Ig G (SP-9002, ZSGB-BIO, Beijing, China) at room temperature for 30 min(Y. Li et al., 2016). Thirdly, avidin-conjugated horseradish peroxidase at room temperature for 30 min, and then peroxidase activity was visualized after an approximately 3-min reaction with DAB Kit (ZLI-9018, ZSGB-BIO, Beijing, China). Finally, the sections were mounted on gelatin-coated slides, dried, dehydrated in increasing concentrations of ethanol, cleared with xylene, and cover-slipped with neutral balsam(Y. . Next, digital images were captured with an ordinary optical microscope (Eclipse E100, Nikon, Nikon Instruments (Shanghai) Co., Ltd., China), modi ed (15 ~ 20% contrast enhancement) with Photoshop CS6 (Adobe Systems, San Jose, CA, USA), and saved as TIFF les(Li et al., 2020).

Statistical analysis
All results in this experiment were expressed as the means ± SEM. The data were statistically assessed via Student's two-tailed t-test and one-way ANOVA using SPSS 18.0 (SPSS Inc., USA). P values of less than 0.05 were accepted as statistically signi cant. All group data in our study are reported in the table or gure legends.

Ethics
All experimental procedures in our study were consistent with the guidelines of the Committee for Animal

Establishment of cocaine self-administration (SA) model for rats
In this experiment, all rats (n = 70) were divided into two groups: the Saline group (n = 8) and the Cocaineaddicted group (n = 62). During cocaine SA training, many rats can be removed due to a number of complications, for example, failed jugular vein catheterization surgery (e.g., leaking, blockage, serious infection, etc.). Overall, 7 of 70 rats in the Saline group (1/8) and the Cocaine-addicted group (6/62) were removed (see Table 1). 0.20 mg/kg β (n = 7) -n (1/6) α presented that n (1/6) rat/rats was/were removed for speci c reasons described in Results.
(0.02, 020, 0.50, 0.10) mg/kg β presented a certain corresponding concentration of SCH 23390/raclopride. SA: Self-administration. In addition, the number of valid nose pokes in the last three days between the Cocaine-addicted group and the Saline group was signi cantly different (t Cocaine−addicted group, Saline group =23.17, P < 0.01; Fig. 3A). Figure 3B showed several original recordings of the experimental rat's behavioral events. Figure 3C and 3D Showed the change for cues before and after valid nose poke. The above results indicated that the Cocaine-addicted group had effectively established a cocaine self-administration model.
After different pharmacological manipulations (intravenous injection, iv) in the dopamine pathway, the rats were placed in the SA apparatus for 2 h. After our study, many coronal brain sections (30 µm) in the target region in the VTA-lesioned group were processed using TH-immunohistochemical staining to con rm the location and range of VTA lesions (Fig. 4). Compare to the unlesioned side, the number of VTA dopamine neurons in the lesioned side reduced signi cantly (n lesioned = 21.67 ± 3.077, n unlesioned = 107.67 ± 9.688, t = 20.72, P < 0.001. not seen in Tables or Figures).
Step 1: We analyzed the rats' behavior during drug self-administration training procedure in every group.  Table 2 and Fig. 7).
However, compared to the number of valid nose pokes in the rst three days, there was a slight decrease in the last three days in the Saline group (t Saline group =2.977, P < 0.01; Table 2 and Fig. 5), and that in the Saline group also remained relatively stable during the last three days of the training period (F Saline group =1.028, P = 0.3777; Table 2 and Fig. 7). In addition, we analyzed the rats' behavior between every Cocaine-addicted group and the Saline group. The number of valid nose pokes in the last three days in every Cocaine-addicted group increased compared to that in the Saline group (t Control group, Saline group =31.38, P < 0.01; t VTA−lesioned group, Saline group =24.38, P < 0.01; t 0.02 mg/kg SCH23390 group, Saline group =28.62, P < 0.01; t 0.20 mg/kg SCH23390 group, Saline group =17.88, P < 0.01; t 0.50 mg/kg SCH23390 group, Saline group =18.99, P < 0.01; t 0.02 mg/kg Raclopride group, Saline group =25.58, P < 0.01; t 0.10 mg/kg Raclopride group, Saline group =23.62, P < 0.01; and Saline group =20.39, P < 0.01; Table 2 and Fig. 8). And there was no signi cant difference in the number of valid nose pokes in the last three days among every Cocaine-addicted group (F = 1.706, P = 0.1111), which indicates that every Cocaine-addicted group had effectively established a cocaine self-administration model.
Step 2: We analyzed the rats' behavior during cue-induced cocaine memory reconsolidation in every group.
At the begining, Our experimental results showed that the number of "valid" nose pokes before and after cue-induced cocaine memory reconsolidation showed a signi cant increase in the Control group compared with the Saline group (t' Control group, Saline group =20.41, P < 0.01; Fig. 9).
These data demonstrated that only a certain high concentration of dopamine D 1 and D 2 receptor antagonists, or VTA lesions, could effectively disturb subsequent cue-induced cocaine SA-related memory reconsolidation drug-seeking behavior in rats. These results indicate that pharmacological interventions for the dopamine motivation system could effectively disturb subsequent cue-induced drug memory reconsolidation.

