para-Chlorophenylalanine induces aggressive behavior by serotonin depletion in male rats and increases tryptophan hydroxylase 2 and GABAA α 1 mRNA expression in the olfactory bulb

Decreased serotonin (5-HT) has long been linked to increased aggressive behavior. Tryptophan hydroxylase (TPH) is an enzyme involved in 5-HT synthesis and para-chlorophenylalanine (pCPA) inhibits its activity. TPH2 mRNA expression presence has been mainly described in the raphe complex rodent’s brain. 5-HT-producing neurons in the raphe project their axons to olfactory bulb, considered to be a relevant structure in rodents for establishing social interactions, including aggressive behavior. However, the relationship between olfactory bulb and aggression in a pCPA 5-HT depletion model has not been studied. Moreover, receptor subunit GABA α 1 has been found in the olfactory bulb and 5-HT depletion could aﬀect GABAA receptor expression in diﬀerent brain areas. Thus, our aim was to evaluate aggressive behavior, serotonergic activity, the TPH2 and GABAA α 1 mRNA expression, in the olfactory bulb, after a single pCPA (300mg/kg) or vehicle i.p. administration in male rats. Aggression was tested using a resident intruder test. Sequentially, the olfactory bulb was obtained, and neurochemical and molecular techniques were used to measure 5-HT, 5-HIAA, TPH2, and GABAA α 1 mRNA expression respectively. pCPA administration increased aggressive behavior parameters, without aﬀecting locomotion, nonsocial or social interaction. 5-HT levels were decreased after pCPA administration, as well as its turnover rate, although there were no signiﬁcant changes in 5-HIAA. TPH2 mRNA expression was increased. GABAA α 1 mRNA expression was increased in the olfactory bulb. Our results apport evidence to the serotonergic deﬁciency hypothesis of aggression and highlight olfactory bulb role as an important structure for understanding aggressive behavior neurobiological complexity.

5-HT is synthesized through the tryptophan hydroxylase (TPH) enzyme (Khan & Thomas, 2004). TPH gene has two isoforms (Walther et al., 2003), while the TPH1 isoform is mostly expressed outside the blood-brain barrier, the TPH2 isoform is expressed in the cerebral tissue and its mRNA expression has so far been mostly described in raphe complex neurons (Malek et al., 2005;Patel et al., 2004;Pelosi et al., 2015;Walther et al., 2003). A human brain post-mortem study, showed TPH2 expression in the cortex, thalamus, hypothalamus, hippocampus, amygdala, cerebellum, and raphe nuclei (Zill et al., 2007). A study using catfishes (Raghuveer et al.; reported TPH2 mRNA expression in OB. Furthermore, Patel et at., (2004) showed that TPH2 mRNA expression in rat brain revealed a very weak signal in the OB.
Principal neurons and local interneurons in OB have GABAA receptors with different subunit components (Panzanelli, 2005). Particularly, the presence of GABAA receptor subunit α1 mRNA in the OB has been described (Zhang et al., 1991) and is the most common GABAA α subunit in adult rodent neurons (Bosman et al., 2002). In addition, it has been reported that 5-HT depletion could affect GABAA receptor expression in different brain areas (Si et al., 2022;Wang et al., 2020).
Our aim was to evaluate aggressive behavior, serotonergic activity in the OB, TPH2, and GABAA α1 mRNA expression in the OB, after a single pCPA i.p administration in male rats.

Methods and Materials
Subjects and housing Animals were housed in a temperature-controlled animal room (22 +/-2°C) on a 12 h light/dark cycle (light on from 07:00 to 19:00), with artificial light (60-70 lumens). Food and water were available ad libitum.
RVI test requires a resident subject and an intruder. Residents were Sprague-Dawley male rats of 60 days and 350 g (average weight). Intruders were Sprague-Dawley male rats of 50 days and 280 g (average weight). Resident subjects were divided into 2 groups (n=10). Experimental subjects were injected with pCPA and returned to their home cage for 6 days; control subjects were injected with a vehicle and returned to their home cage, at the same time. After the RVI test residents were euthanized and OB were removed for posterior analysis (Koolhaas et al., 2013). Intruder subjects remained housed in groups of 3 until the test and they were euthanized after it.

Experimental Design
Between 15:00h and 18:00h animals from the experimental groups received a single pCPA (Sigma Aldrich, 2015) i.p. injection (dose 300 mg/kg), solved in sterile saline. After pCPA administration residents were housed individually in their homecages until RVI test day, which was performed six days later. Then animals were euthanised and OB were obtained for neurochemical and genetic analysis.

