Dysfunctional mesocortical dopamine circuit at pre-adolescence is associated to aggressive behavior in MAO-A hypomorphic mice exposed to early life stress

Abstract

Aggressive behavior (AB) is a multifaceted disorder based on the interaction between genetic and environmental factors whose underlying mechanisms remain elusive. The best-characterized gene by environment (GxE) interaction for AB is the relationship between child neglect/abuse and low-activity alleles of the monoamine-oxidase A (MAOA) gene. MAOA oxidizes monoamines like serotonin and dopamine, whose aberrant signaling at discrete developmental ages plays a pivotal role in the ontogeny of AB. Here, we investigated the impact of this GxE on dopamine function at pre-adolescence by exposing hypomorphic MAOA (MAO^Neo) mice to early life stress (ES) and by performing behavioral and ex vivo electrophysiological analyses in the ventral tegmental area (VTA) and the prefrontal cortex (PFC). MAO^Neo ES mouse dopamine neurons exhibited an enhanced post-synaptic responsiveness to excitatory inputs, aberrant plasticity in the PFC, and an AB. Systemic administration of the selective antagonist at dopamine D1 receptors SCH23390 fully restored PFC function and rescued AB. Collectively, these findings reveal that dysfunctional mesocortical dopamine signaling at pre-adolescence ties to AB in the MAO^Neo ES mouse, and identify dopamine D1 receptor as a molecular target to be exploited for an age-tailored therapy.

1. Introduction

Aggressive behavior (AB) is an innate physiological response required for species survival. However, when AB in adolescence is disproportionate to any provocation and threatens or causes physical harm to others, it is regarded as a key symptom of conduct disorder (CD; DMS-5). CD patients frequently engage in delinquency, and are at greater risk for violent crime and antisocial behavior in adulthood (Farrington, 1990; Henggeler and Sheidow, 2003; Kim-Cohen et al., 2003; Olino et al., 2010; Simonoff et al., 2004). For these reasons, curtailing CD early in life is paramount to prevent the socio-economic impact of violence.

AB has a multi-factorial origin (Caspi et al., 2002; Viding and Frith, 2006) based on the interaction between genetic and environmental factors. The best-characterized gene by environment (G × E) interaction in AB occurs between child neglect/abuse and low-activity alleles of the MAOA gene, which encode the enzyme monoamineoxidase A (MAOA) (Byrd and Manuck, 2014; Caspi et al., 2002; Fergusson et al., 2011; Gorodetsky et al., 2014; Huang et al., 2004; Karere et al., 2009; Kim-Cohen et al., 2006; Viding and Frith, 2006; Williams et al., 2009). MAOA enzyme is the degrading enzyme of monoamines (e.g. serotonin, dopamine), which play age-specific roles in the etiopathogenesis of AB (Yu et al., 2014a).

We recently developed a mouse model of this G × E interaction by subjecting a Maoa hypomorphic mouse (MAOA^Neo) (Bortolato et al., 2011) to an early stressful regimen consisting of maternal separation a daily saline intraperitoneal injections (Godar et al., 2019). MAOA^Neo mice display reduced enzymatic activity of MAOA, but not overt AB (Bortolato et al., 2011). However, MAOA^Neo mice subjected to the aforementioned early stress (ES) and to a few days of social isolation develop AB, as measured by their response to foreign intruders (Godar et al., 2019). Notably, the effects of this G × E interaction rely on the activation of serotonin 5-HT2A during the first week of life. Accordingly, neurobiological studies aimed at disentangling the etiology of AB have shown that monoamine signaling during discrete developmental periods are critically involved in AB onset (de Almeida et al., 2005a; Rebello et al., 2014; Yu et al., 2014a). Hence, imbalances in dopaminergic and serotonergic signaling during different critical windows of development are implicated in aggression (Comai et al., 2012a, 2012b; Yu et al., 2014a). Particularly, increased serotonergic signaling during the first two weeks of postnatal life in mice (Godar et al., 2019; Rebello et al., 2014) or optogenetic stimulation of ventral tegmental area (VTA) dopamine cell activity following social isolation during peri-adolescence are causally related to AB (Yu et al., 2014b).

Dopamine signaling is implicated in several brain functions including salience, motivation and reward, decision-making and behavioral flexibility (Fallon et al., 2013; Richter et al., 2013; Shiner et al., 2015; Shohamy et al., 2005). Discrete brain regions, all of which receive strong dopaminergic innervation, support these processes, including the prefrontal cortex (PFC), which integrates emotional and cognitive information and regulates emotional reactivity (Blair, 2013, 2017; Buckholtz and Meyer-Lindenberg, 2008; Meyer-Lindenberg et al., 2006). Notably, aberrant PFC functioning appears as a system archetype for AB (Anderson et al., 1999; Choy et al., 2018; Halasz et al., 2006; Raine et al., 2000). Indeed, deviant PFC function is exemplified by the impulsivity, poor self-regulation and hostile attributional bias leading to AB (Gansler et al., 2011; Lee et al., 2011; Mason et al., 2014; Yang and Raine, 2009). Pyramidal neurons within the PFC integrate multiple synaptic signals from different brain areas (Goldman-Rakic, 1996; Goldman-Rakic et al., 2000), and project to the main components of the limbic-subcortical circuit that regulate negative affect and aggression. Important nodes within this network include the ventral tegmental area (VTA), amygdaloid nuclei, medial hypothalamus and dorsal periaqueductal gray (Gregg and Siegel, 2001).

