Maturation of Nucleus Accumbens Synaptic Transmission Signals a Critical Period for the Rescue of Social Deficits in a Mouse Model of Autism Spectrum Disorder

doi:10.21203/rs.3.rs-2588108/v1

Download PDF

Research Article

Maturation of Nucleus Accumbens Synaptic Transmission Signals a Critical Period for the Rescue of Social Deficits in a Mouse Model of Autism Spectrum Disorder

https://doi.org/10.21203/rs.3.rs-2588108/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 24 May, 2023

Read the published version in Molecular Brain →

You are reading this latest preprint version

Social behavior emerges early in development, a time marked by the onset of neurodevelopmental disorders featuring social deficits, including autism spectrum disorder (ASD). Although social deficits are at the core of the clinical diagnosis of ASD, very little is known about their neural correlates at the time of clinical onset. The nucleus accumbens (NAc), a brain region extensively implicated in social behavior, undergoes synaptic, cellular and molecular alterations in early life, and is particularly affected in ASD mouse models. To explore a link between the maturation of the NAc and neurodevelopmental deficits in social behavior, we compared spontaneous synaptic transmission in NAc shell medium spiny neurons (MSNs) between the highly social C57BL/6J and the idiopathic ASD mouse model BTBR T+Itpr3tf/J at postnatal day (P) 4, P6, P8, P12, P15, P21 and P30. BTBR NAc MSNs display increased spontaneous excitatory transmission during the first postnatal week, and increased inhibition across the first, second and fourth postnatal weeks, suggesting accelerated maturation of excitatory and inhibitory synaptic inputs compared to C57BL/6J mice. BTBR mice also show decreased optically evoked medial prefrontal cortex-NAc synaptic transmission at P15 and P30. These early changes in synaptic transmission are consistent with a potential critical period, which could maximize the efficacy of rescue interventions. To test this, we treated BTBR mice in either early life (P4-P8) or adulthood (P60-P64) with the mTORC1 antagonist rapamycin, a well-established intervention for ASD-like behavior. Rapamycin treatment rescued social interaction deficits in BTBR mice when injected in infancy, but not in adulthood.

Development

social interaction

rapamycin

BTBR

electrophysiology

Social behaviors, which include social communication, interaction, parental and reproductive behaviors^1,2, enable intraspecies interactions necessary to find food, mate and avoid predation³. Consistent with their key role in survival, early deficits in the development of social interaction and communication have devastating consequences⁴⁵, and are a hallmark of several neurodevelopmental disorders⁶, including Fragile X syndrome⁷, schizophrenia⁸, and autism spectrum disorder (ASD)⁹. ASD encompasses a set of heterogeneous neurodevelopmental disorders affecting an estimated 1% of the world population⁹, and features impairments in social cognition, communication and social perception¹⁰. Even though deficits in social interaction and communication are at the core of clinical diagnoses for neurodevelopmental disorders such as ASD¹¹, very little is known about the neural basis of such deficits, particularly at the time of clinical onset and diagnosis¹².

Evidence points to conserved brain regions underlying social processing across species¹³. In particular, the rodent nucleus accumbens (NAc) and its projections^14,15 have been extensively implicated in multiple facets of social behavior, including social interaction and motivation^16–18, social cognition¹⁹, social approach^20,21, social memory^22–24, social-spatial representations²⁵ and susceptibility to social stress^26–30. Consistent with its important role in social behavior, imaging studies of ASD patients show abnormal activity in NAc^31,32, as well as changes in functional connectivity within frontostriatal^33–35 and striatal³⁶ circuits. Accordingly, ASD mouse models display altered NAc synaptic plasticity and transmission^37–39, as well as changes in gene expression of inhibitory and excitatory markers⁴⁰ in adult animals. Importantly, manipulations targeted to the NAc can mimic⁴¹ or rescue behavior deficits in a range of ASD mouse models^37,42–44, further implicating the NAc in ASD pathophysiology.

Recent evidence has highlighted the value and long-term benefits of early ASD interventions^45,46, but the precise time window of optimal efficacy for these interventions has not been clearly defined. In the developing sensory cortex, plasticity in response to external stimuli is curtailed within critical periods⁴⁷, suggesting that similar mechanisms could guide the efficacy of rescue interventions in other brain regions. As with sensory cortex development, the rodent NAc also undergoes early postnatal changes in synaptic transmission^48–52 and plasticity⁵³, in the levels of dopamine receptors^54–57 ⁵⁸, dopamine/serotonin transporters^59,60, stimulus-evoked dopamine transients⁶¹, complement C3⁵⁸ and microglia⁶², which stabilize around the start of adolescence. To determine whether the timing of these developmental changes defines a window of increased efficacy for interventions, they must be examined in animal models featuring early social deficits, paralleling the clinical presentation of ASD.

Several studies have identified rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), as an effective pharmacological intervention in ASD animal models^63–66. mTOR is a critical regulator of cell growth, proliferation, and metabolism^67,68, as well as synaptic plasticity and memory consolidation^69–71. mTOR is part of two structurally and functionally distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), of which only mTORC1 shows sensitivity to acute inhibition by rapamycin⁶⁸. Importantly, impaired mTORC1 signaling has been implicated in ASD and related neurodevelopmental disorders^67,72–74, consistent with the success of rapamycin treatment in rescuing social behavior deficits in ASD mouse models^75–77.

Here, we compared the maturation of spontaneous excitatory and inhibitory synaptic transmission of NAc shell medium spiny neurons (MSNs) of C57BL/6J mice and a mouse model of idiopathic ASD, the BTBR T⁺ Itpr3^tf/J (BTBR)^78,79 mouse strain. BTBR mice display face validity to all three core ASD behavioral symptoms: deficits in social communication^80–82, deficits in social interaction^79,83–85 and repetitive behaviors^79,84–86. Specifically, when compared to the highly social C57BL/6J strain, BTBR mice display changes in ultrasonic vocalizations, a well-established metric of social communication^87,88, starting from infancy, with a remarkably reduced repertoire of calls as early as postnatal day (P)8⁸⁰, matching the early social deficits seen in ASD patients⁶. We found that both strains show age-dependent increases in NAc shell MSN spontaneous excitatory and inhibitory transmission, particularly at the start of the second postnatal week. Compared to C57BL/6J, BTBR mice show changes in spontaneous transmission from as early as P4, with increased excitation during the first postnatal week and increased inhibition in the first, second and fourth weeks. Additionally, infralimbic cortex (IL) optically evoked transmission onto BTBR NAc MSNs is decreased at P15 and P30 as revealed by paired pulse ratios. Based on these data, we next tested the efficacy of targeting treatment with the mTORC1 inhibitor rapamycin to the age of onset of maturational changes in NAc synaptic transmission. We found that rapamycin treatment was successful at rescuing BTBR deficits in social interactions when applied in infancy, but not in adulthood, suggesting that early changes in synaptic transmission may act as a marker for a critical period maximizing the efficacy of therapeutic interventions.

Animals
C57BL/6J (Jackson Laboratory) and BTBR T+ Itpr3tf/J (Jackson Laboratory; referred to as BTBR for simplicity) mouse strains were bred at the University of Toronto Scarborough and kept on a 12h light/dark cycle (lights on at 07:00 h) with access to food and water ad libitum. Date of birth was assigned P0. Approximately equal numbers of females and males were used for social behavior, and males were used for slice electrophysiology experiments. Offspring were weaned with same-sex siblings on P21 (2-4 mice per cage). All animal procedures were approved by the Animal Care Committee at the University of Toronto.

Stereotaxic Surgery

C57BL/6J and BTBR male mice underwent surgery at P8 or P23. Mice were given an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and placed in a stereotaxic frame (Stoelting). AAV1.CaMKIIa.hChR2(H134R)-eYFP.WPRE.hGH virus (Addgene) was infused bilaterally in the infralimbic cortex (IL; coordinates relative to bregma: P8: AP +1.2 mm, ML ± 0.25 mm, and DV –1.7 mm; P23: AP +1.8 mm, ML ± 0.25 mm, and DV –2.6 mm). Viral particles (0.2 µl/hemisphere) were delivered at a rate of 100 nl/minute by way of a motorized microsyringe. After viral infusion, the needle was left in place for five additional minutes to allow for diffusion of the virus, and then was slowly removed from the injection site. Mice were then returned to their home cages for seven days to allow for recovery and viral expression. Whole-cell patch-clamp recording experiments were performed at P15 or P30.

