Pharmacologic blockade of monoamine oxidase A (MAOA) or serotonin transporter (5-HTT) has antidepressant and anxiolytic efficacy in adulthood. Yet, genetically conferred MAOA or 5-HTT hypoactivity is associated with altered aggression and increased anxiety/depression. Here we test the hypothesis that increased monoamine signaling during development causes these paradoxical aggressive and affective phenotypes. We find that pharmacologic MAOA blockade during early postnatal development (P2–P21) but not during peri-adolescence (P22−41) increases anxiety- and depression-like behavior in adult (>P90) mice, mimicking the effect of P2–21 5-HTT inhibition. Moreover, MAOA blockade during peri-adolescence, but not P2–21 or P182-201, increases adult aggressive behavior, and 5-HTT blockade from P22–P41 reduced adult aggression. Blockade of the dopamine transporter, but not the norepinephrine transporter, during P22–41 also increases adult aggressive behavior. Thus, P2–21 is a sensitive period during which 5-HT modulates adult anxiety/depression-like behavior, and P22–41 is a sensitive period during which DA and 5-HT bi-directionally modulate adult aggression. Permanently altered DAergic function as a consequence of increased P22–P41 monoamine signaling might underlie altered aggression. In support of this hypothesis, we find altered aggression correlating positively with locomotor response to amphetamine challenge in adulthood. Proving that altered DA function and aggression are causally linked, we demonstrate that optogenetic activation of VTA DAergic neurons increases aggression. It therefore appears that genetic and pharmacologic factors impacting dopamine and serotonin signaling during sensitive developmental periods can modulate adult monoaminergic function and thereby alter risk for aggressive and emotional dysfunction.
Most neuropsychiatric disorders have developmental origins, and an emerging model postulates that such developmental vulnerability is often restricted to sensitive periods.1, 2, 3 The concept of sensitive developmental periods for the indelible modulation of complex behaviors is similar to that described for sensory systems (for example, visual cortex, ocular dominance plasticity),4,5 but affected behaviors, modulating factors and underlying mechanisms are much less well understood. Furthering our knowledge of sensitive periods that determine the developmental trajectory of complex behaviors is a necessary step toward improving prevention and treatment approaches for neuropsychiatric disorders. Therefore, we here investigate how genetic and environmental risk factors act during sensitive periods of brain development to alter adult behavior and thereby potentially confer vulnerability to neuropsychiatric disorders.
Monoamine oxidase A (MAOA) inactivates bioamines, including serotonin (5-HT), norepinephrine, dopamine (DA) and trace amines through oxidative deamination. Pharmacologic MAOA blockade is an effective treatment for depression and anxiety disorders in adults.6 Unlike the salutary effects of pharmacologic MAOA inhibition, constitutive mutations of maoa function result in a syndrome characterized by antisocial/aggressive behavior in human males.7 Moreover, low-expressing maoa variants are also associated with aggression and anxiety traits in rhesus macaques and humans.8, 9, 10 Consistent with higher primates, mice with genetic inactivation of maoa exhibit heightened levels of aggression and neophobia.11, 12, 13 The divergent effects of genetic (life-long) mutations versus pharmacologic inhibition (during adulthood) lead us to hypothesize that lower MAOA function during sensitive periods of brain maturation increases the likelihood of aggressive and anxiety traits later in life.
The role of monoamine signaling in brain development has been firmly established. During the embryonic and early postnatal period, monoamines act as trophic factors, modulating neurodevelopmental processes, such as cell division, migration and differentiation.14, 15, 16 In mice, genetic maoa ablation increases brain tissue concentrations of 5-HT, NE and DA during development and early adulthood.11 The most prominent teratogenic consequence of murine maoa ablation, the disruption of barrel fields in the somatosensory cortex, has been linked to increased 5-HT signaling during perinatal development.17, 18, 19 After closure of this sensitive window relating to the establishment of somatosensory cortex organization, increased 5-HT signaling during early postnatal development increases anxiety/depression-like behavior including neophobia in adult mice.20,21 Thus, we hypothesized first that increased 5-HT signaling during early postnatal development causes neophobia in maoa-deficient mice. Secondarily, we predicted that the origins of aggressive behaviors are also developmental and postnatal, because re-expressing maoa from P1 onwards restores normal aggressive behavior in conditional maoa knockout mice.22 To test both hypotheses, we compared the effects of developmental MAOA inhibition and monoamine transporter blockade on adult behavior and monoamine signaling in mice.