Revealing the maladaptive trajectory that could be related to AB (endo)phenotypes might help uncovering novel molecular targets for this condition, whose treatment represents an unmet clinical need (Zuddas, 2014). Further, to the best of our knowledge, no age-specific pharmacological treatment is yet available for this disorder (Balia et al., 2018). The identification of critical windows of vulnerability during brain development suggests that therapeutic interventions during such sensitive periods, typically before or at symptom onset, might prevent derangement and conversion to late-onset of disease (Marin, 2016). These developmental milestones overlap with critical period of brain plasticity, key in the maturation of neural networks, and range from before birth and childhood to adolescence (Borre et al., 2014; Marin, 2016).

Since we have elucidated the role of serotonin signaling early in life in MAOA^Neo ES mice, in the current study we investigated the effects of this G × E interaction on dopamine signaling at pre-adolescence. This allowed us to reveal synaptic changes in both VTA and PFC function, and to disclose potential molecular targets for the treatment of AB before adolescence.

2. Material and methods

2.1. Animals

Male MAOA^Neo mice were generated from 129S6 genetic background by mating primiparous MAOA^Neo heterozygous females with wild-type (WT) sires, as previously described (Bortolato et al., 2011). Since Maoa is a X-linked gene, male offspring of MAOA^Neo heterozygous dams were either MAOA^Neo or WT. Pregnant dams were singly-housed 3 days prior to parturition. Only litters with >4 pups (and at least 2 males) were used, and all litters with more than 8 pups were culled to eight at postnatal day (PND) 1 to assure uniformity of litter size. Litter effects were minimized by using mice from at least six different litters for behavioral and electrophysiological experiments. Bedding was changed in all cages at PND 7 and PND 14, and mice were weaned at PND 21. Animals were housed in a room maintained at 22 °C, on a 12 h/12 h light/dark cycle from 8 a.m. to 8 pm. Food and water were available ab libitum. Behavioral experiments occurred between 11 a.m. and 5 p.m. during the light phase of the light/dark cycle. All experimental procedures were in accordance with the National Institute of Health guidelines and approved by the Animal Use Committees of the Universities of Utah, Kansas and Cagliari. In Italy, all procedures were performed in accordance with the European legislation (EU Directive, 2010/63) and were approved by the Animal Ethics Committees of the University of Cagliari and by Italian Ministry of Health (auth. n. 659/2015-PR and 621/2016-PR). All possible efforts were made to minimize animal pain and discomfort and to reduce the number of experimental subjects.

2.2. Drug treatments

All drugs except for SCH23390 hydrochloride (Tocris Bioscience) were purchased from Sigma-Aldrich. Both alpha-methyl-p-tyrosine (AMPT; 200 mg/kg) and SCH23390 hydrochloride (0.1 mg/kg) were dissolved in 0.9% saline solution and administered via i.p. injections 30 min prior to testing. The injection volume was 10 ml/kg. Control mice were treated with 0.9% saline. For electrophysiological experiments, each slice received only a single drug exposure. Drugs were applied in known concentrations to the superfusion medium. Drugs were dissolved in DMSO when it was needed. The final concentration of DMSO was <0.01%.

2.3. Early stress procedure

Pups were subjected to a stress regimen of maternal separation plus saline injection from PND 1 to 7, heretofore designated as early-life stress (ES). Briefly, maternal separation was performed for 2–3 h/day in a pseudo-random fashion and at different times during the light cycle (Fig. 1A). Specifically, male pups were removed from the home cage and placed into a new cage in a separate temperature-controlled (25 °C) room. Physiological saline injections were performed using a microinjector connected to a Hamilton syringe (10 μL/g body weight). Non-stressed controls were briefly removed from their cages, gently handled for 5 min and returned to their home cage. All males in each litter received the same manipulation. At PND 22, mice were singly housed for 5 days before performing behavioral and electrophysiological experiments. This regimen was implemented to enhance the translational relevance of the animal model (Godar et al., 2019).

Fig. 1. Dopamine synthesis inhibition at puberty prevents the manifestation of aggressive behavior in MAOA^Neo mice subjected to early life stress.

(A) Schematic timeline of experimental procedure to impose early life stress (ES) and to assess changes in social behavior. (B) Acute administration of the tyrosine hydroxylase enzyme inhibitor alpha-Methyl-para-tyrosine (AMPT) (200 mg/kg i.p.) in MAO^Neo ES mice reduced both the duration and the episodes of fighting behaviors but (C) did not affect the latency to the first attack. Data are represented as mean ± s.e.m. with single values (each circle represents an individual value; n_pup = 12 per group). Main effects are not indicated. *p < 0.05 for WT ES-VEH vs MAO^Neo ES-VEH; ###p < 0.001 for MAO^Neo ES-VEH vs MAO^Neo ES-AMPT (genotype × treatment interaction, post-hoc Tukey’s test).

2.4. Resident-intruder aggression

Aggression was tested in the resident-intruder task as previously described (Bortolato et al., 2012). Briefly, animals were singly housed for 5 days from PND 22 to PND 27. During this period, the bedding of the cage was not cleaned to let the resident establish its own territory. This regimen was sufficient to evoke robust reactive aggressive responses in MAOA^Neo mice exposed to ES procedure, but not in WT littermates. Resident mice were then exposed to foreign WT intruder males from different litters, for 10 min (Fig. 1A). The intruder was placed at the opposite side of the resident in a counterbalanced position (right or left). Behavior was video-monitored from an adjacent room, recorded and scored by a trained observer blind to both genotype and treatment. Measures included: a) the social exploration, in terms of frequency and duration of active social approach; b) the latency to first “non fighting” social approach; c) the latency between the introduction of the intruder and the first attack; d) the number and duration of fighting behavior; e) the episodes of tail rattling, a prominent feature of aggressive phenotype in mice; f) the number and duration of chasing behavior of the resident towards the intruder; g) rearing behavior, as index of general motor activity of the resident. At the end of the test, resident and intruders mice were returned to their respective home cages.

