Upregulations of α1 adrenergic receptors and noradrenaline synthases in the medial prefrontal cortex are associated with emotional and cognitive dysregulation induced by post-weaning social isolation in male rats

Early-life social isolation induces emotional and cognitive dysregulation, such as increased aggression and anxiety, and decreases neuron excitability in the medial prefrontal cortex (mPFC). The noradrenergic system in the mPFC regulates emotion and cognitive function via α1 or α2A adrenergic receptors, depending on noradrenaline levels. However, social isolation-induced changes in the mPFC noradrenergic system have not been reported. Here, male Wistar rats received post-weaning social isolation for nine consecutive weeks and were administered behavioral tests (novel object recognition, elevated plus maze, aggression, and forced swimming, sequentially). Protein expression levels in the mPFC noradrenergic system (α1 and α2A adrenergic receptors, tyrosine hydroxylase, and dopamine-β-hydroxylase used as indices of noradrenaline synthesis and release) were examined through western blotting. Social isolation caused cognitive dysfunction, anxiety-like behavior, and aggression, but not behavioral despair. Socially-isolated rats exhibited increased protein levels of the α1 adrenergic receptor, tyrosine hydroxylase, and dopamine-β-hydroxylase in the mPFC; there was no significant difference between the groups in the α2A adrenergic receptor expression levels. Preferential activation of the α1 adrenergic receptor caused by high noradrenaline concentration in the mPFC may be involved in social isolation-induced emotional and cognitive regulation impairments. Targeting the α1 adrenergic receptor signaling pathway is a potential therapeutic strategy for psychiatric disorders with symptomatic features such as emotional and cognitive dysregulation.


Introduction
Individuals exposed to early-life social isolation, a form of neglect, exhibit increased aggression in adulthood [1,2], which has a strong positive correlation with the development of mental illnesses such as depression and anxiety disorders [3,4]. Much evidence indicates that rodents exposed to isolated rearing for several weeks after weaning exhibit abnormal aggressive behaviors concomitant with anxiety-like behavior and cognitive dysfunction [5][6][7]. Therefore, aberrant earlylife social experiences have deleterious effects on brain regions associated with emotional regulation and cognition. Although the exact molecular mechanism is unknown, the medial prefrontal cortex (mPFC) is involved in social isolation-induced emotional and cognitive dysregulation in early life.
The mPFC regulates emotional and cognitive functions [8][9][10]. Emotional and cognitive dysregulation in patients with psychiatric disorders is attributed to impairment of the mPFC [11][12][13]. Social isolation after weaning in mice impacts mPFC neural activity, by decreasing the excitability of mPFC neurons [14]. Furthermore, mice socially-isolated in early life exhibited increased protein levels of ΔFosB in the mPFC, associated with a decrease in the mPFC neural function [15,16]. These observations suggest that early-life social isolation suppresses mPFC activity, potentially leading to emotional and cognitive dysfunction. However, the neurotransmitters affecting the pathogenesis of mPFC Abbreviations: AT, aggression test; DBH, dopamine-β-hydroxylase; EPMT, elevated plus maze test; FST, forced swimming test; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LC, locus coeruleus; mPFC, medial prefrontal cortex; NORT, novel object recognition test; SEM, standard error of the mean; TH, tyrosine hydroxylase. neural dysfunction induced by early-life social isolation are unknown.
Noradrenaline, an essential neurotransmitter in the brain, modulates stress responses [17]. Activation of the brain noradrenergic system during acute stress plays an important role in coping with and adapting to stress [18], while prolonged activation causes maladaptive responses, including the development of psychiatric disorders [17]. Noradrenaline is synthesized by tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) in the locus coeruleus (LC), the primary source of noradrenaline in the brain [19,20]. Noradrenergic neurons in the LC project to the mPFC [21], in which α 1 and α 2A adrenergic receptors regulate neural activity depending on noradrenaline levels. Because noradrenaline has a higher affinity for α 2A than α 1 adrenergic receptors, α 2A adrenergic receptors engage at basal levels of noradrenaline (i.e., non-stress conditions) and strengthen the mPFC neural function.
Noradrenaline preferentially binds to α 1 adrenergic receptors when there are high levels of noradrenaline in the synaptic cleft during stress, resulting in the mPFC functional impairment [22]. Interestingly, socially-isolated rats exhibit increased noradrenaline concentration in the brain [23].
This suggests that changes in the mPFC noradrenergic system are involved in social isolation-induced emotional and cognitive dysfunctions. However, no studies published to date have focused on social isolation-induced changes in the mPFC noradrenergic system. Therefore, we assessed the protein levels of α 1 and α 2A adrenergic receptors, TH, DBH, and ΔFosB in the mPFC of socially-isolated rats to determine whether social isolation affects the mPFC noradrenergic system.