Materials and methods
Mice (129SvEv/Tac) were bred at Columbia Psychiatry, New York State Psychiatric Institute. Mice used for experiments were born to litters containing 4–6 pups. Mice were separated by sex and weaned into groups of five mice per cage at P22. Mice were treated during postnatal development (P2–21, as previously described20,21) or peri-adolescence (P22–41). One additional control group was treated during adulthood (P182-201). Treatment consisted of the following intraperitoneal (i.p.) drug injections: vehicle (VEH, 0.9% NaCl, 5 ml kg−1), fluoxetine (FLX, 10 mg kg−1, 5 ml kg−1, 5-HTT blocker), clorgyline (CLO, 20 mg kg−1, MAOA inhibitor), desipramine (DMI, 20 mg kg−1, NET blocker), or GBR12909 (GBR, 20 mg kg−1, DAT blocker). The dose for each treatment was chosen based on previous experiments: FLX at 10 mg kg−1 per day and DMI at 20 mg kg−1 per day produce serum and brain drug and metabolite levels that are comparable to those with therapeutic efficacy in humans;20,21 GBR at 20 mg kg−1 is a standard dose to achieve functional DAT blockade in mice;23 and a CLO dose of 20 mg kg−1 per day produces partial disruption of MAOA-dependent barrel field formation (to achieve full disruption of barrel field formation, CLO has to be administered every 8 h).19 Treatment assignment was randomized in each cage. Injections were administered daily between 3 and 5 pm. Animals were maintained on a 12-hour light–dark cycle (lights on at 08 hours) and provided with food and water ad libitum. Animal testing was conducted in accordance with the Principles of Laboratory Animal Care National Institute of Health (NIH) guidelines and the institutional animal committee guidelines.
In order to selectively express channelrhodopsin2 (ChR2) in dopaminergic cells we crossed a Dopamine-transporter Cre-driver-line (DATIRESCre,24 ) with a ROSA26-floxedSTOP ChR2-eYFP line (ai32 line25). Under isofluorane surgery, optical fiber ferrules (Ceramic ferrules (OD 1.2 mm): Precision fiber products; Optical fiber (OD 0.2 mm): Thor labs, Newton, NJ, USA), were implanted over the VTA (AP:-3.5; ML:0.5; DV:-4.2). After recovery, mice were subjected to the isolation-induced aggression test as described bellow, but pairing DATIRESCre:ai32 mice with single-mutant controls. During each encounter only one mouse of the pair was stimulated using blue light pulses (473 nm, 20 Hz, 10 ms pulse duration, 8–10 mW) originating from a laser source. Each mouse of the pair was stimulated in consecutive days, counterbalancing the stimulation order by genotype. After behavioral tests were completed, mice were euthanized, and genotypes and fiber placements were confirmed.
For neophobia/anxiety-related behavioral testing, male and female mice were investigated, and all animals were exposed to the same series of behavioral paradigms starting at 3 months of age. The tests were administered in the following order: open field, novelty-suppressed feeding and shock escape with a minimum of 7 days between each test. Aggression and amphetamine response were tested in behavioral naïve independent male cohorts starting at 5 months of age for the P2–21 and P22–41 groups and at 10 months of age for the P182-201 group. All behavioral testing took place during the light cycle between 12 and 7 pm. To eliminate odor cues, each apparatus was thoroughly cleaned after each animal.
Open field test
Exploration and reactivity to a novel environment was assessed using the open field test as previously described.26 In brief, square Plexiglas activity chambers equipped with infrared detectors to track animal movement in the horizontal and vertical planes were used as the novel environment. The conflicting innate tendencies to avoid bright light and open spaces but explore novel environments influence locomotor behavior. Mice were placed into the center of the open field and activity was recorded for 30 min. Testing took place under bright ambient light conditions. Total distance, total ambulatory time and vertical activity were measured.
Novelty-suppressed feeding test
The novelty-suppressed feeding test is a behavior conflict paradigm that is sensitive to chronic but not acute antidepressant treatment as well as acute benzodiazepine treatment. The test was performed as described previously:20 the testing apparatus consisted of a plastic box (50 × 50 × 20 cm). The floor was covered with 2 cm of wooden bedding. Twenty-four hours before behavioral testing, animals were deprived of food in the home cage. At the time of testing, two food pellets were placed on a round filter paper (12 cm diameter) positioned in the center of the box. Animals were placed in a corner of the box and the latency to approach the pellet and begin feeding was recorded (maximum time, 10 min). Each mouse was weighed before food deprivation and before testing to determine the percentage of body weight lost.