2.5. Electrophysiological analysis

We prepared horizontal VTA brain slices (230 μm) or coronal PFC slices (300 μm) from PND 28–30 mice anaesthetized with isoflurane until loss of righting reflex. Solutions were saturated with 95% O₂/5% CO₂. VTA slices were cut in an ice-cold solution containing, in mmol/L: 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.5 CaCl₂, 7 MgCl₂, and 75 sucrose or 126 NaCl, 1.6 KCl, 1.2 NaH₂PO₄, 1.2 MgCl₂, 0.625 CaCl₂, 18 NaHCO₃, and 11 glucose. As previously described (Kasanetz et al., 2013), PFC slices were maintained in a sucrose-based physiological solution at 4 °C (in mM: 87 NaCl, 75 sucrose, 25 glucose, 5 KCl, 21 MgCl₂, 0.5 CaCl₂ and 1.25 NaH₂PO₄). Immediately after cutting, slices were stored for 40 min at 32 °C in an artificial cerebrospinal fluid (aCSF) (in mM): 130 NaCl, 11 glucose, 2.5 KCl, 1.2 MgCl₂, 2.4 CaCl₂, 23 NaHCO₃, 1.2 NaH₂PO₄, and were equilibrated with 95% O₂/5% CO₂. Slices were then stored in ACSF at room temperature until recording. PFC slices recovered >1 h at 32 °C in aCSF, at 300–306 mOsm, and contained, in mmol/L: 126 NaCl, 2.5 or 1.6 KCl, 1.1 NaH₂PO₄, 1.4 MgCl₂, 2.4 CaCl₂, 11 d-glucose, and 26 NaHCO₃. VTA slices were stored in aCSF at 36 °C until recording. Cells were visualized with an upright microscope with infrared illumination (Axioskop FS 2 plus; Zeiss), and whole-cell patch-clamp recordings were made by using an Axopatch 200 B amplifier (Molecular Devices). Voltage-clamp recordings were made with electrodes filled with a solution containing the following (in mm): 117 Cs methanesulfonic acid, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA-Cl, 2.5 Mg₂ATP, and 0.25 Mg₂GTP, pH 7.2–7.4, 275–285 mOsm. Picrotoxin (100 μm) was added to the aCSF for recording, to block GABA_A receptor-mediated IPSCs. Experiments were begun only after series resistance had stabilized (typically 15–40 MΩ). Series and input resistance were monitored continuously on-line with a 5 mV depolarizing step (25 msec). Data were filtered at 2 kHz, digitized at 10 kHz, and collected on-line with acquisition software (pClamp 10; Molecular Devices). Dopamine neurons from the posterior VTA were identified by the presence of a large I_h current that was assayed immediately after break-in, using a series of incremental 10 mV hyperpolarizing steps from a holding potential of −70 mV. Pyramidal neurons were identified by their pyramidal shape, the presence of a prominent apical dendrite, and the distance from the pial surface (layers 5/6).

A bipolar stainless-steel stimulating electrode (FHC) was placed 100 μm rostral to the recording electrode and was used to stimulate at a frequency of 0.1 Hz. NMDA EPSCs were evoked while holding cells at +40 mV. The AMPA EPSC was isolated after bath application of the NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-AP5, 100 μM). The NMDA EPSC was obtained by digital subtraction of the AMPA EPSC from the dual (AMPA + NMDA-mediated) EPSC. The values of the AMPA/NMDA ratio may be underestimated since the experiments were performed in the presence of spermine in the recording pipette. We calculated paired-pulse ratios (PPR), with an interstimulus interval of 50 msec, as the ratio between the second and first EPSC averaged over 5 min. For field excitatory postsynaptic potential (fEPSP), extracellular recording electrodes were filled with aCSF. To evoke synaptic currents in the PFC, stimuli (100 μs duration) were delivered at 0.1 Hz with the stainless steel stimulating electrode placed in layer 2/3 as previously described (Lafourcade et al., 2007). Experiments were performed blind to the experimental group.

2.6. Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 6.01). Kolmogorov-Smirnov and Bartlett’s tests were used to verify normality and homoscedasticity of data. Data were analyzed with two-way ANOVAs and ANCOVAs followed by Tukey’s for post-hoc comparisons. Predicted Gaussian curves were built upon nonlinear regression analysis of frequency distribution. Significance threshold was set at 0.05.

3. Results

3.1. Dopamine synthesis inhibition prevents aggressive behavior in MAOA^Neo mice

To determine if dopamine signaling during pre-adolescence is involved in the development of AB in MAOA^Neo mice subjected to ES (MAOA^Neo ES), animals were acutely administered either the tyrosine hydroxylase enzyme inhibitor alpha-methyl-para-tyrosine (AMPT, 200 mg/kg, i.p.) or saline at puberty. In the resident intruder test, as expected, MAOA^Neo ES mice exhibited an increased number of fighting bouts [Main effect of genotype F (1,44) = 6.90, P < 0.05] and fighting duration [Main effect of genotype: F (1, 50) = 30.57: P < 0.001] (Fig. 1B), and a decreased latency to first attack [Main effect of genotype: F (1,44) = 6.90, P < 0.05] (Fig. 1C). Moreover, a significant treatment effect was detected for all the parameters [bouts, treatment: F (1,44) = 20.60, P < 0.0001; duration, treatment: F (1,44) = 22.84, P < 0.0001; latency, F (1,44) = 20.60, P < 0.0001]. Significant differences in genotype × treatment interactions were detected for the duration [F (1,44) = 4.08, P < 0.05], and number [F (1,44) = 3.96, P < 0.05] of attacks (Fig. 1B), but not for the latency [F (1,44) = 2.08, NS] (Fig. 1C). Multiple comparisons revealed that the administration of the dopamine synthesis inhibitor AMPT significantly dampened both the fighting duration and fighting bouts in MAOA^Neo ES mice (P < 0.001 for comparison between MAOA^NeoES-VEH and MAOA^Neo ES-AMPT, Tukey’s post-hoc test, Fig. 1B). Furthermore, the effect of AMPT is not accompanied by a concomitant decreased overall activity, as no significant interaction treatment x genotype was found for the duration and number of social sniffing as well as the rearing behaviors, two indices of exploratory activity (Supplementary Fig. 1). Accordingly, to further control for potential confounds in the effects of AMPT on aggressive behaviors, the relationship between the numbers of social and aggressive episodes in each group was compared by ANCOVA: significant main effects of treatment [F (1,43) = 18.84, P < 0.00001] and group [F (1,43) = 5.95, P < 0.05], but no significant effect for group × treatment interaction [F (1,43) = 3.44; NS] were detected. Accordingly, none of the linear regressions was significant.