Animals
In this study, 18 male Wistar rats (age at arrival, 3 weeks; weight, 30-40 g; CLEA Japan Inc., Tokyo, Japan) were housed under the following conditions: light-dark cycle, 12:12 h light turned on at 7:00 a. m., temperature, 23 ± 2 • C, and humidity, 60 ± 2%. Food and water was provided ad libitum. For an aggression test (AT), 18 male Wistar rats (age, 9 weeks; CLEA Japan Inc.) were purchased and kept in cages, with three rats in each cage (plastic cages, 30 × 35 × 17 cm). All animal experiments were performed in accordance with regulations established by the Experimental Animal Care and Use Committee of Fukuoka University, which follow the universal principles of laboratory animal care (Nov. 21, 2018; approval number: 1811082).

Experimental design
Initially, the animals were randomly housed in groups of three (plastic cages, 50 × 40 × 20 cm) or isolated socially (plastic cages, 23 × 14 × 12 cm) for 9 weeks. Additionally, opaque blackboards were placed between the cages of the isolated rats to prevent visual contact between them. After rearing, to facilitate the development of territoriality for the AT, all rats were individually housed in plastic cages (30 × 35 × 17 cm).
The rats were administered a battery of behavioral tests sequentially: the novel object recognition test (NORT), elevated plus maze test (EPMT), AT, and forced swimming test (FST). On the day after the FST, the mPFC was quickly collected on an ice-cold plate after decapitation under anesthesia. Protein levels of α 1 and α 2A adrenergic receptors, TH, DBH, and ΔFosB in the mPFC were analyzed. The experimental design is illustrated in Fig 1

Behavioral testing
All behavioral tests were conducted between 9:00 a.m. and 11:30 a. m.; the rats were acclimated to the testing room for at least 60 min prior to testing. The NORT was performed under white-light conditions. Other behavioral tests were performed under red-light conditions. Analyses of all behavioral tests were conducted blindly.

Novel object recognition test (NORT)
Long-term memory was assessed using the NORT [24]. The NORT was conducted as previously described, with minor modifications [25,26]. Briefly, the rats were individually placed in a gray acrylic open field arena (82 × 82 × 45 cm) for 10 min without object presentations. The next day, each animal was allowed to explore two identical objects for 10 min in the same arena. After 24 h, the animals returned to the arena for 5 min for a test trial with one of the objects replaced with a new one. The new ones differed from familiar objects in terms of size, shape, texture, and color. The arena and objects were cleaned with 50% isopropyl alcohol between trials to avoid odor cues. All test sessions were videotaped and the time the animals spent exploring each object (defined as sniffing or touching the object) was measured. The recognition index, used to measure long-term memory performance, was calculated as the time taken by the animals to explore the novel object divided by the total exploration time (novel plus familiar objects).

Elevated plus maze test (EPMT)
To examine anxiety-like behavior, the EPMT was performed as previously described with minor modifications [27,28]. Briefly, the behavioral apparatus consisted of two closed arms (12 × 51 cm) with 40cm-high walls and two open arms of the same size with 1-cm-high walls. The platform height was 40 cm above the room floor, and all four arms were connected to a square zone (12 × 12 cm) with no walls. Animals were individually placed in the central area facing one open arm and were allowed to explore the maze for 5 min. Each trial was video recorded, and the time spent in the closed and open arms was measured as an index of anxiety-like behavior. The maze was then cleaned after each test session.