Behavioral Testing
To assess aggressive behaviors, RVI was used. RVI test was applied according to Koolhaas et al. (2013) with modifications. Briefly, the RVI test consists of the interaction of two subjects: the experimental animal, named the resident, and the interaction animal, named the intruder. RVI test was performed 6 days after pCPA administration, between 15:00h and 18:00h (Corthell et al., 2013). In order to verify that residents were heavier than intruders, the animals were weight before the test. The test was carried out in a wood box with the wood chips from the resident home cage. The box was cleaned after each test, with ethanol (10%) (Casas et al., 2011). All the tests were recorded with the Everio G-series GZ-MG330 JVC camera, from above the field. Behavioral analysis was hand scored watching the videos in blind. RVI test total time was 900 seconds. RVI test total time was divided into two phases, the adaptation phase, and the interaction phase. The first 300 seconds were considered the resident adaptation phase to the environment. 600 seconds remaining were considered the interaction phase. At the beginning of the interaction phases, an intruder was placed on the opposite side of the resident at the end of the adaptation phase and the interaction behaviors were measured. We evaluated the animal locomotor activity as the travel distance during the adaptation phase. In the interaction phase, we evaluated four main groups of behaviors: 1-nonsocial activity, as time grooming, sniffing, and sitting; 2-social activity as the time of heterogrooming and heterosniffing; 3-aggressive behavior, as an event of the chase, moving towards, upright posture and false mount, bite, clinch, clinch attack, lateral threat and keep down were evaluated in the resident during interaction phase; and 4-aggressive latency, as the time preceding the first aggressive behavior. The criteria to discontinue resident interactions with the intruders were bite to delicate body parts e.g. belly, throat and paws.

RNA extraction and Real Time PCR analysis
Total RNA from all left and right OB tissue was extracted using the TRIzol reagent, according to the manufacturer specifications (Invitrogen-Life Technologies, Buenos Aires, Argentina.). mRNA integrity samples were confirmed by 1% agarose gel electrophoresis and staining with Sybr Gold (Invitrogen-Life Technologies, Buenos Aires, Argentina). 10 μg The mRNA levels of TPH2 and GABAA α1 were estimated by RT real-time PCR with a Corbett Rotor-Gene 6000 Real-Time Thermocycler (Corbett Research Pty Ltd (Sydney, Australia) using rat-specific primers and reaction conditions described in Table 1. The PCR reactions were performed using a Corbett Rotor-Gene 6000 Real-Time Thermocycler using Eva-GreenTM (Biotium, Hayward, CA) in a final volume of 20 μL. The reaction mixture consisted of 2 μL of 10×PCR Buffer, 1 μL of 50 mM MgCl2, 0.4 μL of 10 mM dNTP Mix (Invitrogen), 1 μL of 20× Eva Green, 0.25 μL of 5 U/μL Taq DNA Polymerase (Invitrogen) 0.1 μL of each 2.5 mM primer (forward and reverse primers) and 10 μL of diluted cDNA. The PCR reactions were performed under the conditions described in Table 1. Melt curve analysis was used to check that a single specific amplified product was generated. Real-time quantification was monitored by measuring the increase in fluorescence caused by the binding of EvaGreen dye to double-strand DNA at the end of each amplification cycle. According to the manufacturer protocol, the relative expression was determined using the Comparative Quantitation method of normalized samples about the expression of a calibrator sample (Pfaffl, 2001). Each PCR run included a no-template control and a sample without reverse transcriptase. All measurements were performed in duplicate. The reaction conditions and quantities of cDNA added were calibrated such that the assay response was linear concerning the amount of input cDNA for each pair of primers. RNA samples were assayed for DNA contamination by performing the different PCR reactions without prior reverse transcription. Relative levels of mRNA were normalized to the S16 reference gene.
The real-time PCR products were analyzed on 2% agarose gels containing 0.5 mg/mL ethidium bromide and a unique band of the approximately correct molecular weight corresponded with a unique peak in melt curve analysis.
The Real-Time PCR reactions were carried out for 40 cycles with an initial step of 5 min at 95 ºC followed by a three-step scheme: 30 s at 95 ºC, 30 s at the annealing temperature shown above for each primer pair, and a final step at 72 ºC for 30 s. Primer's design was done with Beacon Designer 7.9 software. 5-HT and 5-HIAA content determination by HPLC in the olfactory bulb All OB tissue homogenization was performed according to Chi et al., 1999. Briefly, the tissue was collected in 400 μl of 0.2 N perchloric acid and then homogenized in a glass-glass homogenizer. The homogenate was centrifuged at 12000×g for 15min at 4°C (Hermle LaborTechnik GmbH, model Z233MK-2) and the supernatant was injected into a high-performance liquid chromatography (HPLC) instrument coupled to electrochemical detection, to measure 5-HT, 5-HIAA. The pellet was resuspended in 1N NaOH for protein quantification by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Richmond, CA, USA) using bovine serum albumin as standard. The contents of 5-HT and 5-HIAA were expressed as picograms per milligram of total protein. 10 μl of each supernatant were injected into the HPLC system with the following setting: A isocratic pump, (model PU-2080 Plus, Jasco Co. Ltd., Tokyo, Japan), a UniJet microbore column (MF-8912, BAS, West Lafayette, IN, USA), and an amperometric detector (set at 650 mV, 0.5 nA; model LC-4C, BAS, West Lafayette, IN, USA). The mobile phase, containing 0.05 M NaH2PO4, 1.0 mM 1-octane sulfonic acid, 0.27 mM EDTA, 1.0% (v/v) tetrahydrofuran, and 4.0% (v/v) acetonitrile (CH3CN) (pH adjusted to 2.6) was pumped at a flow rate of 100μl/min. The level of neurotransmitters and metabolites was assessed by comparing the respective peak area and elution time of the sample with a reference standard. The quantification was performed using a calibration curve for each neurotransmitter (Program ChromPass, Jasco Co. Ltd., Tokyo, Japan). Under these experimental conditions, retention times were 33.3 for 5-HT and 25.6 for 5-HIAA. Standards, EDTA, and 1-octane sulfonic acid were purchased from Sigma-Aldrich, Inc. (St Louis, MO, USA), and all other reagents were of analytical grade.

