Neuronal activity and the expression of hypothalamic oxytocin and vasopressin in social versus cocaine conditioning

Accepted Manuscript Title: Neuronal activity and the expression of hypothalamic oxytocin and vasopressin in social versus cocaine conditioning Author: Chaobao Liu Jianli Wang Bo Zhan Guangchao Cheng PII: DOI: Reference:

S0166-4328(16)30276-5 http://dx.doi.org/doi:10.1016/j.bbr.2016.05.010 BBR 10189

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

30-1-2016 1-5-2016 3-5-2016

Please cite this article as: Liu Chaobao, Wang Jianli, Zhan Bo, Cheng Guangchao.Neuronal activity and the expression of hypothalamic oxytocin and vasopressin in social versus cocaine conditioning.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2016.05.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research Highlights

Highlights 1. ICR mice produced CPP when conditioned with unfamiliar conspecific or cocaine alone 2. Subject mice reduced preference for conspecific in conspecific versus cocaine conditioning 3. The expression of c-Fos-IR neurons in conspecific or cocaine conditioning alone differs from that in conspecifics versus cocaine conditioning. 4. The subject mice undergoing different conditioning showed differential expression of OT and AVP in the PVN and SON．

*Manuscript

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Neuronal activity and the expression of hypothalamic oxytocin and vasopressin in social versus cocaine conditioning Chaobao Liu, Jianli Wang, Bo Zhan, Guangchao Cheng College of Biological Sciences and Engineering, Beifang University of Nationalities, Yinchuan, Ningxia 750021, China

Correspondence should be addressed to: Jianli Wang, Tel: +86 951-2067893; Fax: +86 951-2067875; E-mail: [email protected]

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Abstract: Although drug rewards and natural rewards share neural substrates, the neuronal activation patterns and mechanisms behind the interaction between cocaine and social reward are poorly understood. Here, we investigate the conditioned place preference (CPP) in social (conspecific) vs cocaine conditioning, and the expression of central c-Fos, oxytocin (OT) and vasopressin (AVP) in the conditioned ICR mice. We found that the mice produced CPP when conditioned with unfamiliar conspecific or cocaine alone. However, the mice failed to produce CPP when the two stimuli were concurrently conditioned. Compared to conditioning with conspecific alone, mice decreased preference for conspecific when conditioning with social vs cocaine. We observed differential expression of c-Fos-immunoreactive neurons in the ventral anterior cingulate cortex, posterior cingulate cortex, accumbens (shell and core), medial nucleus of the amygdale and the ventral pallidum when comparing the control (CK), social (SC) or cocaine conditioning (CC) group, and social vs cocaine conditioning (SCC) group. Compared to the CK group, the SC or CC group had higher OT expression in the paraventricular nucleus (PVN) and lower AVP expression in the PVN and supraoptic nucleus. The SCC group showed lower OT expression compared to the SC group, and higher OT and AVP expression in the PVN compared to the CC group. These results indicate that cocaine impairs social preference through competing with social reward. The differential activations of neurons within specific reward areas, and differential expression of OT and AVP are likely to play an important role in mediating the interaction between social and cocaine reward. Keywords: Cocaine; Neuropeptide; Conditioned place preference; Reward

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1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

A great deal of research has showed negative consequences of drug abuse on social behavior, such as social investigation, contact behavior, aggression and social bonding [1-3]. On the other hand, it is also known that dyadic social interaction (DSI) is a substantial natural reward. Mice with high levels of sociability spend more time in a compartment containing an unfamiliar mouse compared to an empty compartment or a compartment containing a familiar conspecific [4]. Social interaction influences the effective valence of drug abuse. For example, social interaction with a conspecific can influence the general responsiveness and sensitivity to alcohol, and prevent reinstatement of cocaine-induced conditioned place preference (CPP) [5-8]; the exposure to different peers can alter the abuse potential of opioids [9]. These findings highlight a growing interest in the synergistic interactions between social and drug rewards. Therefore, investigating how the two rewards interact would provide insights for treatment of drug addiction. CPP is a widely used paradigm in studying the behavioral and neural processes involved in drugs and social rewards [10-12]. Social reward-CPP is established via Pavlovian association between the environment and rewarding effects of social interaction, similar to that of drug-CPP [8, 11, 13]. A wealth of data has demonstrated that social reward arising from social interaction modulates drug-CPP [6-8, 14]. An ongoing assumption in the field is that the abuse of drugs activates neural pathways underlying natural rewards. Both social and drug rewards converge on the mesolimbic pathway and activate common mechanism of neural plasticity [15, 16]. Prast et al have demonstrated in rats that the neurons in accumbens corridor medial of the 3