Aggression test (AT)
Aggression was assessed using the AT [29], conducted as previously described, with minor modifications [5]. Group-housed or sociallyisolated rats were used as resident rats, and 9-week-old male unfamiliar conspecific animals were used as intruder rats. Briefly, an intruder rat Fig. 1. Experimental design. Animals housed in groups of three or individually for nine weeks, followed by individual housing to develop territoriality; behavioral tests were conducted. The animals were decapitated 24 h after the FST to evaluate protein levels of α 1 and α 2A adrenergic receptors, TH, DBH, and ΔFosB in the medial prefrontal cortex. AT, aggression test; EPMT, elevated plus maze test; FST; forced swimming test; NORT, novel object recognition test was introduced into the resident rats' home cage for 15 min and then removed following the test session. The test session was video recorded and analyzed. The resident rats' aggression was measured by determining the latency of the first attack (attack latency) and the total attack counts. If a resident rat did not attack, the latency of the first attack was recorded as 900 s (test duration), and the total attack counts were recorded as zero.

Forced swimming test (FST)
The FST was conducted as previously described [30]. Briefly, each rat was forced to swim in a Plexiglas cylinder (50 cm height and 20 cm diameter) filled with water (27 ± 1 • C; 30 cm depth) for 5 min. The test session was conducted 24 h after a 15-min pre-test session. The test sessions were videotaped and analyzed. Each rat was manually evaluated at 5-s intervals, and the predominant behavior was classified as immobility, swimming, or climbing. The frequency of immobility was used as an index of behavioral despair. After each test, the water was changed and the cylinder was rinsed with clean water.

Western blotting analysis
The preparation of the mPFC samples and the protein levels analysis through western blotting were conducted as previously described [30]. Proteins were separated using SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking, the following primary antibodies were used overnight at 4 • C: rabbit polyclonal anti-  Effects of post-weaning social isolation for nine weeks on long-term memory performance, anxiety, aggression, and depression-like behaviors. (a) Novel object recognition test: social isolation significantly decreased the recognition index. (b) Elevated plus maze test: social isolation significantly increased the closed arm time. Aggression test: (c) socially-isolated rats exhibited significantly shorter attack latency and (d) significantly higher total attack counts. (e) Forced swimming test: socially-isolated rats displayed significantly higher climbing frequency and significantly lower immobility. Bars represent mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001 vs. group-housed rats.

Statistical analysis
Data were analyzed using StatView software Ver.5 (HULINKS, Tokyo, Japan) and R software 4.0.1 (Free Software Foundation, Vienna, Austria; R Development Core Team, 2009). The data of each group were normally distributed (Shapiro-Wilk test), except for the attack latency and total attack counts in the AT. Data with a normal distribution exhibited homogeneity of variance (Levene's test), excluding the open arm time in the EPMT and the α 1 adrenergic receptor protein levels in the mPFC. For statistical analysis between the group-housed and isolation-reared rats, an unpaired student's t-test was used to assess the results (e.g., the recognition index in the NORT and the protein expression levels of the DBH in the mPFC). Welch's t-test was performed when the open arm time in the EPMT and α 1 adrenergic receptor protein levels in the mPFC were heterogeneous. The Mann-Whitney U test was used to compare the attack latency and the total attack counts in the AT and ΔFosB protein levels in the mPFC between the groups. All data are presented as the mean ± standard error of the mean (S.E.M). Statistical significance was set at P < 0.05.