Slice Electrophysiology

Electrophysiological recordings were obtained from male C57BL/6J and BTBR mice at different postnatal ages (P4, P6, P8, P12, P15, P21 and P30). Mice were deeply anesthetized using isoflurane and the brain was quickly removed and placed in ice-cold sucrose-based cutting solution containing (in mM): 180 sucrose, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 1 CaCl₂, 2 MgCl₂, 2 Na⁺ pyruvate and 0.4 L-Ascorbic acid with 95% O₂ and 5% CO₂. Coronal brain slices containing the NAc (250 µm- thick) were prepared with a vibratome (Leica VT1000S). Slices were then incubated for 30 min at 30 °C in a recovery solution composed of 50% sucrose-based cutting solution and 50% artificial cerebrospinal fluid (ACSF). The ACSF was composed of (in mM): 120 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 11 glucose, 2 CaCl₂, 1 MgCl₂, 2 Na⁺ pyruvate and 0.4 L-Ascorbic acid with 95% O₂ and 5% CO₂. Slices were then placed in regular ASCF for another 30 min at room temperature before electrophysiology recordings. After recovery, slices were placed in the recording chamber and perfused with 2ml/min oxygenated ACSF at room temperature for recordings.

Whole-cell patch-clamp recordings were obtained from NAc shell MSNs using borosilicate glass pipettes (3-5 ΩM; WPI) filled with intracellular solution containing (in mM): 120 Cs-methanesulfonate, 10 HEPES, 0.5 EGTA, 8 NaCl, 4 Mg-ATP, 1 QX-314, 10 Na-phosphocreatine, and 0.4 Na-GTP, pH 7.3, at 290 mOsmol. Spontaneous excitatory (sEPSCs) and inhibitory (sIPSCs) postsynaptic currents were recorded from the same cell at holding potentials of -60 mV and 0 mV, respectively, and five minutes of stable recordings were analyzed for frequency and amplitude (miniAnalysis; Synaptosoft).

For ex vivo optogenetic experiments, IL axon terminals were stimulated using TTL-pulsed microscope objective-coupled LEDs (460 nm, ∼1 mW/mm2; Prizmatix) and light-evoked EPSCs were recorded from MSNs at holding potential of -60 mV. Glutamate release probability from IL terminals to NAc shell MSNs was assessed by measuring paired-pulse ratio by dividing the amplitude of the second light-evoked EPSC by the amplitude of the first EPSC at inter-stimulus intervals of 250 and 100 ms for P15 and P30 respectively. All electrophysiology recordings were obtained in the absence of synaptic blockers.

Electrophysiology data were acquired using a MultiClamp 700B (Molecular Devices), digitized at 10 kHz using Digidata 1550A and pClamp 10 (Molecular Devices). Recordings were low pass filtered at 4 kHz. Access resistance was regularly assessed during experiments and data were included only if the holding current was stable and access resistance varied <20% of initial value.

Behavior

The three-chamber social interaction and social memory tests were conducted following established protocols from the Crowley group⁸⁹. Tests took place in a 60x20x40cm plexiglass apparatus with three 20x20x40cm chambers interconnected by two 7x40cm vertically removable plexiglass doors. Two 8.5x8.5x40 cm enclosures with three 0.3cm vertical slits per side were placed at the lateral wall of the right and left chambers and used to hold social target animals as required in the tasks. The chamber was elevated 41 cm off the floor and a camera was mounted 75 cm above the chamber on a metal rack.

On the day prior to behavior testing, C57BL/6J and BTBR mice were socially isolated for an hour in a separate room. On the day of testing, mice were socially isolated for an hour, followed immediately by habituation to the apparatus for 10 minutes. Following habituation, mice underwent the three-chamber social interaction test, in which an age-, strain- and sex-matched stranger mouse was placed in one of the enclosures in a counterbalanced manner. Experimental mice were placed in the middle chamber at the start of the experiment, and opening of the doors marked the start of the social interaction test. Mice were able to freely explore the three chambers for a total of 10 minutes.

Social memory tests took place one hour after the social interaction test. For the social memory tests, two mice were placed in the enclosures: the stranger mouse used in the social interaction test (now familiar mouse), and a novel age, strain and sex-matched mouse, that the experimental mouse had never previously encountered (stranger mouse). The experimental mouse was placed in the middle chamber with the doors closed. Following the opening of the doors, the animal was free to explore the three chambers for 10 minutes.

Social behavior was conducted during the late phase of the light cycle. A subset of animals had undergone social interaction and social memory tests in the dark cycle, but similar to what has been described in the literature⁹⁰, we saw no behavioral differences between light and dark cycles for these strains, and therefore consolidated the datasets. Behavior was analyzed using ANY-maze® software, and cross validated through manual scoring by an experimenter blind to experimental conditions.

Drugs

The stock solution of rapamycin (50 mg/kg in 100% ethanol; LC laboratories; Woburn, MA; U.S.A) was stored at -80^oC. Stock solution was diluted in 10% of polyethylene glycol 400 (PEG400) and 10% Tween 80 to a final concentration of 1mg/ml in 2% ethanol and stored at -20^oC ⁶⁶. Mice received an intraperitoneal injection of rapamycin (0.5mg/kg) or vehicle (10% PEG400, 10% Tween; 1mg/kg)⁶⁶ for five consecutive days, from either P4 to P8 or P60 to P64.

Perfusions and Sectioning
BTBR mice were treated with vehicle or rapamycin from P4-P8 and were perfused at P30 for immunohistochemistry against phosphoS6. At P30, behaviorally naïve mice were injected with Avertin (250mg/kg, i.p.) and once deeply anesthetized, were transcardially perfused with 0.1M phosphate buffered saline (PBS), followed by 4 % paraformaldehyde (PFA). The brains were then extracted and stored overnight at 4°C in 4% PFA. The brains were sectioned using a vibratome (VT 1000, Leica) to obtain 50 µm coronal sections, which were stored in a 60% glycerol and 0.01% sodium azide in PBS solution at -20^oC.

Immunohistochemistry

Sections containing the NAc shell were treated with 0.3% Triton X-100 in PBS for 15 minutes, followed by 10% normal goat serum in 0.1% Triton X-100 in PBS for 1 hour. Sections were incubated with rabbit monoclonal anti-phospho-S6S^240/244(pS6; 1:1000; Cell Signaling, Beverly, MA, RRID:AB 10694233) for 48h, followed by secondary antibody incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500; Jackson Immunoresearch Laboratories) and Hoechst dye (1:1000; Thermo Fisher Scientific) for 90 minutes. Sections were mounted on glass slides (Thermo Fisher Scientific) with Permafluor aqueous mounting medium (Thermo Fisher Scientific) and coverslipped for storage at 4°C.

Image Acquisition and Quantification

Image acquisition was performed using a Nikon Eclipse Ni-U epiluorescence microscope. Immunofluorescence was visualized with an LED illumination system (X-Cite 120 LED Boost, Excelitas Technologies) and captured with a Nikon DS-Qi2 digital camera. All images were acquired at 10x magnification using Plan-Apochromat differential interference contrast (DIC) N1. When brightness and/or contrast adjustments were made in a figure, these changes were made equally to all photomicrographs.

Cell counts were done manually using Fiji/ImageJ software (v. 1.53e) by an experimenter blind to treatment conditions. Three to five sections per subject containing the NAc shell were used for cell counts. For each section, the integrated density in the NAc shell was acquired, corrected for background, and averaged to obtain the mean integrated density per animal.

Statistical Analysis
Data are presented as mean ± standard error of the mean. All statistical analyses were performed in Graphpad Prism® version 9. Potential sex differences were first assessed using a two- or three-way, repeated measures ANOVA and given the absence of effects, data were pooled for subsequent analyses. Exploration time in all chambers in the social interaction and social memory tests was analyzed by two-way, repeated-measures ANOVA followed by Sidak’s post-hoc tests. Distance travelled was analyzed using unpaired two-tailed t tests. For electrophysiology experiments and immunohistochemistry, paired t-tests and Wilcoxon signed-rank tests were used for within-group comparisons, unpaired t-tests and one- or two-way repeated measures ANOVA followed by Tukey’s and Sidak’s post-hoc tests respectively were used for normally distributed data and Mann-Whitney Rank Sum test when data did not pass normality. For all analyses, p<0.05 was considered significant.