3.2. Early life stress alters MAOA^Neo mouse post-synaptic responsiveness to excitatory inputs of VTA dopamine neurons

To isolate the potential impact of ES on MAOA^Neo VTA dopamine neurons, we examined the properties of excitatory synapses on these cells. We obtained whole-cell patch-clamp recordings from putative dopamine neurons in acute VTA slices from pre-adolescent (PND 28–30) male WT and MAOA^Neo mice cells and excitatory post-synaptic currents (EPSCs) were evoked by electrical stimulation of rostral afferents and pharmacologically isolated. We examined the current-voltage relationship (I–V) of AMPAR-mediated EPSCs. ES impacted only MAOA^Neo mouse dopamine cells, since MAOA^Neo ES mouse I-V curves are non-linear and exhibit an inward rectification (Fig. 2A, 2-way ANOVA, interaction F (1,24) = 6,8; p < 0.01; genotype F (1,24) = 71,2; p < 0.0001; ES F (1,24) = 20,6; p = 0.0001; Tukey’s post-hoc comparison: MAOA^Neo ES vs WT, p < 0.0001; MAOA^Neo ES vs WT ES, p < 0.0001; MAOA^Neo ES vs MAOA^Neo, p < 0.001) suggestive of expression of calcium permeable AMPARs (Bellone et al., 2011; Hausknecht et al., 2015). However, no differences in synaptic strength elicited by paired stimuli (i.e., paired-pulse ratio, PPR) given at an interval of 50 ms is found between genotypes and ES (Fig. 2B and 2-way ANOVA, interaction F (1,31) = 0,4; p = 0.53). A common assay to quantify the strength of individual synapses is the ratio of the AMPAR-mediated EPSCs to the slow NMDAR-mediated EPSC, which is routinely used to make inferences about the past history of a synapse (Bariselli et al., 2016; Kasanetz et al., 2010, 2013; Saal et al., 2003; Ungless et al., 2001). Putative dopamine neurons from MAOA^Neo ES mice exhibited a higher AMPA/NMDA ratio compared to WT ES and unstressed mice (Fig. 2C and 2-way ANOVA, interaction F (1,26) = 6,1; p = 0.02; genotype F (1,26) = 7,9; p = 0.009; ES F (1,26) = 12,8; p = 0.001; Tukey’s post hoc comparison: MAOA^Neo ES vs WT, p < 0.001; MAOA^Neo ES vs WT ES, p < 0.01; MAOA^Neo ES vs MAOA^Neo, p < 0.01). We also computed NMDAR-mediated EPSC decay time kinetic, measured as weighted tau (τ), and found that it is faster in neurons recorded from MAOA^Neo ES mice, as compared to WT ES and unstressed mice, as shown by both the raw data and the predicted Gaussian curves (Fig. 2D and 2-way ANOVA, interaction F (1,47) = 20,9; p < 0.0001; genotype F (1,47) = 29,2; p < 0.0001; ES F (1,47) = 3,5; p = 0.06; Tukey’s post hoc comparison: MAOA^Neo ES vs WT, p < 0.0001; MAOA^Neo ES vs WT ES, p < 0.0001; MAOA^Neo ES vs MAOA^Neo, p < 0.001). Since NMDAR channel properties depend upon subunit composition and their relative contribution also in VTA dopamine cells (Bellone et al., 2011), these results suggest that the ratio GluN2A/N2B in NMDA receptors on dopamine neurons of MAOA^Neo ES mice increases. Collectively, these data show that this naturalistic mouse model recapitulating the core features of human AB exhibits an enhanced postsynaptic responsiveness to glutamatergic transmission of VTA dopamine neurons.

Fig. 2. Early life stress affects postsynaptic responsiveness of VTA dopamine neurons in MAOA^Neo mice.