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anterior commissure are differentially mediated by cocaine reward vs DSI reward; the time spent in the cocaine associated compartment was strongly correlated with the degree of activation, i.e., expression of the immediate early genes (IEGs) c-Fos and Early Growth Related Protein 1 (EGR1) in the accumbens corridor [17, 18]. However, it is not well known about the extent to which there is overlap or differences in the other neurocircuitry and mechanisms that process social reward versus cocaine reward. Oxytocin (OT) and arginine vasopressin (AVP) are two well-known neuropeptides produced by the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). OT and AVP can facilitate close social attachment and enhance the reward value by co-activating the dopaminergic circuits that are involved in motivation and reward [19-21]. Previous studies demonstrated that OT may play a role in opposite-sex mate or same-sex partner preference formation [22, 23]. In addition, OT and AVP are correlated with cocaine abuse. OT is involved in modulating acute and long-term drug effects and inhibiting addiction-relevant behaviors，such as cocaine-induced exploratory activity, locomotor hyperactivity, and stereotyped behavior [24, 25]. AVP was previously implicated in acquisition of cocaine seeking behavior [26-28] and cocaine induced locomotion [27]. Thus, OT and AVP is an important neuroendocrine factor regulating social facilitation and drug inhibition and it is possible that they are involved in the interaction between social reward and drugs reward. c-Fos is a non-specific marker of neuronal activity and there is a correlation between the cocaine CPP and the density of c-Fos-immunopositive

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neurons in accumbens corridor medial to the anterior commissure [18]. To identify neuronal activation patterns and the role of OT and AVP in the interaction between social and cocaine reward, we investigated the expression of central c-Fos, OT and AVP in social versus cocaine conditioning. 2. Methods 2.1. Subjects Male ICR mice (20-22g) were purchased from Ningxia Medical University Laboratory Animal Center (Yinchuan, China). The animals were housed in groups of four in standard transparent polycarbonate (Makrolon) cages (32  21.5 17 cm). The colony room was illuminated with a 12:12 light-dark cycle (lights on 20: 00 h), and the temperature was maintained at 23 ± 2°C. Food and water were available ad libitum. All animals were treated humanely according to guidelines approved by the Animal Care and Use Committee of Beifang University of Nationalities. 2.2. CPP test The place preference apparatus consisted of two large compartments (34 cm× 25 cm× 32 cm, length × width × height) with different visual cues (one had gray walls and the other had white-black striped walls) separated by a small middle compartment (11 cm× 25 cm× 32 cm, length × width × height). The middle compartment served as the acclimation compartment with a door (7 cm× 9 cm, height × width) in the center of the base. Pre-test: On the day prior to conditioning, all animals were tested to determine whether there were any innate individual preferences to either of the large lateral

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compartments. The mice (n =12) were given free access to each cue-decorated compartment. The time spent in two lateral compartments was recorded for 15 minutes by a camera (Sony, HDR-XR260E) mounted 70 cm above the arena. After each trial, the compartment was thoroughly cleaned using 70% ethanol. Conditioning with unfamiliar conspecifics (social conditioning, SC): The mice showed no inherent preference for either compartment. The subjects were conditioned in an alternate half of day design. In the morning, the subjects (n =10) cohabited with an unfamiliar conspecific for 1h in one of the outer compartments; in the afternoon, they were placed alone in the opposite compartment. Subjects underwent four consecutive days of conditioning, in which they were alternately reinforced with an unfamiliar conspecific or no conspecific for 1 h in the morning or afternoon. The post-test was performed 24 h after the last conditioning trial by placing the subject in the middle (neutral) compartment of the CPP apparatus, and allowing the mouse to move freely between the three compartments. Subjects were given a 15 min post-test without the presence of conspecific. Conditioning with cocaine (cocaine conditioning, CC): Cocaine hydrochloride (Northwest Pharmaceutical, Sinopharm, Xian, China) was dissolved in sterile 0.9% physiological saline. Conditioning sessions and post-test procedure were similar to the social conditioning described above. The mice (n =10) were conditioned by receiving 20 mg/kg intraperitoneally (i.p.) injections of cocaine or saline (4ml/kg). Subjects were conditioned for 1 h per session. The morning session and the afternoon session were at least 6 h apart to allow time for cocaine clearance [10]. Thus, we provided