Shock escape test
Shock escape latency is the primary dependent measure affected by uncontrollable stress and chronic antidepressant treatment. The shock escape test was performed in a two-chambered Plexiglas shuttle box with the two chambers separated by an automated guillotine door as previously described.26 Each apparatus is equipped with infrared detectors to track animal movement and located within a sound-attenuated chamber. At the beginning of each trial, the door was raised and a mild scrambled foot shock (0.2 mA; 10 s duration) was delivered to the subject through the floor bars. The end of a trial was signaled by the closing of the guillotine door and was triggered either by a transition to the opposite chamber or after 10 s. Transition latencies were recorded. If the subject failed to make a transition during the 10-s duration of the foot shock, a maximum latency of 10 s was recorded. A session consisted of 30 trials separated by a 30-s inter-trial interval. Locomotor activity was assessed during the pre-shock habituation phase with the guillotine door open.
Isolation-induced aggression test
We used a rectangular cage that was divided in half by a perforated partition made of clear plastic. A pair of mice with the same treatment was placed in the cage, one in each compartment. The mouse in one compartment is able to see, hear and smell the other mouse through the holes in the plastic divider, but physical interaction is blocked. Mice were housed for at least 10 days before experiment was performed. On the test day, dividers were taken out and mice were allowed to freely interact for 10 min. All behaviors were videotaped. The time spent mounting, tail rattling and biting was scored.
Adult mice were injected with amphetamine (3–5 mg kg−1, i.p.) or VEH and locomotor activity was assessed in open field apparati (see above).
High-pressure liquid chromatography
High-pressure liquid chromatography (HPLC) was carried out on several brain regions as previously described.27 Concentrations of biogenic amines and metabolites were measured using reversed-phase HPLC with electrochemical detection. The dissected brain samples in a 1.5-ml microfuge tube were homogenized in 0.5–1.0 ml of 0.4 M perchloric acid with an Ultra Cell Disruptor (Microson, NY). The homogenate was centrifuged for 5 min at 14 000 g and 4 °C. A quantity of 50 μl of typically fourfold diluted aliquot of the supernatant was injected over the HPLC system. The HPLC system, equipped with Waters Millennium software (Milford, MA, USA), consists of a Waters 515 HPLC pump, a Waters 717 Autosampler, a Varian Microsorb 100–5 C18 reverse-phase column (DYNAMAX 150 × 4.6 mm) attached with a Guard column (4.6 mm) and an ESA Coulochem electrochemical detector (Model 5100A) with a guard cell (Model 5020) and a dual analytical cell (Model 5011A). The electrochemical detector was set at potential of +0.05V for the first cell and +0.5V for the second cell. The mobile phase contained 0.75 mM sodium phosphate (pH 3.1), 1.4 mM 1-Octanesulfonic acid, 10 μM sodium EDTA and 8% acetonitrile. The mobile phase was filtered through a Millipore 0.22 μm filter (type GV) and degassed in vaccuo. The flow rate was maintained at 0.8 ml min−1. A chromatography software package (Waters Millennium) was used for data acquisition and analysis. Values are calculated based on peak area and compared with standard solutions. The inter- and intra-assay coefficients of variation of the assay were each less than 5%. The sensitivity of the assay was less than 0.5 pmol per injection.
For immunohistochemistry, animals were deeply anesthetized with ketamine/xylazine and perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and post-fixed overnight (4% paraformaldehyde in PBS), and subsequently cryoprotected in 30% sucrose and frozen. Coronal sections (50 μm thick) were cut with a cryostat and fluorescence immunohistochemistry was performed on free-floating sections.
Immunohistochemistry was performed using a rabbit primary antibody against GFP (rabbit anti-GFP; 1:1000; Life Technologies, Grand Island, NY, USA). As secondary antibody, we used cy3 donkey anti-rabbit (1:350; Jackson Immunoresearch, West Grove, PA, USA). Antibodies were diluted in blocking solution containing 2% bovine serum albumin and 0.2% Triton X-100 dissolved in PBS. Sections were incubated in primary antibody for 24 h at 4 °C, washed with PBS, and then incubated with secondary antibody for 2 h at room temperature. After washing, sections were mounted with Vectashield mounting medium (Vector Labs, Burlingame, CA, USA).
Statistical analysis was performed using StatView 5.0 software (SAS Institute, Cary, NC, USA). Data were analyzed using Student’s t-test, one-way or two-way ANOVA with Student–Newman–Keuls post hoc testing; survival analysis, Kaplan–Meier, Logrank Mantel–Cox test; or non-parametric Mann–Whitney test as indicated. The criterion for significance for all analyzes was P<0.05. No statistically significant interactions of independent variables and gender were detected and therefore data were collapsed for sex. Results from data analyses are expressed as mean±s.e.m.