Statistical Analysis
All data were analyzed by t-test. Shapiro-Wilks test was previously performed on each group to determine normal distribution. The significance level was set at p <0.05 for all statistical tests. Data were expressed as means ± S.E.M of 10 rats per experimental group. All data were analyzed using Statistica's software application (StatSoft, Krakow, Poland).

Real Time PCR analysis
The pCPA effect on TPH2 and GABAAα mRNA expression in the OB. We found that pCPA single administration significantly increased (p<0.05) TPH2 mRNA expression in OB (Figure 2-A). Also, it significantly increased GABAAα1 mRNA expression (p<0.05) in OB (Figure 2-B).

Discussion
The initiation, maintenance, and termination of aggressive behavior activates complex neurobiological circuits, among which the serotonergic system is strongly involved (Niederkofler et al., 2016;Takahashi & Miczek 2014). In the current study, using a RVI paradigm, we studied aggression behavior induced by pCPA and the relation with serotonergic activity, TPH2 and GABAAα1 mRNA expression in the OB.
It is well accepted that when evaluating social interaction, it becomes necessary to discard locomotor activity alterations. Takahashi & Miczek 2014). A locomotor activity alteration in the resident could prevent the correct measurement of aggressive behavior, in particular the aggression latency parameter. Interestingly, 5,7-dihydroxytryptamine lesions, used to deplete central 5-HT did not affect locomotor activity (Vergner et al., 1988;Hole et al., 1977). Nonetheless, previous works utilizing pCPA 1000 mg/kg doses found a substantial decrease in locomotor activity (Dringenberg et al., 1995;Matte & Tornow, 1978). Significantly reduced locomotor activity was also seen following chronic treatment with lower dosage of pCPA (100 mg/kg) (Keleta et al., 2007Kubala et al., 2008. Our data show that pCPA i.p. did not change locomotor activity. It is possible that an acute administration of pCPA at lower doses does not affect this parameter. According to Koolhaas et al. (2013) and Takahashi & Miczek (2014), to establish an animal aggressive behavior model, species specific qualitative and quantitative parameters of aggression must be considered. Therefore, when establishing the behaviors to evaluate, we took into account parameters that reconciled both characteristics. We measured aggressive behavior as a quantitative parameter and aggressive latency as a qualitative one . Stunder et al. (2015) show decreased aggressive latency, as a parameter of desadaptative aggression, and nonaggressive behaviors in mice treated with pCPA. Our data show that the administration of pCPA increased aggressive behavior and decreased aggressive latency. These findings are consistent with others, in which central 5-HT depletion caused aggression increased (Valzelli et al., 1981;Vergner et al., 1986). This might indicate that our model, with pCPA, was effective to induce aggression, both in a qualitative and quantitative sense. Furthermore, we did not find changes in nonsocial activity and social activity after pCPA administration. 5-HT low-levels might affect social interactions, e.g. in studies where social isolation is used to produce aggressive behavior models (Goodell et al., 2017) or in maternal aggression models (Toth et al., 2012). Thus, this result could be explained by our model and experimental design.
Serotonergic innervation originating in the raphe nuclei towards the different brain structures has one of its main synaptic center in the OB (Locki 1985;Steinfeld et al., 2015). Our results show that after pCPA administration, the concentration of 5-HT in the OB was significantly decreased pointing to higher aggressive behavior. In this way, our model provides evidence to the serotonergic deficiency hypothesis and aggression (Kravitz & Huber, 2003;Miczek et al., 2004;Mongillo et al., 2014;Takahashi et al., 2011;Niederkofler et al., 2016). In addition, 5-HT modification has been associated with changes in its major metabolite, 5-HIAA (Stenfors & Ross, 2004). In humans (Sharma et al., 2021;Stanley et al., 2000) and monkeys (Zajicek et al., 2000), higher aggressiveness and low cerebrospinal 