Maturation of spontaneous excitatory currents of medium spiny neurons of the nucleus accumbens shell of C57BL/6J and BTBR mice.

To examine the maturation of spontaneous excitatory and inhibitory synaptic transmission of the NAc shell in C57BL/6J and BTBR mice, whole cell patch-clamp recordings were obtained from visually identified MSNs (Fig. 1A). In MSNs from both strains, spontaneous excitatory postsynaptic currents (sEPSCs) were observed as early as P4 and developed gradually during the first postnatal month (Fig. 1). In C57BL/6J mice, sEPSC frequency showed a significant increase at P15 and P21 with a further increase at P30 (Fig. 1B-C, one way ANOVA, F_{(6, 64)}= 25.40 p<0.0001, Tukey’s multiple comparison tests: P4 vs P15, p=0.02; P4 vs P21, p=0.0003; P4 vs P30, p<0.0001; P6 vs P15, p=0.04; P6 vs P21, p=0.0004; P6 vs P30, p<0.0001; P8 vs P21, p=0.0017; P8 vs P30, p<0.0001; P12 vs P30, p<0.0001; P15 vs P30, p<0.0001; P21 vs P30, p<0.0001). However, in BTBR mice, a marked increase in sEPSC frequency was observed at P12, with a further increase from P21 (Fig. 1B, D, one way ANOVA, F_{(6, 66)} = 14.86 p<0.0001, Tukey’s multiple comparison tests: P4 vs P12, p=0.007; P4 vs 21 and P30, p<0.0001; P6 vs P21, p=0.0001; P6 vs P30, p<0.0001; P8 vs P21, p=0.0007; P8 vs P30, p<0.0001; P12 vs P30, p=0.0007; P15 vs P21, p=0.004; P15 vs P30, p<0.0001). Interestingly, sEPSC amplitude displayed a distinct maturation profile. While C57BL/6J mice showed increased sEPSC amplitude between P6-P8 and P15 and P30 (Fig. 1E, one way ANOVA, F_{(6, 66)} = 7.429 p<0.0001, Tukey’s multiple comparison tests: P6 vs P15, p=0.0004; P6 vs P30, p=0.0005; P8 vs P15, p=0.0003; P8 vs P30, p=0.0003), the amplitude of sEPSCs in MSNs from BTBR mice was decreased at P8, P12 and P21 relative to P4 (Fig. 1F, one way ANOVA, F_{(6, 70)} = 3.480 p=0.0046, Tukey’s multiple comparison tests: P4 vs P8, p=0.002; P4 vs P12, p=0.03; P4 vs P21, p=0.01). Furthermore, sEPSC time constant displayed a marked increase during the second (P12, P15) and third (P21) postnatal weeks in C57BL/6J mice and similarly during the second (P8) and third (P21) postnatal weeks in BTBR mice (Fig. 1G, one way ANOVA, F_{(6, 67)} = 5.602 p<0.0001, P4 vs P12, p=0.006; P4 vs P15, p=0.0006; P4 vs P21, p<0.0001; Fig. 1H, one way ANOVA, F_{(6, 70)} = 3.121 p=0.0091; P4 vs P8, p=0.04; P4 vs P21, p=0.02). Overall, these data show that MSNs from BTBR mice display slightly earlier increases in sEPSC frequency and distinct age-dependent changes in sEPSC amplitude, but broadly equivalent timeline of maturation of sEPSC kinetics.

We then examined the maturation of inhibitory inputs onto MSNs in both strains. Spontaneous inhibitory postsynaptic current (sIPSC) frequency increased significantly during the second postnatal week for both strains and plateaued from P15 (Fig. 2A-B, C57BL/6J: one way ANOVA, F_{(6, 61)} = 10.07 p<0.0001, Tukey’s multiple comparison tests: P4 vs P15, p<0.0001; P4 vs P21, p=<0.0001; P4 vs P30, p=0.004; P6 vs P15, p=0.0003; P6 vs P21, p=0.0003; P6 vs P30, p=0.01; P8 vs P15, p=0.0004; P8 vs P21, p=0.0005; P8 vs P30, p=0.03; Fig. 2A,C, BTBR, one way ANOVA, F_{(6, 61)} =5.73 p<0.0001, Tukey’s multiple comparison tests: P4 vs P15, p=0.0031, P4 vs P30, p=0.0053; P6 vs P15, p=0.007; P6 vs P30, p=0.011; P12 vs P15, p=0.017, P12 vs P30, p=0.025). In contrast, while sIPSC amplitude increased significantly and plateaued from P15 in C57BL/6J mice (Fig. 2D, C57BL/6J: one way ANOVA, F_{(6, 61)}= 7.931 p<0.0001; Tukey’s multiple comparison tests: P4 vs P15, p=0.0031; P6 vs P15, p<0.0001; P6 vs P21, p=0.0002; P8 vs P15, p=0.0009; P8 vs P21, p=0.033; P12 vs P15, p=0.041), BTBR mice only showed a slight increase at P21 (Fig. 2E, BTBR: one way ANOVA F_{(6, 62)} = 4.04, p=0.0018; Tukey’s multiple comparison tests: P8 vs P21, p=0.023; P15 vs P21, p=0.044). Interestingly, sIPSC time constant displayed a distinct maturation profile between MSNs from C57BL/6J and BTBR mice. While in C57BL/6J mice sIPSC time constant was stable across all ages tested (Fig. 2F; one way ANOVA, F_{(6, 60)} = 2.186 p=0.0567), sIPSC tau showed a significant increase between P4 and P30 in MSNs from BTBR mice (Fig. 2G; one way ANOVA, F_{(6, 65)} = 3.481 p=0.0048, Tukey’s multiple comparison tests: P4 vs P30, p=0.01). Overall, these data show broadly synchronous developmental changes in sIPSC frequency between strains, with BTBR mice showing slightly delayed increases in sIPSC amplitude and distinct maturation of sIPSC decay time.

To better understand strain differences in NAc shell MSN spontaneous excitatory and inhibitory transmission in early life, we directly compared MSNs sEPSC and sIPSC parameters between C57BL/6J and BTBR mice across ages (Fig. 3). We found that BTBR mice exhibited reduced sEPSC frequency at P30 (Fig. 3A; two way ANOVA, significant effects of age F_{(6, 130)} = 39.16, p<0.0001, strain F_{(1, 130)}=5.65, p=0.02, and age x strain interaction F_{(6, 130)}=4.9, p=0.0002; Sidak’s multiple comparisons P30, p<0.0001) but increased sEPSC amplitude at P4 and P6 relative to C57BL/6J mice (Fig. 3B; two way ANOVA, significant effects of age F_{(1, 136)}=6.27, p<0.0001, and age x strain interaction F_{(6, 136)}=4.36, p=0.0005; Sidak’s multiple comparisons P4, p=0.01; P6, p=0.04). Comparison of spontaneous inhibitory synaptic transmission revealed that BTBR mice showed higher sIPSC frequency at P8 and sIPSCs amplitude at P6, P12 and P30 compared to C57BL/6J mice (Fig. 3C-D; sIPSC frequency: two way ANOVA, significant effects of age F_(6,122)=12.90, p<0.0001, and strain F_{(6, 122)}=14.2, p=0.0003; Sidak’s multiple comparisons P8, p=0.02; sIPSC amplitude: two way ANOVA, significant effects of age F_{(6, 123)}=7.08, p<0.0001, strain F_{(6, 123)}=4.55, p<0.0001, and age x strain interaction F_{(6, 123)}=4.55, p=0.0003; Sidak’s multiple comparisons P6, p=0.004, P12, p=0.01, P30, p=0.009). Consistent with this, comparison of within-cell differences between the frequency of spontaneous excitation and inhibition showed a shift toward synaptic inhibition at P30 in BTBR mice relative to age-matched C57BL/6J mice (Fig. 3E; two way ANOVA, significant effects of age F_{(6, 120)}=5.51, p<0.0001, strain F_{(6, 120)}=23.47, p<0.0001, and age x strain interaction F_{(6, 120)}=3.74, p=0.0019; Sidak’s multiple comparisons P30, p<0.0001). In conclusion, these data show that MSNs from BTBR mice display higher excitatory inputs during the first postnatal week, which is reduced by P30. Inhibitory synaptic inputs onto MSNs from BTBR mice were stronger during the first, second and fourth postnatal weeks than onto MSNs from C57BL/6J mice, suggesting differences in the maturation of excitatory and inhibitory transmission in MSNs of BTBR mice. At P30, C57BL/6J and BTBR mice show opposite profiles of excitation-inhibition balance, with predominant excitation over inhibition for C57BL/6J and inhibition over excitation for BTBR mice. The temporal profile of changes in NAc synaptic transmission in BTBR mice coincides with the onset of social deficits in this strain⁸⁰, suggesting these electrophysiological differences might contribute to the early changes in behavior.