(A) Current-voltage relationship (I–V) curves of AMPA EPSCs recorded from dopamine neurons of MAOA^Neo ES (n_cells = 10, n_pup = 8), WT ES (n_cells = 6, n_pup = 4), WT (n_cells = 7, n_pup = 4) and MAOA^Neo (n_cells = 6, n_pup = 3) mice. Data are represented as mean ± s.e.m. Insets show representative traces of AMPA EPSCs recorded at −70 and + 40 mV from WT ES and MAOA^Neo ES mice. Calibration bar: 10 ms, 50 pA. (B) Bar graph summarizing the effects of early life stress (ES) on paired-pulse ratio (EPSC2/EPSC1) of AMPA EPSCs recorded from WT (n_cells = 5, n_pup = 3), MAOA^Neo (n_cells = 10, n_pup = 5), WT ES (n_cells = 7, n_pup = 4) and MAOA^Neo ES (n_cells = 11, n_pup = 8) slices. Data are represented as mean ± s.e.m. with single values (each circle represents a recorded cell). Numbers in parenthesis represent the number of animals used. (C) Top, Representative traces of AMPA and NMDA EPSCs recorded from dopamine neurons held at +40 mV in slices from WT ES and MAOA^Neo ES mice. Calibration bar: 20 ms, 100 pA. Bottom, Quantification of the data summarizing ES effect on AMPA/NMDA ratio recorded from WT (n_cells = 7, n_pup = 4), MAOA^Neo (n_cells = 6, n_pup = 3), WT ES (n_cells = 7, n_pup = 4) and MAOA^Neo ES (n_cells = 10, n_pup = 8) slices. Data are represented as mean ± s.e.m. with single values (each circle represents a recorded cell). Numbers in parenthesis represent the number of animals used. (D) Left hand, Quantification of the data showing NMDA EPSC decay time kinetic (weighted tau, τ) in MAOA^Neo ES (n_cells = 14, n_pup = 8), WT ES (n_cells = 13, n_pup = 7), MAOA^Neo (n_cells = 13, n_pup = 7) and WT (n_cells = 11, n_pup = 5) slices. Data are represented as mean ± s.e.m. with single values (each circle represents a recorded cell). Numbers in parenthesis represent the number of animals used. Insets show representative traces of NMDA EPSCs recorded at +40 mV in slices from WT ES and MAOA^Neo ES mice. Right hand, a predicted Gaussian showing distribution of NMDAR EPSCs τ in the different groups of mice. Asterisks in the figure refer to the interaction revealed by 2-way ANOVA analyses.

3.3. Early life stress affects MAOA^Neo mouse PFC function

Imbalances in information processing within the PFC largely depends upon changes in monoamine levels (Levy, 2004; Stahl, 2009a, b) and NMDAR function (Homayoun et al., 2005; Jackson et al., 2004), which enable temporal and spatial summation of incoming inputs and allow for a dynamic integration of information processing (Miller and Cohen, 2001; WA et al., 2010). To investigate potential impact of ES on MAOA^Neo PFC pyramidal neurons, we examined excitatory synaptic properties of layer 5/6 pyramidal neurons at pre-adolescence. We found no effect of either genotype or ES on I-V relationship of AMPAR-mediated EPSCs (Fig. 3A, 2-way ANOVA, interaction F (1,24) = 2,5; p = 0.13). However, genotype affected the impact of ES on PPR of AMPAR-mediated EPSCs recorded from mouse pyramidal neurons (Fig. 3B, 2-way ANOVA, interaction F (1,25) = 17,7; p = 0.0003; genotype F (1,25) = 5,9; p = 0.02; ES F (1,25) = 0,1; p = 0.81; Tukey’s post hoc comparison: MAOA^Neo ES vs WT ES, p < 0.001; WT ES vs WT, p < 0.05; MAOA^Neo ES vs MAOA^Neo, p < 0.05), but no interaction was revealed for the AMPA/NMDA ratio (Fig. 3C, 2-way ANOVA, F (1,23) = 2,6; p = 0.11), although ES affected both genotypes (2-way ANOVA, F (1, 23) = 45,15; p < 0.0001; Tukey’s post hoc comparison: MAOA^Neo ES vs WT ES, p < 0.05; WT ES vs WT, p < 0.0001; MAOA^Neo ES vs MAOA^Neo, p < 0.01). Notably, ES prolonged the decay time of NMDAR-mediated EPSCs exclusively in MAOA^Neo mouse pyramidal neurons (Fig. 3D, 2-way ANOVA, F (1,41) = 11,2; p = 0.0018; genotype F (1,41) = 17,5; p = 0.0001; ES F (1,41) = 7,6; p = 0.008; Tukey’s post hoc comparison: MAOA^Neo ES vs WT ES, p < 0.0001; WT ES vs WT, p < 0.0001; MAOA^Neo ES vs MAOA^Neo, p < 0.001) as shown by both the raw data and the predicted Gaussian curves. Consistently, GluN2B-specific antagonist RO25698 (0.03–1 μM) more potently reduced NMDAR-mediated EPSC amplitude in MAOA^Neo ES mouse PFC pyramidal neurons when compared to WT ES and naïve mice (Fig. 3E, 2-way RM ANOVA, interaction F (9,57) = 6,4; p < 0.0001; 0.03 μM, WT ES vs MAOA^Neo ES: Tukey’s post hoc comparison: MAOA^Neo ES vs WT ES, p < 0.01; WT ES vs WT, p < 0.0001; MAOA^Neo ES vs MAOA^Neo, p < 0.0001).

Fig. 3. Early life stress changes synaptic properties and plasticity of PFC in MAOA^Neo mice.