5-HTT and MAOA blockade during early postnatal development increase adult anxiety/depression-like behavior
Early postnatal 5-HTT blockade increases anxiety/depression-like behavior in adult mice.20,21 Because 5-HTT and MAOA blockade both increase 5-HT signaling, we hypothesized that early postnatal MAOA blockade would also increase adult anxiety/depression-like behavior. To test this hypothesis, we administered the 5-HTT blocker fluoxetine (FLX, 10 mg kg−1 per day, i.p.), the MAOA inhibitor clorgyline (CLO, 20 mg kg−1 per day, i.p.), or vehicle (VEH, 0.9% NaCl, i.p.) to mice in the early postnatal (PN) period, from postnatal day 2–21 (P2–P21) and assessed emotional behavior in adulthood (P90). To investigate developmental specificity, we also tested mice that received FLX, CLO and VEH in the peri-adolescent period (ADO), from postnatal day 22–41 (P22–P41). To control for the effects of the injection procedure, we included naïve littermates in our behavioral tests.
Using the novel open field, we assessed neophobic behaviors by measuring exploratory activity. We observed that FLX and CLO treatment from P2–P21 decreased total ambulatory time (treatment effect: F(2,127)=6.225, P=0.0026) and total time spent rearing (treatment effect: F(2,127)=5.42, P=0.0055; developmental timing effect: F(1,127)=13.56, P=0.0003) when compared with VEH-treated mice (Figure 1a and b). Neither FLX nor CLO treatment from P22–P41 altered exploratory behavior compared with VEH-treated mice. Behaviors in VEH-treated mice and naïve littermates did not differ in either measure.
To further probe neophobia phenotypes, we used the novelty-suppressed feeding paradigm, which assesses approach-avoidance behavior when a food-deprived animal is presented with a familiar food pellet placed in the center of a brightly lit novel arena. Mice exposed to FLX or CLO from P2–P21 exhibited longer latencies to approach and feed when compared to PN–VEH controls (treatment effect: P=0.0001, survival analysis, Kaplan–Meier, Logrank Mantel–Cox) (Figure 1c). However, neither FLX treatment nor CLO treatment from P22–P41 produced such behavioral effect. No effect for treatment, developmental timing or treatment x developmental timing was detected for weight loss during food deprivation (Figure 1d). Behavior between VEH-treated mice and naïve littermates did not differ in either measure.
To assess behavioral response to stress, we examined escape latency in the shock escape paradigm. Consistent with our findings in tests of neophobia, mice exposed to FLX or CLO from P2–P21 but not P22–P41 exhibited significantly increased escape latencies when compared with PN–VEH controls (treatment effect: F(2,133)=21.106, P<0.0001; developmental timing effect: F(1,133)=46.749, P<0.0001; treatment x developmental timing interaction: F(2,133)=24.613, P<0.0001) (Figure 1e). No effect of treatment, developmental timing or treatment x developmental timing was detected for pre-shock activity in the dark chambers with the door open (Figure 1f), indicating that the effect on escape latencies was not caused by reduced overall activity. Behavior between VEH-treated mice and naïve littermates did not differ in either measure.
Peri-adolescent MAOA blockade increases and 5-HTT blockade reduces adult aggressive behavior
Next we investigated aggressive behaviors in adult mice that had received FLX, CLO or VEH during early postnatal or peri-adolescent development using the isolation-induced aggression paradigm. We measured the time mouse dyads spent engaged in aggressive behaviors consisting of mounting, tail rattling or biting.
We found that CLO treatment from P22–P41 but not from P2–P21 increased aggressive behavior, when compared with VEH controls (effect of treatment: F(2,69)=7.258, P=0.0014; effect of developmental timing: F(1,69)=4.737, P=0.033; treatment x developmental timing interaction: F(2,69)=3.828, P=0.0265) (Figure 2a). FLX treatment reduced aggressive behavior when compared to VEH treatment, with no indication of developmental timing specificity. Aggressive behavior between VEH-treated mice and naïve littermates did not differ. Next we investigated period specificity for the effect of transient CLO treatment on adult aggression, using mice that had received VEH or CLO treatment during adulthood (P182–P201) and were tested 4 months later (P300). We detected no effect of treatment on aggression (Figure 2b).
Peri-adolescent MAOA blockade reduces the metabolism of 5-HT, NE and DA
The differential consequences of P22–P41 5-HTT and MAOA blockade on adult aggressive behavior suggest that non-5-HT effects of peri-adolescent MAOA blockade increase adult aggression. In peri-adolescence, maoa is expressed in 5-HTergic, DAergic and NEergic neurons.28 To determine the consequences of P22–P41 CLO treatment on brain monoamine signaling, we assessed tissue monoamine and metabolite levels at P23 (24 h after treatment initiation) and at P42 (24 h after treatment cessation). Specifically, we quantified 5-HT and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA), DA and its two main metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and NE. These measures serve as a proxy for enzyme activity.