Discussion
This study examined the effect of early-life social isolation on the noradrenergic system in the mPFC, which is associated with emotional and cognitive regulation and presents two major findings. First, social isolation rearing induced cognitive dysfunction, anxiety-like behavior, and abnormal aggression. Second, socially-isolated rats exhibited increased protein expression levels of α 1 adrenergic receptors in the mPFC.
Here, social isolation after weaning decreased cognitive function and increased anxiety-like and aggressive behaviors, consistent with previous studies indicating post-weaning social isolation results in the development of psychiatric symptoms such as cognitive dysfunction, anxiety, and abnormal aggression [5][6][7]. Our study also indicated that early-life social isolation led to decreased immobility in the FST, differing from previous findings that socially-isolated rats exhibited depression-like behavior [31]. An increase in FST immobility is commonly used as an indicator of depression-like symptoms, such as behavioral despair in rodents [32,33]. Though the FST is highly effective Fig. 3. Effects of post-weaning social isolation on the noradrenergic system protein levels in the medial prefrontal cortex (mPFC) of rats. The socially-isolated group showed significantly higher α 1 -AR protein expression levels; no significant difference in α 2A -AR expression levels was found between the groups. High expression levels of TH, DBH, and ΔFosB in the mPFC were observed in the isolation-reared compared to the group-housed rats. Bars represent mean ± S.E.M. *P < 0.05, **P < 0.01 vs. group-housed rats. α 1 -AR, α 1 adrenergic receptor; α 2A -AR, α 2A adrenergic receptor; DBH, dopamine-β-hydroxylase; TH, tyrosine hydroxylase in detecting the efficacy of antidepressants in animals at a basal emotional state [34], it might be inadequate for detecting depressionlike states in the presence of elevated anxiety. This perspective is supported by studies demonstrating that FST immobility behavior was decreased by anxiogenic drugs (e.g. pentylenetetrazole) or social isolation [35][36][37]. Here, isolated rats exhibited a high anxiety state, possibly interfering with depressive behavior, as evaluated by the FST. In future studies, we will conduct novel experiments to elucidate the effects of early-life social isolation on other depression-related behaviors using additional tests, including sucrose preference and nesting behavior.
Because the mPFC plays an essential role in cognitive and emotional regulation, including aggression [8,9], emotional and cognitive dysregulation is associated with mPFC neural dysfunction [11][12][13]. Here, socially-isolated rats exhibited significantly increased ΔFosB protein levels in the mPFC, consistent with a recent study [15]. ΔFosB, a member of the Fos family of transcription factors, exhibits expression levels elevated by chronic stress [38]. Overexpression of ΔFosB by stress and genetic manipulation in the mPFC can downregulate c-Fos levels, a neural activation marker in this brain region, and mediate stressinduced changes in emotional behaviors [16]. Indeed, social isolation after weaning in mice decreases the excitability of the mPFC neurons [14]. Thus, our results suggest that post-weaning social isolation impairs mPFC neural function, potentially inducing cognitive dysfunction, anxiety-like behavior, and abnormal aggression.
Based on this, we examined the effects of social isolation on the noradrenergic system in the mPFC. The mPFC noradrenergic system is regulated by α 1 and α 2A adrenergic receptors, depending on noradrenaline levels in the synaptic cleft [22]. Our study showed for the first time that socially-isolated rats exhibited increased protein levels of α 1 adrenergic receptors in the mPFC, but not in those of α 2A adrenergic receptors. In the mPFC, noradrenaline preferentially binds to α 2A adrenergic receptors, which enhances neural function. Alternatively, α 1 adrenergic receptors are activated by the presence of a large amount of noradrenaline in the synaptic cleft (e.g., during stressful conditions), which weakens mPFC neural function. Pharmacological activation of the α 1 adrenergic receptor leads to neural function impairment in the prefrontal cortex, including the mPFC [39][40][41]. Our study revealed that socially-isolated rats exhibited significantly increased TH and DBH protein levels in the mPFC. Because TH and DBH are rate-limiting enzymes involved in noradrenaline synthesis in LC noradrenergic neurons that project to the mPFC, these enzymes are considered key factors that determine the amount of noradrenaline synthesis and release [19][20][21]42]. Thus, our results suggest that noradrenaline levels increased in the mPFC of isolated rats, consistent with a previous finding showing that social isolation increased noradrenaline levels in the brain [23]. Additionally, we confirmed that social isolation increased climbing behavior in the FST, further supporting this interpretation concerning the effects of increased noradrenaline levels. Detke et al. (1995) showed that climbing behavior involved noradrenergic neurotransmission in the brain, since noradrenaline reuptake inhibitors increased climbing behavior and decreased immobility behavior in the FST [43,44]. We speculate that post-weaning social isolation induces the preferential activation of α 1 adrenergic receptors in the mPFC through increased expression levels of α 1 adrenergic receptors and high noradrenaline concentrations, potentially leading to neural dysfunction of the mPFC. Stress-induced excessive activation of the noradrenergic system in the prefrontal cortex is associated with the development of psychiatric disorders, such as attention deficit hyperactivity disorder and post-traumatic stress disorder, which are characterized by emotional and cognitive dysfunctions [45,46].

Conclusions
Early-life social isolation may impair emotional and cognitive regulation through preferential activation of the α 1 adrenergic receptor in the mPFC. The α 1 adrenergic receptor is an important potential target for new therapeutic strategies for psychiatric disorders with primary symptomatic features including emotional and cognitive dysregulation.

Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
No data was used for the research described in the article.