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four associative pairings with cocaine and saline: two stimuli per day in an alternating counterbalanced sequence for 4 days. On the fifth day, post-test was performed in a drug- and saline- free state for 15 min. Conditioning with conspecifics versus cocaine (social vs cocaine conditioning, SCC): Since the subjects established a preference for conspecific or cocaine, we compared the relative reinforcing strengths of conspecific and cocaine. An unfamiliar conspecific and a 20 mg/kg i.p. injection of cocaine were alternately presented in different compartments for 1 h. Thus, the subjects experienced opposing reinforcing stimuli to determine which is more rewarding. Conditioning sessions and post-test procedures were similar to that described above. 2.3. Tissue collection and immunochemistry We used a different subset of mice conditioning for the c-Fos, OT and AVP immunoreactive (IR) neurons test. Subject mice were assigned to one of four treatment groups: SC group (n = 6), CC group (n = 6), SCC group (n = 6), and the control (CK) group (n = 6), which received no unconditioned stimulus-cue conditioning, only being exposed to cues that served as the conditioned stimuli for the experimental group. Animals were exposed to each cue-decorated compartment for 1 h in an alternating sequence, matching the parameters for the treatment of the experimental group [29]. Fifty min after the start of CPP test, animals were deeply anesthetized and perfused with 0.1 M phosphate buffer solution (PBS, pH 7.4) and 4% paraformaldehyde in 0.1 M PBS. The brain was removed, and placed in 4%

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paraformaldehyde overnight. Prior to the dissection, brains were immersed in 30% sucrose until saturated. Coronal sections (40μm) were cut on a cryostat and consecutive sections were collected in three vials containing 0.01 M PBS for c-Fos, OT and AVP immunohistochemical staining. The following rabbit polyclonal antibodies were used to detect the proteins: c-fos (sc-52, Santa cruz); OT (AB911; Upstate, Lake Placid, USA), with less than 1% cross-reactivity with arginine AVP; AVP (AB1565; Upstate, Lake Placid, USA), with less than 1% cross-reactivity with arginine OT. Floating sections were processed using Streptavidin/Peroxidase methods (Bioss Company, Beijing, China). Each vial was incubated for 10 min with 3% H2O2, and then washed for 4 × 5 min with 0.01 M PBS. Sections were preincubated for 90 min with normal goat serum (SP-0023), and incubated at 4 °C overnight in primary antibody (c-Fos, 1:100; OT:1: 5000; AVP,1:5000) diluted in antibody diluent (0.01 M PBS containing 20% bovine serum albumin and 1.7% Trition-X-100). Following the primary antibody incubation, sections were washed 4 × 5 min with 0.01 M PBS, and then incubated for 60 min in a 37 °C water bath with biotinylated goat anti-rabbit antibody (SP-0023), followed by another round of 4 × 5 min 0.01 M PBS washes. After 60 min of incubation with S-A/HRP and 4 × 10 min washes with 0.01 M PBS, stains were visualized with 3, 30-diaminobenzidine tetrahydrochloride. In addition to the PVN and SON, we focused on the following regions of the brain that are thought to be involved in reward or motivation [6, 30-34]: cingulated cortex (CG) including ventral anterior cingulate cortex (ACv) and posterior cingulate

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cortex (PCg), nucleus accumbens (NAC) including the medial core (NACc) and lateral shell (NACs), central nucleus of the amygdala (CE), medial nucleus of the amygdala (MeA), bed nucleus of the stria terminalis (BNST), and the ventral pallidum (VP). An Olympus microscope was used to count stained nuclei. Slides were randomized and coded for microscopic analysis, so that counters were blind to experimental treatment. The number of immunoreactive neurons was quantified by eye with the aid of a reticle placed in one ocular lens. For each brain area investigated, three representative sections from anterior to posterior that were anatomically matched between subjects were chosen and counted to minimize variability. Individual means were obtained by counting positive neurons bilaterally in three sections from each nucleus. Each hemisphere was counted separately, and results were averaged between hemispheres. Sections were chosen by correspondence to the reference atlas plate, not by the level or intensity of immunoreactive neurons labeling. Chosen sections were imaged with a camera attached to a microscope. 2.5. Statistical analysis Statistical analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, USA). All data were checked for normality using a one-sample Kolmogorov–Smirnov test, and found to be normally distributed. CPP test measures were compared using paired-samples t-tests. Independent sample t-tests were performed to determine whether there were significant differences in time spent by mice in the conspecifics or cocaine

cues-associated

compartment

9

following

different

conditioning.