Evidence suggests specialization in the contribution of NAc input and output connections to social processing^20,91–93, with optogenetic manipulation of medial prefrontal cortex (PFC)-NAc projections in particular modulating different aspects of social behavior^23,27,30,94. To test whether PFC-NAc transmission might be altered in BTBR mice, we injected AAV-ChR2 virus into the infralimbic cortex (IL) subdivision of the PFC of C57BL/6J and BTBR mice one week prior to conducting patch clamp recordings from NAc shell MSNs at P15 and P30 (Fig. 4A). IL-expressing ChR2 terminals onto MSNs were stimulated using a brief light-pulse (5ms) through the objective, and glutamate release probability at IL-NAc synapses was examined by measuring paired-pulse ratios (PPR). Light-evoked EPSCs from MSNs of BTBR mice showed higher PPR at both P15 and P30 compared to C57BL/6J mice, indicating reduced presynaptic glutamate release probability at IL-NAc synapses (Fig. 4B-C; unpaired t tests, P15: t=2.13, p=0.04; P30: t=5.91, p<0.0001).

Given the temporal coincidence of alterations in NAc synaptic transmission and behavior in the BTBR strain, targeting intervention strategies to this window may improve treatment outcomes. Informed by our electrophysiological data showing changes in NAc spontaneous transmission as early as P4 (Figs 1-3), we decided to target a well-established rescue strategy for animal models of ASD, the mTORC1 antagonist rapamycin⁹⁵, to early development, starting at P4. We first confirmed that BTBR mice displayed social interaction deficits in our lab by testing them on a three-chamber social interaction test, followed by a social memory test. We tested animals at P30, a crucial time in social development⁹⁶. In the social interaction test, BTBR mice spent less time with the social target and more time in the empty compartment, compared to C57BL/6J mice (Fig. 5A-B; two-way RM ANOVA, significant effect of chamber F_{(1.536, 90.61)}= 38.79, p<0.0001 and chamber x strain interaction F_{(2, 118)}= 16.71, p<0.0001; Sidak’s multiple comparisons test, empty p=0.0074, middle p=0.0030, and social p=0.0006 chambers).

While BTBR social interaction deficits consistent with ours have been widely reported^79,97, social memory deficits in the BTBR strain have a more conflicting literature^75,98,99. We found no differences in social memory, i.e. preference for the novel, unfamiliar mouse, between C57BL/6J and BTBR mice (Fig. 5C-D; two-way RM ANOVA, significant effect of chamber F_{(2, 110)}= 39.63, p<0.0001 and strain F_{(1, 55)}= 10.23 p=0.0023; Sidak's multiple comparisons test showed significant differences between strains only in the middle chamber t=2.775 p=0.0268; p>0.84 for other chambers). Importantly, although our behavioral data showed no sex differences in social interaction (p>0.64) or social memory (p>0.11), replicating a widely reported absence of sex differences in social behavior in these strains^79,84,85,89, we presently cannot exclude the possibility of sex differences in the underlying maturation of NAc synaptic transmission.

To test whether the increased synaptic excitation and inhibition onto MSNs from BTBR mice starting at the first postnatal week could signal the start of a sensitive period in the BTBR strain, we targeted rapamycin treatment, a drug known to rescue social deficits in other mouse models of ASD^65,66, from P4 to P8, and tested animals for social preference at P30 (Fig. 6A). Rapamycin treatment led to a significant increase in time spent in the social chamber (Fig. 6B; two-way RM ANOVA, significant effect of chamber F_{(2, 48)}= 22.42, p<0.0001, and chamber x treatment interaction F_{(2, 48)}= 9.436, p<0.0003; Sidak's multiple comparisons test show significant differences between vehicle and rapamycin treatment in the empty p=0.0293 and social p=0.0002 chambers), indicating a rescue of social preference in BTBR mice treated with rapamycin from P4-P8. We saw no difference in distance travelled between vehicle- and rapamycin-treated groups (Fig. 6C; unpaired t test, t₁₉=0.2272, p=0.8227), suggesting that the social interaction effects did not stem from changes in motor activity. These data indicate that early rapamycin treatment was able to rescue BTBR social deficits at adolescence. As an additional validation, we confirmed that early rapamycin treatment decreased phosphorylation of S6 ribosomal protein, a commonly used measure of mTOR pathway activity¹⁰⁰, in P30 BTBR mice (Additional File 1, Supplementary Fig. 1A-B; unpaired t test, t₆=3.2, p=0.01).

A study by Burket and colleagues had shown that a four-day rapamycin treatment starting at P28 also rescued social deficits in BTBR mice tested 1h after the last injection¹⁰¹. To test whether the efficacy of the rapamycin rescue was tied to its timing during early life, we injected an additional cohort of BTBR mice with the same rapamycin regimen and interval between treatment and testing, but starting at P60 (Fig. 6D). We found that rapamycin treatment starting at early adulthood did not affect the time spent in the social chamber (Fig. 6E; two-way RM ANOVA, significant effect of chamber F_{(2, 34)}= 11.32, p=0.0002; Sidak's multiple comparisons test show no significant differences between vehicle and rapamycin treatment in any of the chambers) or distance travelled (Fig. 6F; unpaired t test, t₁₇=0.052, p=0.9591). Overall, these data indicate that P60 rapamycin treatment did not rescue social interaction deficits in BTBR mice, suggesting that early intervention with rapamycin is necessary for improving social interaction in the BTBR strain.

In this study, we compared the maturation of NAc shell MSN spontaneous synaptic transmission in C57BL/6J and BTBR strains from infancy to adolescence. In both strains, MSNs displayed excitatory and inhibitory spontaneous currents by P4, suggesting the presence of functional excitatory and inhibitory synapses in the NAc early during development. Excitatory and inhibitory inputs onto MSNs developed gradually up to P12 and underwent a marked maturation by P15, when levels plateaued. These data suggest that synaptic inputs onto NAc MSNs establish early during development with a slow maturation during the first 10-12 postnatal days, followed by a potentiation of synaptic inputs onto MSNs by P15. This is consistent with other reports showing stable NAc paired pulse ratio (PPR) of AMPA-mediated EPSCs from juvenility⁵⁰, and changes in AMPAR- and NMDAR-mediated EPSCs up to the second postnatal week (but decrease of NMDA-EPSC later during development)⁵².

Overall, we found age-dependent changes in the maturation of spontaneous excitatory and inhibitory transmission within and between strains, with BTBR mice showing increased excitation during the first postnatal week, which decreased by P30, as well as increased spontaneous inhibition across early development. Additionally, BTBR mice display IL-NAc paired-pulse facilitation at P15 and P30 compared to C57BL/6J mice, suggesting reduced presynaptic glutamate release from IL terminals onto MSNs in BTBR mice. The difference in PPR between C57BL/6J and BTBR mice at P15 and P30 was observed using long (250 ms) and short (100 ms) interstimulus intervals, respectively, which might be influenced by developmental differences in presynaptic short-term plasticity, as reported previously^102–104

In the Shank3B^-/- mouse model of ASD, an accelerated maturation of striatal spiny projecting neurons (SPNs) of the dorsomedial striatum (DMS) was observed during early developmental stages (third and fourth postnatal weeks), followed by a decrease in corticostriatal activity during adulthood (P60)^105,106 - a deficit that was attributed to an overactivation of cortical circuits. Moreover, the authors found that increasing anterior cingulate cortex (ACC) synaptic drive to SPNs of DMS at P8 significantly increased SPN mEPSC frequency, whereas increasing ACC activity from P15 and older reduced SPN mEPSC frequency¹⁰⁶, suggesting a developmental switch in the effect of cortical hyperactivity on SPN synaptic transmission. While our study showed that MSNs from BTBR mice receive weaker synaptic inputs from IL at P15 and P30 relative to MSNs from C57BL/6J mice, it remains unknown whether a similar switch occurs in their response to IL hypofunction.