CLO treatment significantly increased 5-HT and reduced 5-HIAA levels in the brainstem at P23 (F(1,12)=51.972, P<0.0001 and F(1,12)=31.92, P=0.0001, respectively) and P42 (F(1,11)=62.475, P<0.0001 and F(1,11)=18.8, P=0.0012, respectively), when compared with VEH-treated controls (Figures 3a and d). Norepinephrine levels in the brainstem were also increased by CLO treatment at P23 and P42 (F(1,12)=68.717, P<0.0001 and F(1,11)=40.45, P<0.0001, respectively) (Figures 3e and f). Striatal DA levels were not affected at P23, but significantly increased at P42 after chronic CLO treatment when compared with VEH-treated controls (F(1,12)=0.033, P=0.8587 and F(1,11)=61.745, P<0.0001, respectively) (Figures 3g and h). Striatal DOPAC levels were lower at P23 and P42 (F(1,12)=30.292, P=0.0001 and F(1,11)=13.444, P=0.0037, respectively), while HVA levels were unchanged (F(1,12)=0.003, P=0.9586 and F(1,11)=2.41, P=0.1489, respectively) (Figures 3i and l). These data demonstrate that MAOA blockade using CLO treatment during peri-adolescence inhibits 5-HT, NE and DA metabolism and raises levels of 5-HT, DA and NE.
Peri-adolescent DAT but not NET blockade increases adult aggressive behavior
Next we sought to identify the monoamine component in P22–P41 MAOA blockade, which leads to increased adult aggressive behavior. Because peri-adolescent MAOA blockade robustly increases central 5-HT, NE and DA levels, we examined whether increased peri-adolescent NE or DA signaling could increase adult aggressive behavior. To selectively and transiently increase NE or DA signaling, we administered drugs blocking the NE transporter (NET) or the DA transporter (DAT). We used desipramine (DMI, 20 mg kg−1 per day) to block the NET and GBR12909 (GBR, 20 mg kg−1 per day) to block the DAT. DMI treatment from P22–P41, either alone or in combination with FLX (10 mg kg−1 per day) treatment, did not result in increased aggressive behavior in adulthood (Figure 4a). GBR treatment from P22–P41, however, leads to increased time spent in active aggressive behaviors (Mann–Whitney, P=0.0104) (Figure 4b).
Peri-adolescent MAOA but not 5-HTT blockade reduces adult brainstem 5-HIAA levels and increases striatal DA and DOPAC levels
To start investigating the underlying neural substrate that gives rise to altered adult aggressive behavior after peri-adolescent MAOA, 5-HTT and DAT blockade, we next examined if these developmental interventions produce long-lasting changes in brain monoamine or metabolite levels. P22–P41 CLO treatment significantly reduced 5-HIAA levels in the brainstem at P180 when compared with either VEH or FLX treatment (F(2,15)=8.505, P=0.0034) (Table 1a). P22–P41 CLO treatment also significantly increased striatal DA (F(2,15)=4.085, P=0.0384) and DOPAC levels (F(2,15)=5.847, P<0.0133) at P180 when compared with VEH treatment and VEH or FLX treatment, respectively (Table 1a). However, peri-adolescent FLX or GBR treatment did not produce significant changes in monoamines or their metabolites in the brainstem or striatum at P180, when compared with VEH-treated controls (Table 1a and b). Taken together, this dissociation indicates that while altered tissue monoamine levels might be sufficient to alter adult aggressive behavior, they are not necessary.
Peri-adolescent MAOA or DAT blockade increases while 5-HTT blockade reduces the behavioral response to amphetamine in adulthood
Dopaminergic hyperactivity has been implicated in aggression.29 We find a positive association between aggression and striatal DA and DOPAC levels after peri-adolescent MAOA, but not 5-HTT or DAT blockade. Here we further investigate the hypothesis that dysfunction of the DAergic system might underlie altered aggression, using behavioral response to amphetamine challenge (as opposed to total monoamine tissue levels) as a proxy for DAergic function.