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Immunoreactive neurons were analyzed using one-way ANOVA. Group differences were compared using post-hoc tests. All data are presented as mean ± standard error (SE) and alpha was set at 0.05. 3. Results 3.1. CPP test Pre-test: In the pre-test, mice exhibited approximately equal time in the two lateral compartments (t (11) = -1.073, P = 0.306) (Fig. 1a), suggesting that the apparatus is an unbiased environment. Post-test: After 1 h of conditioning with unfamiliar conspecific or cocaine, mice developed a preference for compartment associated with the conspecific cues (t -5.773, P < 0.001) or cocaine cues (t

(9)

(9)

=

= -4.704, P = 0.001) (Fig.1b-c). The SCC

group did not exhibit a particular preference for compartment associated with either conspecific or cocaine (t

(7) =

0.961, P = 0.362) (Fig.1d). Compared to the SC group,

the SCC group spent less time in compartment associated with the conspecific cues (t (7)

= 3.449, P = 0.003) (Fig. 2). However, the time mice spent in compartment

associated with cocaine cues were not different between the CC group and the SCC group (t (7) = 1.961, P = 0.066) (Fig. 2). 3.2. The number of c-Fos-IR neurons Compared to the CK group, the SC group showed more c-Fos-IR neurons in the PCg (mean difference = 5.517, P = 0.001), NACc (mean difference =11.333, P < 0.001), NACs (mean difference = 2.400, P = 0.015) and MeA (mean difference = 5.217, P = 0.001). Similarly, the CC group also showed significantly more c-Fos-IR in the PCg (mean difference = 5.250, P = 0.001) and NACs (mean difference = 3.688, P 10

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= 0.001) compared to the CK group. Compared to the SC group, the SCC group had significantly more c-Fos-IR neurons in the ACv (mean difference = 4.833, P = 0.001), PCg (mean difference = 5.600, P = 0.001) and NACs (mean difference =2.667, P = 0.008). Compared to the CC group, the SCC group had significantly more c-Fos-IR neurons in the ACv (mean difference = 3.74, P = 0.006), PCg (mean difference = 5.867, P < 0.001), NACc (mean difference =7.267, P < 0.001), VP (mean difference = 2.410, P = 0.003), and fewer c-Fos-IR neurons in the NACs (mean difference = -3.955, P < 0.001). We did not find a significant difference in the number of c-Fos-IR neurons in the CE and BNST among these comparisons (P > 0.05, data not shown) (Fig. 3a-b, Fig. 4). 3.3. The number of OT- and AVP-IR neurons Compared to the CK group, the SC group displayed more OT-IR neurons (mean difference =17.600, P < 0.001) in the PVN and fewer AVP-IR neurons in the PVN (mean difference = 17.200, P = 0.005) and SON (mean difference = 9.42, P = 0.04). In comparison to the CK group, the CC group showed an increased OT-IR neurons in the PVN (mean difference = 4.831, P = 0.006) and a decreased AVP-IR neurons in the PVN (mean difference = 21.407, P < 0.001) and SON (mean difference =11.74, P = 0.013). The SCC group had fewer OT-IR neurons in the PVN (mean difference = 9.233, P < 0.001) than the SC group. In comparison to the CC group, the SCC group had significantly more OT-IR neurons (mean difference = 3.535, P = 0.035) and AVP-IR neurons (mean difference =10.640, P = 0.03) in the PVN (Figs.5-7). 4. Discussion 4.1. CPP in social vs cocaine conditioning 11