Given the developmental timeline of NAc shell MSN spontaneous transmission, we hypothesized that early postnatal development might constitute a critical period for the efficacy of rescue manipulations correlating with improved outcomes. To test this, we subjected BTBR mice to rapamycin treatment in infancy (P4-P8) or adulthood (P60-64) and tested its effect on social preference in a three-chamber social interaction test. We found that rapamycin treatment in infancy, but not adulthood, reversed the social interaction deficits in BTBR mice. Our data point to a critical period for the efficacy of rapamycin treatment in BTBR mice from P4-P8 which closes by P60. Work by Burket and colleagues showed that rapamycin rescues BTBR social interaction deficits when administered between P28-P31, with animals tested one hour after the last injection¹⁰¹. While this study did not probe the long-term effects of rapamycin treatment on social behavior, it reinforces the value of rapamycin as an effective rescue intervention in BTBR mice. Furthermore, it points to a longer window of efficacy for rapamycin treatment in this strain, likely spanning from infancy to early adolescence. Consistent with this, we found differences in NAc spontaneous transmission between BTBR and C57BL/6J mice up to P30. In the rat, changes in NAc intrinsic excitability and excitatory transmission predominantly occur within the first three postnatal weeks, reaching adult levels by P35 or earlier^50,51. A study by Zhang and Warren saw changes in rat NAc excitatory transmission stabilizing by P15, with the amplitude of NMDA responses peaking from P9-12, which they propose is a critical period for this brain region⁵². Additionally, Liu and colleagues reported higher sEPSC frequency and amplitude, as well as lower halfwidth, rise and decay time in the NAc shell of P21-P28 rats compared to adults¹⁰⁷, further pointing to the first postnatal month as a developmental window of heightened changes in NAc synaptic transmission.

Nardou and colleagues had described a critical period for social reward learning in the NAc¹⁰⁸, which peaks at P42, and closes by P98¹⁰⁸. This learning window coincides with age-dependent changes in oxytocin-induced long-term synaptic depression in the NAc^16,108. Interestingly, NAc oxytocin signaling also modulates social approach²¹, suggesting its signaling and developmental time course might also influence social interaction. Another NAc microcircuit regulator, μ-opioid receptor (MOR), modulates social interaction and NAc synaptic transmission¹⁰⁹, as well as social behavior in juveniles such as play behavior^110,111 and social preference¹¹². Furthermore, mice lacking a functional MOR gene (Oprm1^-^/^- mice), an ASD mouse model, display increased oxytocin receptor expression in the NAc, with their social deficits reversed by oxytocin treatment¹¹³. NAc MOR levels^114,115 and function¹¹⁶ also change across development, suggesting that the interplay between NAc MOR and oxytocin might contribute to the shaping of social behavior in early life.

While a wide array of acute or short-term interventions rescue social interaction deficits in adult ^{75,97,117–140} and young^141–146 BTBR mice, very few probed their long-term efficacy. Pharmacological rescue interventions yielding long-term rescue of BTBR social deficits predominantly target mice up to the third postnatal week^147–151, with effects lasting until adulthood. To our knowledge, no other study compared the efficacy of a rescue intervention across different ages in BTBR mice. In an interesting study⁴¹, deletion of Shank3 in the NAc at a similar time period to ours (<P6) triggered social deficits in adulthood that were not replicated when the manipulation started at P90, corroborating the existence of a critical period for NAc-associated behavior and susceptibility to long-lasting interventions. Notably, the NAc is not the only region contributing to the regulation of social behavior in the BTBR strain, as seen with adult manipulations of cerebellum¹⁵² and PFC¹⁵³, as well as changes in spontaneous transmission in hippocampus¹³³. While we saw decreased presynaptic efficacy in the PFC-NAc pathway of BTBR mice, its precise contribution to behavioral deficits in this strain remains unclear.

Our data delineate the maturation of spontaneous synaptic transmission in the NAc shell across early postnatal development, uncovering distinct early maturation signatures between C57BL/6J and BTBR mice, strains that display distinct patterns of NAc-mediated social behavior. These synaptic changes coincide with key developmental changes in social communication, play, affiliative and social approach behavior^154–158, and abridge a period in which environmental conditions and stress bear a disproportionate impact in shaping future social behavior^159–163. Accordingly, we revealed a restricted time window for the rescue of social interaction deficits in BTBR mice by rapamycin treatment. These findings emphasize the importance of matching experimental conditions to clinically relevant timelines, and highlight the power of leveraging research in early development toward precisely targeted interventions that maximize positive outcomes.

AMPA – α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

ACC - Anterior cingulate cortex

ASD – Autism spectrum disorder

DMS - Dorsomedial striatum

IL – Infralimbic cortex

MOR - μ-opioid receptor

MSN – Medium spiny neuron

mTOR - Mammalian target of rapamycin

NAc – Nucleus accumbens

NMDA – N-methyl-D-aspartate

PFC – Medial prefrontal cortex

sEPSC – Spontaneous excitatory postsynaptic current

sIPSC – Spontaneous inhibitory postsynaptic current

SPN - Striatal spiny projecting neurons

Ethics approval and consent to participate

All animal procedures were approved by the Animal Care Committee at the University of Toronto.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information file.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by a University of Toronto Scarborough Postdoctoral Fellowship to MM, and grants from the SickKids Foundation and Canadian Institutes of Health Research (CIHR) – Institute of Human Development, Child and Youth Health (NI19-1132R), CIHR (PJT 399790), Human Frontier Science Program Organization (CDA00009/2018 and RGY0072/2019), and Natural Sciences and Engineering Research Council of Canada (RGPIN-2017-06344) to MAC. A Canada Foundation for Innovation grant (#493864) was used to establish the Centre for the Neurobiology of Stress (CNS) Core Facility.

Author Contributions:

MM, AK, CJS and MAC designed research. MM, AK, CJS, MD, UM and MAC performed research. MM, CJS, AK, MD and MAC analyzed/interpreted data. AK and MAC wrote the paper. All authors reviewed and approved the manuscript.

Acknowledgements

We thank Mehreen Inayat and Ryan Appings for help with mouse breeding and Jennifer Wilkin for help with the design of figure schematics. We acknowledge resources and support from the Centre for the Neurobiology of Stress (CNS) Core Facility at the University of Toronto Scarborough.