(A) Current-voltage relationship (I–V) curves of AMPA EPSCS recorded from Layer 5/6 pyramidal neurons in WT (n_cells = 7, n_pup = 3), MAOA^Neo (n_cells = 5, n_pup = 2), WT ES (n_cells = 7, n_pup = 3) and MAOA^Neo ES (n_cells = 7, n_pup = 4) mice. Data are represented as mean ± s.e.m. Insets show representative traces of AMPA EPSCs recorded at −70 and + 40 mV from WT ES and MAOA^Neo ES mice. Calibration bar: 10 ms, 50 pA. (B) Bar graph summarizing the effects of ES on paired-pulse ratio (EPSC2/EPSC1) of AMPA EPSCs recorded from WT (n_cells = 7, n_pup = 4), MAOA^Neo (n_cells = 7, n_pup = 4), WT ES (n_cells = 7, n_pup = 5) and MAOA^Neo ES (n_cells = 8, n_pup = 6) slices. Data are represented as mean ± s.e.m. with single values (each circle represent a single cell recorded). Numbers in parenthesis represent the number of animals used. Insets show representative traces of paired AMPA EPSCs. Calibration bar: 20 ms, 100 pA. (C) Quantification of the data summarizing ES effect on AMPA/NMDA ratio recorded from WT (n_cells = 7, n_pup = 3), MAOA^Neo (n_cells = 5, n_pup = 2), WT ES (n_cells = 7, n_pup = 3) and MAOA^Neo ES (n_cells = 8, n_pup = 4) slices. Data are represented as mean ± s.e.m. with single values (each circle represent a single cell recorded). Numbers in parenthesis represent the number of animals used. Insets show representative traces of AMPA and NMDA EPSCs recorded from Layer 5/6 pyramidal neurons held at +40 mV in slices from WT ES and MAOA^Neo ES mice. Calibration bar: 10 ms, 100 pA. (D) Top, quantification of the data showing NMDAR EPSC decay time kinetics (weighted tau, τ) in MAOA^Neo ES (n_cells = 14, n_pup = 13), WT ES (n_cells = 13, n_pup = 12), MAOA^Neo (n_cells = 8, n_pup = 6) and WT (n_cells = 10, n_pup = 8) slices. Data are represented as mean ± s.e.m. with single values (each circle represent a single cell recorded). Numbers in parenthesis represent the number of animals used. Bottom, a predicted Gaussian showing the frequency distribution of NMDA EPSCs τ in the four groups of mice. (E) Dose-response curves for percentage inhibition in NMDA EPSC amplitude by the GluN2B selective antagonist RO256981 in MAOA^Neo ES (n_cells = 5, n_pup = 3), WT ES (n_cells = 6, n_pup = 3), WT (n_cells = 6, n_pup = 3) and MAOA^Neo (n_cells = 6, n_pup = 3). Each symbol represents the averaged value (±s.e.m.) obtained from different cells. Insets show representative traces of paired NMDA EPSCs. Calibration bar: 100 ms, 200 pA. (F) Theta burst stimulation (TBS: 50 Hz, 100 pulses, four times at 0.1 Hz, at the arrow) induced long-term potentiation of extracellular field potentials (fEPSPs) recorded from PFC layer 5/6 only in MAOA^Neo ES (n_cells = 5, n_pup = 4) slices, whereas it did not affect fEPSP amplitude inWT ES (n_cells = 6, n_pup = 3), WT (n_cells = 5, n_pup = 3) and MAOA^Neo (n_cells = 5, n_pup = 3) slices. Data are represented as mean ± s.e.m. Asterisks in the figure refer to the interaction revealed by 2-way ANOVA analyses.

Expression of GluN2B-containing NMDARs favors the induction of synaptic plasticity bi-directionally (Barria and Malinow, 2005). Particularly, in the PFC insertion of GluN2B enhances synaptic responses to presynaptic stimulation and facilitates long-term potentiation (LTP) (Cui et al., 2011; Ruan et al., 2014), similarly to the effects of a background dopamine signal (Matsuda et al., 2006; Ruan et al., 2014) via D1 receptor activation (Lewis and O’Donnell, 2000; Ruan et al., 2014). We, therefore, hypothesized that genetic background might affect PFC output function in response to ES. We recorded field potentials in layer 5/6 evoked by stimulating layer 2/3 and applied a theta burst stimulation protocol (TBS; 50 Hz, 100 pulses 4 times at 0.1 Hz) that induces LTP only when slices are primed with dopamine (Matsuda et al., 2006). As predicted, LTP could be induced only in MAOA^Neo-ES mouse PFC (Fig. 3F, 2-way RM ANOVA, interaction F (72,408) = 3,4; p < 0.0001; genotype × ES F (3,17) = 13,93; p < 0.0001).

3.4. Blockade of dopamine D1 signaling restores PFC function and behavior

Phasic dopamine release in the PFC following bursts of VTA dopamine cell activity modulates membrane potential of pyramidal neurons via D1 receptor activation (Lewis and O’Donnell, 2000), an effect that might enable input-target association and rule activity-dependent synaptic plasticity (Ruan et al., 2014). To address the role of D1 receptor activation in TBS-induced LTP in MAOA^Neo ES mouse PFC, we pharmacologically blocked them in vivo since dopamine receptors are tonically stimulated in the intact brain. Mice were administered the D1 receptor antagonist SCH23390 (0.1 mg/kg i.p.) and acute PFC-containing slices were prepared 30 min after. This manipulation fully blocked TBS-induced LTP in MAOA^Neo-ES mice (Fig. 4A, 2-way RM ANOVA, Interaction F (72,432) = 2,3; p < 0.0001; SCH23390 on genotype × ES, F (3,18) = 16,0; p < 0.0001) without affecting fEPSP in WT-ES mice.

Fig. 4. In vivo blockade of dopamine D1 receptors prevents aggressive behavior and restore PFC plasticity in MAOA^Neo mice subjected to early life stress.

(A) In vivo administration of the D1 receptor antagonist SCH23390 (0.1 mg/kg, 30 min before slice preparation) prevented theta burst stimulation (TBS: 50 Hz, 100 pulses, four times at 0.1 Hz, at the arrow) -induced long-term potentiation of extracellular field potentials (fEPSPs) recorded from PFC layer 5/6 in MAOA^Neo ES (n_cells = 6, n_pup = 6) slices, whereas it did not affect fEPSP amplitude in WT ES (n_cells = 6, n_pup = 6) slices. Gray and red lines represent time course of the effects shown in Fig. 3F for WT ES and. MAOA^Neo ES mice, respectively. Data are represented as mean ± s.e.m. Significance refers to the interaction revealed by 2-way ANOVA. The high levels of aggression in MAO^Neo-ES mice were abolished by the selective blockade of D1 receptor: SCH23390 markedly decreased (B) fighting duration, (C) fighting bouts, (D) the episodes of tail rattling, (E) the duration and (F) number of chasing behavior of the resident towards the intruder mice. Data are represented as mean ± s.e.m. with single values (each circle represents an individual value; n_pups = 11–18 per group). Main effects are not indicated. ***p < 0.001 for WT ES-VEH vs MAO^Neo ES-VEH comparison; ###p < 0.001 for MAO^Neo ES-VEH vs MAO^Neo ES-SCH comparison (genotype × treatment interaction, post-hoc Tukey’s test).