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We found that subject mice developed preference for conspecific or cocaine when the either was presented alone. Although no CPP was produced when the two stimuli were concurrently provided, the time mice spent in the conspecific associated compartment decreased when compared with conspecific conditioning alone, indicating cocaine impaired the motivation for social interaction during social vs cocaine conditioning, Social activity itself is rewarding [4, 10]. The time the animals spend in the compartments with stimulus-associated cues indicate motivation or desire for the stimulus. Novelty is a powerful reward and has to be considered an aspect of DSI [8, 35]. Here, subject mice spent more time in the compartment containing unfamiliar conspecific compared to an empty compartment, suggesting that social interaction with unfamiliar conspecifics is rewarding. Similar to the effects of cocaine on other strains of mice [36-38], ICR mice showed robust preferences for the cocaine-paired compartment when conditioned with cocaine alone. In contrast, the mice did not exhibit a particular preference for either when social vs cocaine conditioning, suggesting that social reward does influence the effects of cocaine. Similarly, exposure to a conspecific or methylphenidate alone produces CPP in adolescent rats, whereas no CPP is produced when these stimuli are combined [39]. Pairing a conspecific with one compartment and cocaine with the other compartment fails to produce CPP, even though either reward alone produces CPP [35, 40]. These findings, together with the present results, support the view that that social reward competes with drug reward, and there is an inhibitory interaction between social reward and drug [35]. The present results were inconsistent with some reports

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showing that only four 15-min episodes of inter-male interaction can completely reverse cocaine CPP, and even prevent reacquisition of cocaine CPP [7，41]. It is believed that social interaction may lose much of its attractiveness after the first 15 min experiencing DSI for 30 min so as to induce no preference for DSI in individually housed rats, whereas a 15 min DSI may induce a preference [8, 35]. Thus, one potential explanation for this discrepancy is that we extended the duration of conditioning sessions to 1h, which may have decreased the attractiveness of social interaction. In addition, the CPP for, or aversion to, social interaction is considered the sum total of the appetitive and aversive components of the handling and i.p.saline injection administered immediately before placing the animals together within the confines of the CPP apparatus minus the sum total of the appetitive and aversive aspects of the alternative stimulus (i.e. the handling and i.p. injection of saline alone or of cocaine) [8]. Here, we did not inject the mice in the conspecific condition but did so in the cocaine condition, which would likely affect the preference. Another possibility may reside in differences in the susceptibility to cocaine across species and the cocaine dose used [36, 42]. The effectiveness of alternative reinforcing stimuli may be greatest among individuals prone to addiction [35]. For example, novel stimulation decreases amphetamine self administration to a greater extent in HR rats than in LR rats [43]. 4.2. Neuronal activation in social vs cocaine conditioning There were distinct patterns of neuronal responses in subject mice undergoing different conditioning regimes. The induction of IEGs may be the initial step in the

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neurobehavioral plasticity involved in addiction [31 44, 45]. c-Fos expression was used here to detect neuronal responses to drug and social reward, as well the interaction between social and cocaine reward. There were more c-Fos -IR neurons in the PCg, NACc, NACs and MeA in mice that formed conspecific CPP, suggesting increased neuronal activity in these regions in response to social reward. These responses may lead to the synaptic plasticity processes in the brain areas associated with reward. The NAC are key components of the mesolimbic DA pathway, and are thought to be involved in the reinforcing or rewarding effects of natural stimuli and drugs of abuse [45-47]. Our results support previous findings that social interaction conditioning increases the spike frequency in the NAC [48]. MeA and other nuclei of the limbic system play important roles in regulating social contact [49-50]. Our findings show that these regions are involved in mediating motivational processing of social interaction. Both drug and natural rewards may be processed in similar brain regions [34, 48]. We observed that specific brain regions regulating reward-response exhibit increased neuronal activation when the subjects were presented with conspecific- or cocainepaired cues. Thus, the activation of these neurons may be involved in forming associations between reinforcers’ stimulant and the environment in which the conditioning occurred [51]. In contrast to the CK group, mice in the CC group showed increased c-Fos expression in the PCg and NACs. In a previous study using rats or mice, it was shown that expression of cocaine CPP is associated with increased c-Fos expression in the NACc [52] or NACs [44, 53]. Combined with the results as shown