Ko, J. Neuroanatomical Substrates of Rodent Social Behavior: The Medial Prefrontal Cortex and Its Projection Patterns. Front Neural Circuits 11, 41 (2017).
Robinson, G. E., Fernald, R. D. & Clayton, D. F. Genes and social behavior. Science (80-. ). 322, 896–900 (2008).
Chen, P. & Hong, W. Neural Circuit Mechanisms of Social Behavior. Neuron 98, 16–30 (2018).
Rutter, M. et al. Quasi-autistic patterns following severe early global privation. English and Romanian Adoptees (ERA) Study Team. J. Child Psychol. Psychiatry. 40, 537–549 (1999).
Volkmar, F. R. & Pauls, D. Autism. Lancet 362, 1133–1141 (2003).
Ozonoff, S., Heung, K., Byrd, R., Hansen, R. & Hertz-Picciotto, I. The Onset of Autism: Patterns of Symptom Emergence in the First Years of Life. Autism Res. 1, 320–328 (2008).
Garber, K., Visootsak, J. & Warren, J. Fragile X syndrome. Eur. J. Hum. Genet. 1, 666–672 (2008).
Penn, D. L., Corrigan, P. W., Bentall, R. P., Racenstein, J. M. & Newman, L. Social Cognition in Schizophrenia. Psychol. Bull. 121, 114–132 (1997).
Levy, S. E., Mandell, D. S. & Schultz, R. T. Autism. Lancet 374, 1627–1638 (2009).
Lai, M., Lombardo, M. V & Baron-cohen, S. Autism. Lancet 383, 896–910 (2014).
Constantino, J. N. & Charman, T. Diagnosis of autism spectrum disorder : reconciling the syndrome , its diverse origins , and variation in expression. Lancet Neurol. 15, 279–291 (2016).
Courchesne, E. et al. Mapping Early Brain Development in Autism. Neuron 56, 399–413 (2007).
Insel, T. R. & Fernald, R. D. How the brain processes social information : Searching for the Social Brain. Annu. Rev. Neurosci. 27, 697–722 (2004).
Beckstead, R. M. An Autoradiographic Examination of Corticocortical and Subcortical Projections of the Mediodorsal-Projection ( Prefrontal ) Cortex in the Rat. J Comp Neurol 43–62 (1979).
Oades, R. D. & Halliday, G. M. Ventral tegmental ( A10) system : neurobiology . 1 . Anatomy and connectivity. Brain Res. Rev. 12, 117–165 (1987).
Dölen, G., Darvishzadeh, A., Huang, K. W. & Malenka, R. C. Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin. Nature 501, 179–184 (2013).
Gunaydin, L. A. et al. Natural neural projection dynamics underlying social behavior. Cell 157, 1535–1551 (2014).
Yizhar, O. et al. Neocortical excitation / inhibition balance in information processing and social dysfunction. Nature 477, 171–178 (2011).
Bicks, L. K., Koike, H., Akbarian, S. & Morishita, H. Prefrontal Cortex and Social Cognition in Mouse and Man. Front. Psychol. 6, 1–15 (2015).
Rogers-Carter, M. M., Djerdjaj, A., Gribbons, K. B., Varela, J. A. & Christianson, J. P. Insular cortex projections to nucleus accumbens core mediate social approach to stressed juvenile rats. J. Neurosci. 39, 8717–8729 (2019).
Williams, A. V. et al. Social approach and social vigilance are differentially regulated by oxytocin receptors in the nucleus accumbens. Neuropsychopharmacology 45, 1423–1430 (2020).
Okuyama, T., Kitamura, T., Roy, D., Itohara, S. & Tonegawa, S. Ventral CA1 neurons store social memory. Science (80-. ). 353, 1536–1541 (2016).
Park, G. et al. Social isolation impairs the prefrontal-nucleus accumbens circuit subserving social recognition in mice. Cell Rep. 35, 109104 (2021).
Xing, B. et al. A Subpopulation of Prefrontal Cortical Neurons Is Required for Social Memory. Biol. Psychiatry 89, 521–531 (2021).
Murugan, M. et al. Combined Social and Spatial Coding in a Descending Projection from the Prefrontal Cortex Combined Social and Spatial Coding in a Descending Projection from the Prefrontal Cortex. Cell 1–15 (2017) doi:10.1016/j.cell.2017.11.002.
Heshmati, M. et al. Depression and social defeat stress are associated with inhibitory synaptic changes in the nucleus accumbens. J. Neurosci. 40, 6228–6233 (2020).
Bagot, R. C. et al. Ventral hippocampal afferents to the nucleus accumbens regulate susceptibility to depression. Nat. Commun. 6, 7062 (2015).
Nam, H. et al. Reduced nucleus accumbens enkephalins underlie vulnerability to social defeat stress. Neuropsychopharmacology 44, 1876–1885 (2019).
Chaudhury, D. et al. Rapid regulation of depression-related behaviours by control of midbrain dopamine neurons. Nature 493, 532–6 (2013).
Vialou, V. et al. Prefrontal Cortical Circuit for Depression- and Anxiety- Related Behaviors Mediated by Cholecystokinin : Role of ⌬ FosB. J. Neurosci. 34, 3878–3887 (2014).
Dichter, G. S. et al. Reward circuitry function in autism spectrum disorders. SCAN 160–172 (2012) doi:10.1093/scan/nsq095.
Nehrkorn, B. et al. Reward system dysfunction in autism spectrum disorders. SCAN 565–572 (2013) doi:10.1093/scan/nss033.
Zeeland, A. A. S., Dapretto, M., Ghahremani, D. G., Poldrack, R. A. & Bookheimer, S. Y. Reward Processing in Autism. Autism Res. 3, 53–67 (2010).
D’Cruz, A.-M., Mosconi, M. W., Ragozzino, M. E., Cook, E. H. & Sweeney, J. A. Alterations in the functional neural circuitry supporting fl exible choice behavior in autism spectrum disorders. Transl. Psychiatry 1–9 (2016) doi:10.1038/tp.2016.161.
Langen, M. et al. Fronto-striatal circuitry and inhibitory control in autism : Findings from diffusion tensor imaging tractography. Cortex 48, (2012).
Martino, A. Di et al. Aberrant Striatal Functional Connectivity in Children with Autism. BPS 69, 847–856 (2010).
Rothwell, P. E. et al. Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors. Cell 158, 198–212 (2014).
Neuhofer, D. et al. Functional and structural deficits at accumbens synapses in a mouse model of Fragile X. Front. Cell. Neurosci. 9, 1–15 (2015).
Platt, R. J. et al. Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits. Cell Rep. 19, 335–350 (2017).
Bukatova, S. et al. Shank3 Deficiency is Associated With Altered Profile of Neurotransmission Markers in Pups and Adult Mice. Neurochem. Res. 46, 3342–3355 (2021).
Tzanoulinou, S. et al. Inhibition of Trpv4 rescues circuit and social deficits unmasked by acute inflammatory response in a Shank3 mouse model of Autism. Mol. Psychiatry (2022) doi:10.1038/s41380-021-01427-0.
Walsh, J. J. et al. 5-HT release in nucleus accumbens rescues social deficits in mouse autism model. Nature 560, 589–594 (2018).
Folkes, O. M. et al. An endocannabinoid-regulated basolateral amygdala-nucleus accumbens circuit modulates sociability. J. Clin. Invest. 130, 1728–1742 (2020).
Choe, K. Y. et al. Oxytocin normalizes altered circuit connectivity for social rescue of the Cntnap2 knockout mouse. Neuron 110, 795-808.e6 (2022).
Pickles, A. et al. Parent-mediated social communication therapy for young children with autism ( PACT ): long-term follow-up of a randomised controlled trial. Lancet 388, 2501–2509.
Estes, A. et al. Long-Term Outcomes of Early Intervention in 6-Year-Old Children With Autism Spectrum Disorder. J. Am. Acad. Child Adolesc. Psychiatry 54, 580–587 (2015).
Hensch, T. K. Critical period plasticity in local cortical circuits. Nat. Rev. Neurosci. 6, 877–88 (2005).
Huppe-Gourgues, F. & O’Donnell, P. Periadolescent changes of D(2) -AMPA interactions in the rat nucleus accumbens. Synapse 66, 1–8 (2012).
Huppé-Gourgues, F. & O’donnell, P. D 1-NMDA receptor interactions in the rat nucleus accumbens change during adolescence. Synapse 66, 584–591 (2012).
Kasanetz, F. & Manzoni, O. J. Maturation of excitatory synaptic transmission of the rat nucleus accumbens from juvenile to adult. J. Neurophysiol. 101, 2516–2527 (2009).