First, we analyzed the behavioral response to amphetamine (3 mg kg−1, i.p.) and VEH challenge in adult mice (P180), which had received VEH or CLO during P22–P41. Adult VEH injection did not alter behavior in P22–P41 VEH or CLO-treated mice (Figure 5a); adult amphetamine injection increased ambulatory activity in P22–P41 VEH and CLO-treated mice (effect of time in the amphetamine treated group: F(16,416)=18.812, P<0.0001, Figure 5a); In comparison with P22–P41 VEH-treated mice, P22–P41 CLO-treated mice showed a significantly increased response to amphetamine (effect of peri-adolescent treatment: F(1,26)=6.576, P=0.0165; effect of peri-adolescent x adult treatment interaction: F(16,416)=3.810, P<0.0001; Figure 5a). Analysis of only post-challenge activity confirmed these results: We detected an effect of peri-adolescent treatment, an effect of challenge and an interaction between both (F(1,26)=6.768, P=0.0165, F(1,26)=45.932, P<0.0001 and F(1,26)=5.549, P=0.0263, respectively; Figure 5b). To test for period specificity, we next investigated the consequences of transient CLO treatment during adulthood on behavioral response to amphetamine later in life. Specifically, mice, which had received VEH or CLO during P182–P201, were challenged with amphetamine (3 mg kg−1, i.p.) or VEH 4 months later. We detected an effect of amphetamine but no effect of peri-adolescent treatment or peri-adolescent x adult treatment interaction (F(1,44)=34.464, P<0.0001; Figure 5c). Next, we investigated the behavioral response to amphetamine challenge in adult mice, which had received VEH or FLX during P22–P41. We detected an effect of peri-adolescent treatment, an effect of amphetamine and a peri-adolescent x adult treatment interaction (F(1,18)=4.719, P=0.0434, F(1,18)=4.480, P=0.0485 and F(1,18)=4.484, P=0.0484, respectively; Figure 5d). To our surprise, P22–P41 FLX treated mice displayed no behavioral response to amphetamine, while P22–P41 VEH-treated littermates did respond as expected (Figure 5d). We hypothesized that peri-adolescent FLX treatment shifts the amphetamine dose–response curve as opposed to making mice generally insensitive to amphetamine. Hence we tested the effect of amphetamine challenge at 5 mg kg−1 in adult mice, which had received VEH, CLO or FLX during P22–P41. Analyzing behavior as a function of amphetamine dose, we detected an effect of peri-adolescent treatment and an effect of amphetamine dose (F(2,30)=6.364, P=0.005, F(2,60)=58.639, P<0.0001, respectively; Supplementary Figure S1). Post hoc analysis revealed that FLX treated mice do behaviorally respond to amphetamine (F(2,8)=6.259, P=0.0231), but that their response is diminished (Supplementary Figure S1). Finally, we tested if peri-adolescent GBR treatment impacts adult amphetamine response. Strengthening the positive correlation between aggressive behavior and a sensitized/hyperactive DA system, we found that P22–P41 GBR-treated mice display an exaggerated response to adult amphetamine challenge when compared with adult VEH injection or P22–P41 VEH-treated mice (effect of peri-adolescent treatment: F(1,21)=9.273, P=0.0062; effect of amphetamine challenge: F(1,21)=40.730, P<0.0001; peri-adolescent treatment x adult amphetamine challenge interaction: F(1,21)=6.847, P=0.0161, Figure 5e). These data demonstrate that P22–P41 CLO, GBR and FLX treatment differentially impact the sensitivity of the adult DA system. Furthermore, the behavioral sensitivity to amphetamine correlates positively with levels of adult aggression.
Optogenetic stimulation of VTA DAergic neurons increases aggressive behavior
Peri-adolescent monoamine levels impact adult amphetamine response and aggressive behavior, suggesting that altered DAergic sensitivity is determining aggression levels. Alternatively, DAergic activity may be correlated with but not causally related to aggressive phenotypes. To investigate these alternative hypotheses, we studied the direct casual relationship between DAergic neuronal activity and aggressive behavior using optogenetics.
To selectively target the dopaminergic system, we crossed mice expressing Cre-recombinase under the control of the dopamine transporter promoter (DATIRESCre)24 with a ROSA26-reporter line conditionally expressing ChR2-eYFP in a Cre-dependent fashion (ai32 line)25 (Figure 6a). Optical fibers were implanted over the VTA region in double-mutant mice (DATIRESCre:ai32) and in single-mutant controls (Figures 6b and c). In order to assess aggressive behavior, we used the isolation-induced aggression paradigm, pairing DATIRESCre:ai32 mice with single-mutant controls. Blue light pulses (473 nm, 20 Hz, 10 ms pulse duration) were delivered to the VTA for the duration of the test. Only one mouse of the pair was stimulated during each 10 min encounter. Under these conditions, we detected an effect of genotype on aggressive behavior (F(1,27)=5.146, P=0.0315), with stimulation of DatIRESCre:ai32 mice leading to significantly longer time engaging in aggressive contacts, when compared with stimulation of single-mutant controls (Figure 6d).