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in the SC group, we proposed that there were some subtle differences in the neuronal activity elicited by cocaine and social conditioned response in reward areas. Cocaine paired cues activated similar, though not identical, pathways to those activated by conspecifics paired cues in conditioned response. Compared to the SC group, we observed greater activation of neurons in the ACv, PCg and NACs in the SCC group. Additionally, in comparison to the CC group, the SCC group showed significantly higher c-Fos expression in the ACv, PCg, NACc, MeA and VP, and fewer c-Fos-IR neurons in the NACs. These results indicate that the neuronal activation patterns induced by social vs cocaine conditioning are different from conspecifics or cocaine conditioning alone. Social and drug reward have reciprocal interactions that act on the same brain mechanisms, and if one of the two rewards is experienced first, it can occlude the other through neural alterations [14, 34]. Social conditioning when competing with cocaine conditioning impacted neuronal activation induced by cocaine. We observed activation in the Acv and VP neurons, suggesting that synaptic plasticity in these brain regions play a role in interaction of conspecific and cocaine reward. As a subregion of the medial prefrontal cortex, the CG (including ACv and PCg) is involved in the detection and recognition of different rewards, and the execution of unconditioned and conditioned responses. The CG integrates memory with the response, suggesting that this subregion may mediate a more general natural reward response and recognition capacity [29]. The VP is a major projection field for the NAC, and the both regions are involved in context/cue induced cocaine seeking [34, 53, 54]. The inactivation of c-Fos in the

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NACs may be due to the social rewards inhibit brain activation that motivates cocaine-seeking behavior [35]. Thus, it is possible that the differential neuronal responses in these brain areas determined the preferences and motivation when social reward vs cocaine occurred. There were nonsignificant conditional activation within the BNST and CE, leading us to believe that these brain areas were not probably involved in the conditioned response. 4.3. The expression of OT and AVP in social vs cocaine conditioning The subject mice undergoing different conditioning showed differential expression of OT and AVP. Compared to the CK group, the SC group had more OT-IR neurons in the PVN, and fewer AVP-IR neurons in the PVN and SON, indicating that OT and AVP have different conditioned responses to social reward. Previous studies indicate that OT’s primary mode of action is thought to increase affiliation during social contact and promote reward value associations with social behaviors [55-57]. This effect of OT may be associated with enhanced responses and functional connectivity in emotional memory and reward processing regions [57].We found that the formation of social CPP up-regulates hypothalamic OT expression. AVP is capable of moderating social contact [22, 58]. We observed lower PVN AVP in conspecifics conditioning group, which was surprising, because social interaction was positively correlated with AVP mRNA expression in the PVN [59]. Previous report suggests that OT is involved in augmenting the rewarding effects of social interaction, while AVP does not condition social CPP [60]. Additionally, PVN AVP often is associated with stressful circumstance or high conditioned anxiety [61-63]. It is

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possible that daily conditioning in the control group was perceived as a stressful event for the subjects. When animals are subjected to stress, AVP is released from the PVN. This is a possible reason why SC group has fewer AVP-IR neurons compared to the CK group. We speculate that the stress response of AVP to CPP paradigm may contribute to forming the preference. Consistent with CK group, CC group increased OT expression in the PVN, and reduced the AVP expression in the PVN and SON. These results support the hypothesis that there are functional differences between these two neuropeptides when it comes to cocaine addiction [26]. Some studies have shown that that cocaine may inhibit central OT production [25, 64]. However, others have found an increase in OT levels within some central region following acute and chronic cocaine exposure in rats. For instance, acute cocaine treatment increased OT contents in the hypothalamus and in the hippocampus [65]; gestational cocaine treatment resulted in significant increases in OT mRNA levels in the PVN of cocaine-treated dams [66]; acute and chronic cocaine exposure increased OT mRNA levels within the NAC in rats [67]. From these findings we learn that these effects of OT are an integral aspect of its effects on cocaine exposure. Here, OT exhibited an increased expression in response to cocaine conditioning, suggesting that different OT responses are involved in the unconditioned and conditioned effects of cocaine. Previous studies have suggested that AVP may decrease the reinforcing efficacy of cocaine during acquisition of drug self-administration [68]. Our results are in accordance to the findings that cocaine treatment decreases hypothalamic AVP levels [64, 65], and AVP

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is down regulated during early and mid acquisition of environment-elicited cocaine conditioning [26], but differ from other studies that show that chronic cocaine treatment increases AVP release within the anterior hypothalamus [69]. These finding indicates that AVP may play a specific role in regulating cocaine CPP. Compared to the SC group, SCC group reduced the expression of OT in the PVN. Brain