Next, to ascertain whether D1 receptor blockade could prevent AB in MAOA^Neo ES mice, SCH23390 (0.1 mg/kg, i.p.) or vehicle (VEH) was administered 30 min before behavioral test. First, we confirmed that at pre-adolescence MAOA^Neo ES mice manifest robust AB, as revealed by the increased in fighting duration and bouts [Fig. 4B and C, Main effect of genotype for duration: F (1,54) = 75.55, P < 0.001; bouts: F (1,54) = 60.47]. In addition, in MAOA^Neo ES, we observed other aggressive phenotypes, such as a marked change in the number of episodes of tail rattling (Fig. 4D), as well as the duration and frequency of chasing behavior (Fig. 4E and F), of the resident toward the intruder mice [Main effect of genotype for tail rattling episodes: F (1,54) = 98.06, P < 0.001; chasing duration: F (1,54) = 18.16, P < 0.001; chasing bouts: F (1,54) = 61.06, P < 0.001]. Furthermore, a main effect of treatment as well as a significant treatment × genotype interaction were also found for these parameters [Main effect of treatment for duration: F (1,54) = 66.28, P < 0.001; bouts: F (1,54) = 49.93, P < 0.001]; tail rattling: F (1,54) = 86.22, P < 0.001; chasing duration: F (1,54) = 29.95, P < 0.001; chasing bouts: F (1,54) = 74.38, P < 0.001]; [treatment × genotype interaction, fighting duration: F (1,54) = 67.23, P < 0.001; fighting bouts: F (1,54) = 48.75, P > 0.001; tail rattling: F (1,54) = 84.92, P < 0.001; chasing duration: F (1,54) = 67.23, P < 0.001; chasing bouts: F (1,54) = 60,91 P < 0.001]. Of note, post-hoc analyses revealed that the D1 receptor blockade profoundly abolishes all these AB phenotypes in MAOA^Neo ES mice (Fig. 4B–F; P < 0.001 for comparison between MAOA^Neo-VEH and MAOA^Neo-SCH23390, Tukey’s test). Accordingly, SCH23390 did not modify the duration and number of social sniffing, thus indicating that the effects of this drug on AB were not associated to a decreased exploratory activity [duration of social approach: F (1,54) = 0.06, P = 0.79, NS; number of social sniffing: F (1,54) = 2.06,P = 0.15, NS; rearing duration: F (1,54) = 0.24, P = 0.62, NS; rearing bouts: F (1,54) = 2.43,P = 0.12, NS]. Accordingly, ANCOVA analyses detected significant main effects for treatment [F (1,53) = 71.58, P < 0.00001] and group [F (1,53) = 54.34, P < 0.00001], as well as interaction treatment X genotype [F (1,53) = 57.61, P < 0.00001]; post-hoc analysis revealed significant differences between Neo-sal and all other groups (Ps < 0.001). Notably, the regression line for MAOA^Neo-VEH shows a negative slope (B = −0.99 ± 0.22), thus indicating that the number of social interactions is inversely correlated to the number of attacks.

4. Discussion

Pinpointing neurobiological differences between individuals biologically at risk, and those not at risk, for AB may provide effective strategies to manage AB at pre-adolescence in subjects exposed to ES and social isolation. By taking advantage of the first mouse model mimicking a well-established interaction (Caspi et al., 2002; Viding and Frith, 2006) between a specific genetic risk factor (i.e., low activity MAOA) and early environmental adversity predisposing to an overt AB (Godar et al., 2019), we demonstrated an association between mesocortical dopamine circuit function at pre-adolescence and AB in MAOA^Neo mice subjected to ES.

The present study supports the notion that adversities in early childhood, such as physical and/or sexual abuse, as well as parental neglect, may lead to AB only in a segment of population featuring genetic susceptibility. Accordingly, the strongest robust association between genetic variation and aggression was observed for MAOA (McDermott et al., 2009). For instance, in humans, a rare X-linked point-mutation in the gene’s eighth exon resulting in a complete inactivation of MAOA was associated with violent impulsive criminal behavior (Brunner et al., 1993). Accordingly, in rodents, ablation of MAOA gene elicits similar aggressive phenotypes (Godar et al., 2016). Clinical studies also show an association between low-expressing functional polymorphism of MAOA in the promoter region and increased sensitivity to early-life adversity and consequent development of AB (Guo et al., 2008). In addition, hypermethylation of a region proximal to the transcription start site of MAOA that may contribute to a hypomorphic modulation of this enzyme was strongly associated with antisocial behavior (Checknita et al., 2015).