These data demonstrate that VTA DAergic neuronal activity is causally related to aggressive behaviors.
Our experiments demonstrate the existence of two developmental periods during which early-life perturbation of monoamine signaling alters adult behavior: an early postnatal (P2–P21) 5-HT-sensitive period that affects anxiety - and depression-related behaviors and a later peri-adolescent (P22–P41) 5-HT and DA-sensitive period altering aggression. Serotonergic and dopaminergic signaling perturbations during peri-adolescence also exert profound and opposite effects on the behavioral response to psychostimulant challenge in adulthood, suggesting that altered DA function underlies the changes in aggressive behaviors. In support of this hypothesis, we demonstrate that direct optogenetic stimulation of VTA DAergic neurons increases aggressive behavior.
Our findings can explain the paradoxical high neophobia/anxiety phenotypes of 5-htt−/− mice,26,30 maoa−/− mice11, 12, 13 and pre-synaptic htr1a deficient mice,31 because these mouse lines lack their respective gene products throughout life, including the sensitive P2–P21 period. Likewise, the model can explain the increased aggression seen in constitutive maoa, dat and comt loss-of-function mouse lines.11,12,32,33 The moderating effect of developmental 5-HTT blockade on adult aggressive behavior does not follow the strict timing specificity (5-HTT blockade during P2–P21 or P22–P41 both decreased adult aggression), but nonetheless supports a developmental mechanism for the low-aggression phenotype of 5-htt−/− mice.34 Because we performed developmental manipulations in wild-type mice but not in the respective mutant mouse lines, we can only compare phenotypes qualitatively but not quantitatively. Hence, we cannot draw conclusions as to whether the phenotypes we have observed are fully or only partially recapitulating the magnitude of behavioral consequences elicited by the life-long genetic mutations. Furthermore, we cannot exclude the involvement of off-target effects or downstream developmental effects in contributing to the phenotypes examined here. For example, even though clorgyline preferentially inhibits MAOA, it might also inhibit MAOB in our experiments. To overcome this drawback, we base our conclusions not on single drug effects but on the convergence of multiple pharmacological interventions that resonate with a large literature on genetic effects in both mice and humans. For example, our conclusion that increased DAergic activity during P22–41 increases adult aggression and the behavioral response to amphetamine, is supported by our finding that we can mimic the effect of CLO through DAT but not 5-HTT or NET blockade.
Our findings comport with human vulnerabilities to anxiety, depression and aggression conferred by functional genetic polymorphisms. For example, endophenotypes of affective disorders have been associated with low-expressing alleles for the 5-htt,35, 36, 37, 38, 39 maoa9,40 and presynaptic htr1a.41 Likewise, aggressive behavior has been associated with loss-of-function and low-expressing maoa alleles,7,8,42,43 the 10R variant of dat1,44,45 and the low activity met allele of the comt.46 Our model predicts that these risk alleles act primarily during sensitive developmental periods to alter brain maturation and circuit formation leading to altered behaviors. The temporal dynamics of transcription in the human brain and their genetic moderation support this hypothesis.47 An important aspect to consider when comparing our interventions or gene ablation studies in mice to genetic risk factors in humans is that human polymorphisms rarely lead to complete loss of function whereas the murine interventions often do. Thus, it is possible that in humans multiple risk factors that impact monoamine signaling during adolescence must converge to elicit a phenotype.