OT

systems

exhibit

profound

neuroplasticity

and

undergo

major

neuroadaptations as a result of drug exposure. Cocaine causes long-term changes in markers of OT function and this may be linked to enduring deficits in social behavior in laboratory animals repeatedly exposed to these drugs [24]. By reducing OT, SCC group might thus attenuate the preference of the conspecifics. Similarly, previous finding showed that on postpartum day 5, decreased OT expression in the medial preoptic area was associated with altered preference-like behavior in cocaine-exposed dams [70]. Compared to the CC group, SCC group showed an increased expression of OT and AVP in the PVN. These increased expressions are likely to be caused by the conspecifics stimuli in this condition. Taken together, these findings indicate that there are interactions between cocaine and social rewards, and mutually affected each other’s AVP and OT expression. The differential plasticity of the OT-ergic and AVP-ergic neurotransmissions is likely to affect the preferences for conspecifics or cocaine. In summary, our results provide evidence for the difference in the expression of c-Fos, OT and AVP in social and cocaine reward, as well as the interaction between

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social and cocaine rewards. Cocaine can reduce social seeking behaviors, and this reduction in social motivation appears to be associated with the differential activation of specific brain regions implicated in reward including CG, NAC, MeA, and VP. In addition, the synergistic reaction of OT and AVP might play an important role in mediating the formation of cocaine or social preference. The differential responses of OT and AVP may help improving our understanding of how social reward can protect against drug abuse. Further neuroendocrinological analyses will be useful for understanding the fundamental importance of social reward in reducing drug dependence.

Acknowledgments This research was supported by the National Natural Science Foundation of China (Grants 31260513 and 31460565).

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Figure legends 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Fig.1. Time spent by mice in the apparatus compartments (a) during the pretest, (b) following social conditioning, (c) following cocaine conditioning, (d) following social vs cocaine conditioning. Values are mean ± SE, and * indicates significant differences (P < 0.05).

Fig.2.The change in time mice spent in the conspecific cues- or cocaine cues-associated compartment following different conditioning. Values are mean ± SE, and * indicates significant differences (P < 0.05). SC, social conditioning; CC, cocaine conditioning; SCC, social vs cocaine conditioning.

Fig.3. The differences in the number of c-Fos-IR neurons in the different conditioning regimes. Values are mean ± SE. Groups not sharing same letters are significantly different (P < 0.05). (a) Acv, ventral anterior cingulate cortex; PCg, posterior cingulate cortex; NACc, accumbens core; NACs, accumbens shell. (b) CE, central nucleus of the amygdale; MeA, medial nucleus of the amygdala; BNST, bed nucleus of the stria terminalis; VP, the ventral pallidum. CK, control, SC, social conditioning; CC, cocaine conditioning; SCC, social vs cocaine conditioning.

Fig.4. c-Fos-immunopositive staining in the different conditioning treatments. Bar = 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

200 um. CK group (a, e, i ); SC group (b, f, j); CC group (c, g, k); SCC group (d, h, l); CK and SC groups (m, n); CC and SCC groups (o, p). PVN, paraventricular nucleus; SON, supraoptic nucleus; ACv, ventral anterior cingulate cortex; PCg, posterior cingulate cortex; NACc, accumbens core; NACs, accumbens shell; MeA, medial nucleus of the amygdala; VP, the ventral pallidum;

Fig.5. The number of OT-IR neurons in the PVN and SON in the different conditioning regimes. Values are mean ± SE. Groups not sharing same letters are sigfinicantly different. PVN, paraventricular nucleus, SON, supraoptic nucleus. CK, control; SC, social conditioning; CC, cocaine conditioning; SCC, social vs cocaine conditioning.

Fig.6. The number of AVP-IR neurons in the PVN and SON in the CK, SC, CC and SCC groups. Values are mean ± SE. Groups not sharing same letters are significantly different (P < 0.05).

Fig.7 Immunopositive staining against (a - d) OT and (e - l) AVP in the CK, SC, CC and SCC groups. CK group (a, e, i); SC group (b, f, j); CC group (c, g, k); SCC group (d, h, l). Scale bar = 200 um. PVN, paraventricular nucleus, SON, supraoptic nucleus.

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Figure1

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Figure7