Discussion
In our study, a close correlation between drug (cocaine) and valid nose poke in the presence of drugpaired environmental stimuli was effectively established using the classic drug self-administration model in rats, and drug memory reconsolidation could be strongly reactivated by the drug-paired environmental stimuli alone. Using a biological behavior method, we explored the role of the dopamine system in the cue-induced cocaine memory reconsolidation process. The main results in our study were as follows. First, dopamine played an important role in cue-induced cocaine SA-related memory reconsolidation. Second, pharmacological interventions on the dopamine motivation system could effectively disturb subsequent cue-induced cocaine SA-related memory reconsolidation drug-seeking behavior after reexposure to drug-paired environmental stimuli. Third, only a certain high dose of dopamine D 1 and D 2 receptor antagonists, or VTA lesions, could effectively disturb subsequent cue-induced cocaine SA-related memory reconsolidation behavior in rats. The above results strongly indicated that pharmacological interventions on the dopamine motivation system could effectively disturb subsequent cue-induced drug memory reconsolidation. Authors' contribution Yang Li, Nan Li and Liang Qu equally contributed to this work including animal surgeries, drug selfadministration training, data collection, immunohistochemical staining, manuscript draft and so on.
Xue-lian Wang was involved in study concept and design, and also provided funding.
Shun-nan Ge helped design the primary study, provided advice on the data analysis, and edited the manuscript.
Xin Wang and Ping Wang processed data and conducted literature searches.
Jian Fu, Yu-kun Chen and Jian-cai Wang provided advice on the data analysis and implemented statistical analysis. Figure 1 The experimental process.

Figure 2
The cocaine self-administration training procedure (A and D  Before valid nose poke, only green light is on (C). When the experimental rat nished a valid nose poke, the system will pump drug once (T=1.25 s, ν=1.60 mL/min), accompanied with several changes in environmental cues such as signal lights (Green light is off, and red light is on for 20 s), valid nose poke light (T=20 s), and sound (buzzer, T=1.25 s) (D). SA: Self-administration. Data were presented as means ± SEM, **P<0.01.

Figure 4
The location of VTA lesion. The gure "A" and "B" were coronal schematic diagrams in the VTA level (Bregma -5.20 mm). The gure "D" was a coronal real diagram in the VTA level. The gure "C" was the coronal real diagram in the VTA level by TH-immunohistochemical staining. The two framed areas in "C" were magni ed in "E" and "F", respectively. The yellow arrows showed the injection site of 6-OHDA by a 1 μL syringe. "*" presented the tip of syringe. The Green arrows showed many dopamine neurons labelled by TH. There was almost no dopamine neuron labelled by TH on the left side, although a lot of dopamine neurons labelled by TH existed in the right side. 6-OHDA: 6-hydroxydopamine; TH: tyrosine hydroxylase; VTA: ventral tegmental area; MT: medial terminal nucleus of the accessory optic tract; fr: fasciculus retro exus. Scale bar could be seen in the corresponding gures, respectively.

Figure 5
The cocaine self-administration training procedure in every subgroup. The number of valid nose poke in every Cocaine-addicted group (Control group, VTA-lesioned group, 0.02 mg/kg SCH 23390 group, 0.20 mg/kg SCH 23390 group, 0.50 mg/kg SCH 23390 group, 0.02 mg/kg Raclopride group, 0.10 mg/kg Raclopride group and 0.20 mg/kg Raclopride group) increased signi cantly during cocaine SA training period and kept relatively stable during the last three days of training period. However, there was a little decreasing for that in Saline group and the number of valid nose poke reached a relatively stable level during the last three days of training period. Data were presented as means ± SEM.

Figure 6
The number of valid nose poke for the last three days in every