Belleau, M. L. & Warren, R. A. Postnatal development of electrophysiological properties of nucleus accumbens neurons. J. Neurophysiol. 84, 2204–2216 (2000).
Zhang, L. & Warren, R. A. Postnatal development of excitatory postsynaptic currents in nucleus accumbens medium spiny neurons. Neuroscience 154, 1440–1449 (2008).
Schramm, N. L., Egli, R. E. & Winder, D. G. LTP in the mouse nucleus accumbens is developmentally regulated. Synapse 45, 213–219 (2002).
Stanwood, G. D., McElligot, S., Lu, L. & McGonigle, P. Ontogeny of dopamine D3 receptors in the nucleus accumbens of the rat. Neurosci. Lett. 223, 13–16 (1997).
Tarazi, F. I. & Baldessarini, R. J. Comparative postnatal development of dopamine D1, D2, and D4 receptors in rat forebrain. Int. J. Dev. Neurosci. 18, 29–37 (2000).
Leslie, C. A., Robertson, M. W., Cutler, A. J. & Bennett, J. P. Postnatal development of D 1 dopamine receptors in the medial prefrontal cortex, striatum and nucleus accumbens of normal and neonatal 6-hydroxydopamine treated rats: a quantitative autoradiographic analysis. Dev. Brain Res. 62, 109–114 (1991).
Teicher, M. H., Andersen, S. L. & Hostetter, J. C. Evidence for dopamine receptor pruning between adolescence and adulthood in striatum but not nucleus accumbens. Dev. Brain Res. 89, 167–172 (1995).
Kopec, A. M., Smith, C. J., Ayre, N. R., Sweat, S. C. & Bilbo, S. D. Microglial dopamine receptor elimination defines sex-specific nucleus accumbens development and social behavior in adolescent rats. Nat. Commun. 9, (2018).
Tarazi, F. I., Tomasini, E. C. & Baldessarini, R. J. Postnatal development of dopamine and serotonin transporters in rat caudate-putamen and nucleus accumbens septi. Neurosci. Lett. 254, 21–24 (1998).
Coulter, C. L., Happe, H. K. & Murrin, L. C. Postnatal development of the dopamine transporter: A quantitative autoradiographic study. Dev. Brain Res. 92, 172–181 (1996).
Robinson, D. L., Zitzman, D. L., Smith, K. J. & Spear, L. P. Fast dopamine release events in the nucleus accumbens of early adolescent rats. Neuroscience 176, 296–307 (2011).
Hope, K. T., Hawes, I. A., Moca, E. N., Bonci, A. & De Biase, L. M. Maturation of the microglial population varies across mesolimbic nuclei. Eur. J. Neurosci. 52, 3689–3709 (2020).
Ehninger, D. et al. Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis. Nat Med 14, 843–848 (2008).
Sato, A. et al. Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex. Nat Commun 3, 1292 (2012).
Xing, X. et al. Suppression of Akt-mTOR pathway rescued the social behavior in Cntnap2-deficient mice. Sci Rep 9, 3041 (2019).
Amegandjin, C. A. et al. Sensitive period for rescuing parvalbumin interneurons connectivity and social behavior deficits caused by TSC1 loss. Nat. Commun. 12, 1–18 (2021).
Costa-Mattioli, M. & Monteggia, L. M. mTOR complexes in neurodevelopmental and neuropsychiatric disorders. Nat. Neurosci. 16, 1537–1543 (2013).
Laplante, M. & Sabatini, D. M. MTOR signaling in growth control and disease. Cell 149, 274–293 (2012).
Tischmeyer, W. et al. Rapamycin-sensitive signalling in long-term consolidation of auditory cortex-dependent memory. Eur. J. Neurosci. 18, 942–950 (2003).
Dash, P. K., Orsi, S. A. & Moore, A. N. Spatial memory formation and memory-enhancing effect of glucose involves activation of the tuberous sclerosis complex-mammalian target of rapamycin pathway. J. Neurosci. 26, 8048–8056 (2006).
Tang, S. J. et al. A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proc. Natl. Acad. Sci. U. S. A. 99, 467–472 (2002).
Hooshmandi, M., Wong, C. & Khoutorsky, A. Dysregulation of translational control signaling in autism spectrum disorders. Cell. Signal. 75, (2020).
Costa, V. et al. MTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis. Cell Rep. 15, 86–95 (2016).
Huber, K. M., Klann, E., Costa-Mattioli, M. & Zukin, R. S. Dysregulation of mammalian target of rapamycin signaling in mouse models of autism. J. Neurosci. 35, 13836 (2015).
Steinmetz, A. B., Stern, S. A., Kohtz, A. S., Descalzi, G. & Alberini, C. M. Insulin-like growth factor II targets the mTOR pathway to reverse autism-like phenotypes in mice. J. Neurosci. 38, 1015–1029 (2018).
Tsai, P. et al. Autistic-like behavior and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature 488, 647–651 (2012).
Pagani, M. et al. mTOR-related synaptic pathology causes autism spectrum disorder-associated functional hyperconnectivity. Nat. Commun. 12, (2021).
Meyza, K. Z. & Blanchard, D. C. The BTBR mouse model of idiopathic autism – Current view on mechanisms. Neurosci. Biobehav. Rev. 76, 99–110 (2017).
Mcfarlane, H. G., Kusek, G. K. & Yang, M. Autism-like behavioral phenotypes in BTBR T 1 tf / J mice. Genes, Brain Behav. 7, 152–163 (2008).
Scattoni, M. L., Gandhy, S. U., Ricceri, L. & Crawley, J. N. Unusual Repertoire of Vocalizations in the BTBR T + tf / J Mouse Model of Autism. PLoS One 3, 48–52 (2008).
Scattoni, M. L., Ricceri, L., Crawley, J. N., Elena, V. R. & Superiore, I. Unusual repertoire of vocalizations in adult BTBR T + tf / J mice during three types of social encounters. Genes, Brain Behav. 10, 44–56 (2011).
Wohr, M., Roullet, F. I. & Crawley, J. N. Reduced scent marking and ultrasonic vocalizations in the BTBR T + tf / J mouse model of autism. Genes, Brain Behav. 10, 35–43 (2011).
Bolivar, V. J., Walters, S. R. & Phoenix, J. L. Assessing autism-like behavior in mice : Variations in social interactions among inbred strains. 176, 21–26 (2007).
Yang, M., Zhodzishsky, V. & Crawley, J. N. Social deficits in BTBR T + tf / J mice are unchanged by cross-fostering with C57BL / 6J mothers. Int. J. Devl Neurosci. 25, 515–521 (2007).
Yang, M., Clarke, A. M. & Crawley, J. N. Postnatal lesion evidence against a primary role for the corpus callosum in mouse sociability. Eur. J. Neurosci. 29, 1663–1677 (2009).
Silverman, J. L., Tolu, S. S., Barkan, C. L. & Crawley, J. N. Repetitive Self-Grooming Behavior in the BTBR Mouse Model of Autism is Blocked by the mGluR5 Antagonist MPEP. Neuropsychopharmacology 35, 976–989 (2009).
Scattoni, M. L., Crawley, J. & Ricceri, L. Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders. Neurosci Biobehav Rev 33, 508–515 (2009).
Segbroeck, M. Van et al. NeuroResource MUPET — Mouse Ultrasonic Profile ExTraction : A Signal Processing Tool for Rapid and Unsupervised Analysis of Ultrasonic Vocalizations NeuroResource MUPET — Mouse Ultrasonic Profile ExTraction : A Signal Processing Tool for Rapid and Unsuper. Neuron 94, 465-485.e5 (2017).
Silverman, J. L., Yang, M., Lord, C. & Crawley, J. N. Behavioural phenotyping assays for mouse models of autism. Nat. Rev. Neurosci. 11, (2010).
Yang, M. et al. Social approach behaviors are similar on conventional versus reverse lighting cycles, and in replications across cohorts, in BTBR T+ tf/J, C57BL/6J, and vasopressin receptor 1B mutant mice. Front. Behav. Neurosci. 1, 1–9 (2007).
Folkes, O. M. et al. An endocannabinoid-regulated basolateral amygdala-nucleus accumbens circuit modulates sociability. J Clin Invest 130, 1728–1742 (2020).
Park, G. et al. Social isolation impairs the prefrontal-nucleus accumbens circuit subserving social recognition in mice. Cell Rep. 35, (2021).
Qi, J. et al. VTA glutamatergic inputs to nucleus accumbens drive aversion by acting on GABAergic interneurons. Nat. Neurosci. 19, 725–733 (2016).
Murugan, M. et al. Combined Social and Spatial Coding in a Descending Projection from the Prefrontal Cortex. Cell 171, 1663-1677.e16 (2017).