5-HIAA levels were associated. Furthermore, mutual decreased 5-HT and 5-HIAA were observed in models of pCPA aggression (Keleta et al., 2007;Kubala et al., 2008). However, the decrease in 5-HT concentration, following local or systemic administration of substances that affect its release from nerve terminals, does not always affect 5-HIAA concentration in the same way (Auerbach et al., 1989;Kalén at al., 1988). In our model, pCPA administration did not cause significant modifications in the concentration of 5-HIAA. It is possible that systemic depletion did not affect the metabolite as well as 5-HT, because others (Dringenberg et al., 1995;Matte & Tornow, 1978) administered higher doses or performed a chronic treatment (Keleta et al., 2007;Kubala et al., 2008). We also showed that serotonergic metabolism (5-HIAA/5-HT) decreased in treated animals, which is in accordance with previous reported data (Hritcu et al., 2007;Koe & Weissman, 1966). These findings suggest that pCPA may cause alterations in the OB serotonergic innervation, enhancing aggression in our model.
Since pCPA animals exhibited increased aggression and 5-HT decreased, we hypothesized that these differences are accompanied by differences in a key serotonergic gene, TPH2. TPH2 mRNA expression is frequently found in raphe complex neurons in rodents (Malek et al., 2005;Patel et al., 2004;Pelosi et al., 2015;Walther et al., 2003) and a very low expression has been found in other rats' brain areas, (Patel et al., 2004). Interestingly, we found that pCPA animals showed increased TPH2 mRNA expression in the OB. Therefore, our results provide evidence for TPH2 mRNA expression presence in the OB. It has been also found that after postnatal programming with pCPA, TPH2 mRNA expression decreases in raphe nuclei (Trujillo et al., 2021). In this way, despite that our results are opposite they could be indicating a compensatory mechanism in a brain area receiving 5-HT depleted innervation.
In addition, pCPA decreases the protein expression of GABAA α1 receptors (Wang et al., 2020) and GABAAα1 is expressed in OB (Panzanelli et al., 2005). Interestingly, our results showed a higher expression of GABAAα1 subunit after pCPA administration. These results may indicate increased levels of GABAAα1 subunit mRNA in OB, due to its high synthesis demand caused by the serotonergic decrease as a consequence of pCPA administration.

Conclusion
In this work, we studied aggressive behavior in a rat male model. We concluded that a single and acute pCPA administration produces an increase in aggressive behavior, without affecting locomotion, nonsocial and social activity. We study the OB, a highly innervated serotonergic and GABAergic structure. The pCPA single and acute administration affects the serotonergic activity in OB. We verify TPH2 mRNA expression presence in OB. Moreover, increased GABAAα1 subunit expression mRNA in the OB, may suggest a high synthesis demand, as an alterat result in the OB serotonergic function. Thus, our data provide evidence for the serotonergic deficiency hypothesis of aggression and show OB as a relevant structure to understand neurobiological complexity in aggressive behavior.

Compliance with Ethical Statement
All procedures were made following the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and the EU (Eighth Edition, 2011) and approved by the Comité Institucional para el Cuidado y Uso de Animales de Laboratorio de la Universidad Nacional de Cuyo (CICUAL UNCuyo) (Aval 82/2016), Mendoza, Argentina.

Limitations
Other brain areas relate to aggressive behavior are not included in the present study.

Data Accessibility Statement
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are included in the paper. Table 1: Real Time PCR Primer Design.

Figures and Tables Legends
Specific primers designed for rat gene sequence amplification were selected for real-time PCR assay. Locomotor activity results are expressed as mean ± SEM of distanced travel in centimeters. Nonsocial activity and social activity results are expressed in mean ± SEM of time in seconds. Control (N=10) and pCPA (N=10). Table 3: pCPA effects in 5-HT, 5-HIAA and turnover %5-HIAA/5-HT.