Based on our data, pharmacologic interventions during sensitive periods of human development would be predicted to impact affective and aggressive behavior. The murine P2–P21 period roughly corresponds to the third trimester of human gestation and early childhood, while P22–P41 corresponds to peri-adolescent development.48 Thus, SSRIs taken by pregnant mothers might impact fetal brain maturation and adult mood and anxiety, while stimulant exposure during peri-adolescence could alter adult aggressive behavior. Indeed, chronic stimulant exposure increases aggressive behavior in rodents, non-human primates and humans,49, 50, 51 even in abstinent stimulant users.52 The long-term effects of gestational SSRI exposure on affect, aggression and cognition are unknown,15,53 but prenatal SSRI exposure has been associated with a twofold increased risk of autism spectrum disorder.54
Our findings support the fundamental notion that neural circuitry and consequently behavior are most vulnerable to long-lasting modulation during periods of maturation and high plasticity.4,55 The 5-HT sensitive period described here coincides with the emergence of anxiety- and fear-related behaviors in rodents and the maturation of the underlying circuitry. For example, at around P10, the consequence of odor shock conditioning switches from preference to aversion, with amygdala engagement and hypothalamic–pituitary–adrenal axis integration.56,57 Limbic structures including amygdala, prefrontal cortex and hippocampus are maturing during postnatal development and human s allele carriers as well as 5-htt−/− mice exhibit disrupted mPFC-amygdala functional connectivity that likely arises during early circuit maturation.58,59 The DA-sensitive, peri-adolescent period coincides with the developmental onset of play fighting in rodents60,61 and non-human primates,62 and human adolescence is characterized by dynamic changes in aggressive behavior.63
Insight into how developmental monoamine signaling establishes long-lasting alterations in brain function and behavior remains scarce. The best-studied example is the establishment of the cyto- and circuit-architecture in the somatosensory cortex of mice. MAOA and 5-HTT deficient mice lack topographically defined barrel fields.17, 18, 19,64 This deficit is caused by increased 5-HT signaling through 5-HT1B receptors located on thalamocortical axon terminals, which may inhibit the glutamate release17 necessary for the formation of barrel columns.65 The sensitive period for this developmental process starts at P0 and ends at P4,19 and therefore partially dissociates from the 5-HT and fully dissociates from the DA-sensitive period under investigation here. Thus, thalamocortical wiring abnormalities are unlikely to contribute to the emotional and aggressive phenotypes. Focusing on the peri-adolescent period, we identified changes in monoamine levels and more importantly, changes in behavioral reactivity to amphetamine challenge that correlate with aggressive behavior. Specifically, we report that increased adult aggressive behavior elicited by peri-adolescent MAOA or DAT blockade correlates with increased locomotor-stimulating effects of amphetamine challenge in adulthood. Conversely, reduced adult aggressive behavior elicited by peri-adolescent 5-HTT blockade correlates with decreased locomotor-stimulating effects of amphetamine challenge in adulthood. Supporting phylogenetic conservation, human individuals with antisocial traits also show mesolimbic dopamine hypersensitivity to amphetamine, as impulsivity is positively correlated with the magnitude of amphetamine-induced DA release in the striatum.66 Highlighting the notion that altered DAergic signaling impacts adult behavior as a function of developmental timing, transient pre-adolescent methylphenidate exposure decreases responsiveness to cocaine’s locomotor-activating effects in adult rats.67 In contrast, transient adult methylphenidate exposure does not alter cocaine’s locomotor-activating effects in later adulthood.67
The positive correlation between levels of aggression and behavioral sensitivity to amphetamine across our peri-adolescent treatment regimes comports with the DA hypothesis of aggression, which states that DAergic hyperfunction increases aggression.29,68 However, the DA hypothesis of aggression is largely based on correlative data and has thus far not been tested using direct and specific activation of DAergic neuronal activity. We here therefore performed such an experiment using optogenetics and demonstrate the existence of a direct causal relationship between DAergic neuronal activity and aggressive behavior. Hence, our data support a model in which monoamine signaling during peri-adolescence permanently alters the sensitivity/activity of DAergic neurons/pathways to impact adult aggression. However, many questions remain unanswered: are DAergic changes located pre-synaptically or post-synaptically? Do they occur at the cell-intrinsic level or do they involve altered micro or macrocircuitry? How do DA and 5-HT exert their opposing effects on behavior and how is DA dominance established (aggressive MAOA deficient mice have both, elevated 5-HT and DA levels)? Future studies examining these questions will be critical to understand both the mechanism for the establishment of DAergic sensitivity levels during development as well as the nature of alterations in adult DA-related circuit function.
Taken together, our findings support the existence of sensitive periods that influence life-long vulnerability to anxiety, depression, aggression and substance abuse. Such sensitive periods have been most extensively characterized for sensory systems (for example, visual cortex), but conceptually similar principles may apply to the development and organization of brain circuitry that mediate the more complex behaviors described here. With increased knowledge about sensitive periods during which genetic and environmental factors act to confer risk for neuropsychiatric disorders, we will not only be able to improve classification and diagnosis, but also treatment and prevention.
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We thank D Lin, A Teissier, and C Kellendonk for their critical review of the manuscript. This work was supported by the Sackler Institute for Developmental Psychobiology and the National Institute of Mental Health (R01 MH080116; R00 MH083044; MH062185).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Molecular Psychiatry website
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Yu, Q., Teixeira, C., Mahadevia, D. et al. Dopamine and serotonin signaling during two sensitive developmental periods differentially impact adult aggressive and affective behaviors in mice. Mol Psychiatry 19, 688–698 (2014). https://doi.org/10.1038/mp.2014.10
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