Our findings extend previous preclinical studies that highlight critical development timelines in the manifestation of AB, during which the deviations in the mechanisms regulating monoamine homeostasis can result in opposite behavioral outcome (Comai et al., 2012a; Godar et al., 2019; Rebello et al., 2014; Yu et al., 2014a). In particular, we found that aberrant dopamine signaling during pre-adolescence in the MAOA^Neo ES mouse is accompanied by an aggressive phenotype, as exemplified by its behavioral repertoire displayed in the resident intruder test. Accordingly, both inhibition of dopamine synthesis and blockade of D1 receptors, with AMPT and SCH23390, respectively, prevented the manifestation of AB, suggesting a common target with the signal encoded by dopamine at this age. This is in line with both the evidence that perturbations of dopaminergic signaling during this critical window of development induces AB (Comai et al., 2012a, 2012b; Yu et al., 2014a), and with the hyperdopaminergic hypothesis of aggression (de Almeida et al., 2005b; Seo et al., 2008). Hypothesis that has been substantiated by a direct causal relationship between optogenetic stimulation of dopamine neurons of the VTA following social isolation and increased AB (Yu et al., 2014b). While our data cannot fully substantiate a causal nexus between aberrant dopamine signaling and aggressive phenotypes, it is worth noting that putative dopamine neurons of the VTA exhibited an increased post-synaptic responsiveness to excitatory stimuli, a phenomenon that is key to coding emotional memory formation and adaptive behavior (Pignatelli and Bonci, 2015), and that might be involved in shaping erroneous signal-to-noise ratios within the target region of the PFC, thus resulting in inadequate behavioral responses. When probing synaptic function of PFC pyramidal neurons in MAOA^Neo ES mice, we observed an increased probability of presynaptic glutamate release associated to NMDA receptors containing an enhanced GluN2B/GluN2A ratio along with a form of LTP. These phenomena might affect the regulation of social processing and threat assessment of MAOA^Neo ES mice, thereby leading to the derangement of social responses and to the AB exhibited by these mice.

Disturbances of emotional and executive function, such as hostile attributional bias, lead to a misinterpretation of social cues and AB in children. Given the role of the PFC in both social cognition and the initiation of appropriate behavioral responses, it is tempting to speculate that the imbalances in PFC↔VTA circuit function here reported might contribute to an out-of-tune of executive function in MAOA^Neo ES mice. In fact, dysregulation of such a function is involved in the etiopathogenesis of different forms of aggression, as underscored by impulsivity and poor self-regulation (Ellis et al., 2009; Goldstein et al., 2007; Lewis et al., 2008; Lösel et al., 2007; Seguin et al., 2002). Since AB is one of the most common child problems requiring neuropsychiatric intervention (Burke et al., 2002), the identification of cognitive derangement that feature children engaging in significant AB is paramount for early intervention. Additionally, AB can often be comorbid with other behavioral problems, especially in school-aged children (Lahey et al., 2002; Stahl, 2009a, b). Hence, aberrant function in cortical and limbic networks may underlie the wide array of comorbid symptoms in AB that is important to recognize (Burke et al., 2002; Lahey et al., 2002; Loeber et al., 2000). In particular, abnormalities in PFC-limbic networks appear not only to be involved in aggressive and destructive symptoms of conduct disorder, and in disobedient and oppositional symptoms of oppositional defiant disorder, but also in ADHD, mood instability of bipolar disorders, and irrational fears of anxiety disorders (Burke et al., 2002; Lahey et al., 2002; Loeber et al., 2000; Lösel et al., 2007; Stahl, 2009a, b).

Our findings highlight that early life adversity is a pivotal risk factor to social information processing deficits when interacts with genetic risk factors such as low activity of MAOA to predict AB. In fact, both ES during first postnatal week and post-weaning social isolation are sufficient and necessary to induce AB in MAOA^Neo mice (Godar et al., 2019), whereas they are detrimental to the development of such behavior at this age in male mice (Tsuda et al., 2011) or in adult female mice (Gunaydin et al., 2014). In this framework, the alterations in PFC↔VTA circuit function selectively observed in pre-adolescent MAOA^Neo ES mice, along with their phenotype associated to an altered dopaminergic activity, allowed us to develop a viable pharmacological intervention that proved effective in both rescuing normal PFC circuit function and restoring a proper interpretation of social cues. Hence, one interpretation of our findings is that early life adversities (ES and post-weaning social isolation) may induce aberrant dopamine signaling at pre-adolescence, which is sufficient for PFC to attribute hostile bias and to impair planning ability, thereby leading to overt AB selectively in MAOA^Neo mice. Consistently, early life adverse events have long-term effects on dopamine systems (Ironside et al., 2018; Pizzagalli, 2014) resulting in heightened PFC response and enhanced functional connectivity within the PFC, cingulate and striatal networks (Grandjean et al., 2016). Such changes may override top-down control of response inhibition subsequent to deviations in social cue coding. Notably, the abovementioned deficits in executive functions correlate with one manifestation of AB termed reactive aggression (Crick and Dodge, 1996; McAuliffe et al., 2006).

A contemporary research goal in this field is to be able to predict and treat different subtypes of AB (Zuddas, 2014). Current pharmacological strategies are neither specific for AB nor age-tailored and effective, with methylphenidate and risperidone showing the largest effects in randomized controlled trials (Balia et al., 2018; Zuddas, 2014). Hence, age-specific medication of AB still represents a current unmet clinical need. Our findings suggest a potential molecular target (i.e., D1 receptor) that could be readily exploited. In fact, although SCH23390 is not a FDA-approved drug, another D1 receptor antagonist may be a valid therapeutic avenue to suppress AB, particularly, ecopipam, which is currently under investigation in clinical trials for the treatment of other disorders in children.

Several limitations of the present study should be acknowledged. First, our behavioral analysis of social interaction was curtailed to the resident intruder test. Second, AMPT lacks specificity for dopamine. In fact, AMPT also depletes norepinephrine, another substrate of MAOA enzyme. Although in both rodents and humans AMPT depletes more dopamine than norepinephrine (Booij et al., 2003; Flexner and Goodman, 1975), we cannot rule out the contribution of noradrenergic system to behavioral outcome observed in the resident intruder test. Third, systemic administration with SCH23390 might block D1 receptors also located in other limbic structures involved in aggressive and antisocial behavior (Rosell and Siever, 2015).

These limitations notwithstanding, our results gain insights into developmental mechanisms for the etiology and pathophysiology of AB in a segment of at-risk population, and identify a viable molecular target that could prove effective in age-specific management of AB.