Rapamycin, R. & Lamming, D. W. Inhibition of the Mechanistic Target of. Cold Spring Harb. Perspect. Med. 6, 1–14 (2016).
Sachser, N., Zimmermann, T. D., Hennessy, M. B. & Kaiser, S. Sensitive phases in the development of rodent social behavior. Curr. Opin. Behav. Sci. 36, 63–70 (2020).
Ruskin, D. N. et al. Ketogenic Diet Improves Core Symptoms of Autism in BTBR Mice. PLoS One 8, 4–9 (2013).
Cope, E. C. et al. Atypical perineuronal nets in the CA2 region interfere with social memory in a mouse model of social dysfunction. Mol Psychiatry 27, 3520–3531 (2022).
Zilkha, N., Kuperman, Y. & Kimchi, T. High-fat diet exacerbates cognitive rigidity and social deficiency in the BTBR mouse model of autism. Neuroscience 345, 142–154 (2017).
Biever, A., Valjent, E. & Puighermanal, E. Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function. Front Mol Neurosci 8, 75 (2015).
Burket, J. A., Benson, A. D., Tang, A. H. & Deutsch, S. I. Rapamycin improves sociability in the BTBR T+Itpr3tf/J mouse model of autism spectrum disorders. Brain Res. Bull. 100, 70–75 (2014).
Bornschein, G., Brachtendorf, S. & Schmidt, H. Developmental Increase of Neocortical Presynaptic Efficacy via Maturation of Vesicle Replenishment. Front. Synaptic Neurosci. 11, 1–10 (2020).
Kim, J. & Alger, B. E. Paired-Pulse Facilitation. J. Neurosci. 21, 9608–9618 (2001).
Speed, H. E. & Dobrunz, L. E. Developmental changes in short-term facilitation are opposite at temporoammonic synapses compared to schaffer collateral synapses onto CA1 pyramidal cells. Hippocampus 19, 187–204 (2009).
Peixoto, R. T., Wang, W., Croney, D. M., Kozorovitskiy, Y. & Sabatini, B. L. Early hyperactivity and precocious maturation of corticostriatal circuits in Shank3B(-/-) mice. Nat Neurosci 19, 716–724 (2016).
Peixoto, R. T. et al. Abnormal Striatal Development Underlies the Early Onset of Behavioral Deficits in Shank3B(-/-) Mice. Cell Rep 29, 2016-2027 e4 (2019).
Liu, X. D. et al. Different physiological properties of spontaneous excitatory postsynaptic currents in nucleus accumbens shell neurons between adult and juvenile rats. Neurosci. Lett. 496, 80–83 (2011).
Nardou, R. et al. Oxytocin-dependent reopening of a social reward learning critical period with MDMA. Nature 569, 116–120 (2019).
Toddes, C., Lefevre, E. M., Brandner, D. D., Zugschwert, L. & Rothwell, P. E. Μ-Opioid Receptor (Oprm1) Copy Number Influences Nucleus Accumbens Microcircuitry and Reciprocal Social Behaviors. J. Neurosci. 41, 7965–7977 (2021).
Trezza, V., Damsteegt, R., Marijke Achterberg, E. J. & Vanderschuren, L. J. M. J. Nucleus accumbens μ-opioid receptors mediate social reward. J. Neurosci. 31, 6362–6370 (2011).
Manduca, A. et al. Interacting cannabinoid and opioid receptors in the nucleus accumbens core control adolescent social play. Front. Behav. Neurosci. 10, 1–17 (2016).
Smith, C. J. W., Wilkins, K. B., Li, S., Tulimieri, M. T. & Veenema, A. H. Nucleus accumbens mu opioid receptors regulate context-specific social preferences in the juvenile rat. Psychoneuroendocrinology 89, 59–68 (2018).
Gigliucci, V. et al. Region specific up-regulation of oxytocin receptors in the opioid Oprm1 -/- mouse model of autism. Front. Pediatr. 2, 1–12 (2014).
Chang, L. et al. Early life stress alters opioid receptor mRNA levels within the nucleus accumbens in a sex-dependent manner. Brain Res. 1710, 102–108 (2019).
Albores-Garcia, D., McGlothan, J. L., Bursac, Z. & Guilarte, T. R. Chronic developmental lead exposure increases μ-opiate receptor levels in the adolescent rat brain. Neurotoxicology 82, 119–129 (2021).
Talbot, J. N., Happe, H. K. & Murrin, L. C. μ Opioid receptor coupling to Gi/o proteins increases during postnatal development in rat brain. J. Pharmacol. Exp. Ther. 314, 596–602 (2005).
Deng, W. et al. Effect of metformin in autistic BTBR T + Itpr3tf/J mice administered a high-fat diet. Brain Res. Bull. 183, 172–183 (2022).
Lv, H. et al. Nanoformulated Bumetanide Ameliorates Social Deficiency in BTBR Mice Model of Autism Spectrum Disorder. Front. Immunol. 13, 1–13 (2022).
Wei, H. et al. Inhibition of IL-6 trans-signaling in the brain increases sociability in the BTBR mouse model of autism. Biochim. Biophys. Acta - Mol. Basis Dis. 1862, 1918–1925 (2016).
Albekairi, N. A. et al. Pharmacology , Biochemistry and Behavior CXCR2 antagonist SB332235 mitigates deficits in social behavior and dysregulation of Th1 / Th22 and T regulatory cell-related transcription factor signaling in male BTBR T + Itpr3 tf / J mouse model of autism. 217, (2022).
Kazdoba, T. M., Hagerman, R. J., Zolkowska, D., Rogawski, M. A. & Crawley, J. N. Evaluation of the neuroactive steroid ganaxolone on social and repetitive behaviors in the BTBR mouse model of autism. Psychopharmacology (Berl). 233, 309–323 (2016).
Chadman, K. K. Fluoxetine but not risperidone increases sociability in the BTBR mouse model of autism. Pharmacol. Biochem. Behav. 97, 586–594 (2011).
Yoshimura, R. F. et al. Allosteric modulation of nicotinic and GABAA receptor subtypes differentially modify autism-like behaviors in the BTBR mouse model. Neuropharmacology 126, 38–47 (2017).
Jayaprakash, P. et al. Curcumin potentiates α7 nicotinic acetylcholine receptors and alleviates autistic-like social deficits and brain oxidative stress status in mice. Int. J. Mol. Sci. 22, 1–17 (2021).
Chao, O. Y. et al. Altered dopaminergic pathways and therapeutic effects of intranasal dopamine in two distinct mouse models of autism. Mol. Brain 13, 1–16 (2020).
Wei, D. et al. Enhancement of Anandamide-Mediated Endocannabinoid Signaling Corrects Autism-Related Social Impairment. Cannabis Cannabinoid Res. 1, 81–89 (2016).
Mahmood, H. M. et al. The Role of Nicotinic Receptors in the Attenuation of Autism-Related Behaviors in a Murine BTBR T + tf/J Autistic Model. Autism Res. 13, 1311–1334 (2020).
Wang, L. et al. Modulation of social deficits and repetitive behaviors in a mouse model of autism: The role of the nicotinic cholinergic system. Psychopharmacology (Berl). 232, 4303–4316 (2015).
Gould, G. G. et al. Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice. Prog. Neuro-Psychopharmacology Biol. Psychiatry 38, 260–269 (2012).
Karvat, G. & Kimchi, T. Acetylcholine elevation relieves cognitive rigidity and social deficiency in a mouse model of autism. Neuropsychopharmacology 39, 831–840 (2014).
Silverman, J. L. et al. GABA B Receptor Agonist R-Baclofen Reverses Social Deficits and Reduces Repetitive Behavior in Two Mouse Models of Autism. Neuropsychopharmacology 40, 2228–2239 (2015).
Kratsman, N., Getselter, D. & Elliott, E. Sodium butyrate attenuates social behavior deficits and modifies the transcription of inhibitory/excitatory genes in the frontal cortex of an autism model. Neuropharmacology 102, 136–145 (2016).
Han, S., Tai, C., Jones, C. J., Scheuer, T. & Catterall, W. A. Enhancement of inhibitory neurotransmission by GABAA receptors having α2,3-subunits ameliorates behavioral deficits in a mouse model of autism. Neuron 81, 1282–1289 (2014).
Cai, Y. et al. Citalopram attenuates social behavior deficits in the BTBR T+Itpr3tf/J mouse model of autism. Brain Res. Bull. 150, 75–85 (2019).
Venkatachalam, K. et al. The histamine H3R and dopamine D2R/D3R antagonist ST-713 ameliorates autism-like behavioral features in BTBR T+tf/J mice by multiple actions. Biomed. Pharmacother. 138, (2021).
Eissa, N. et al. Simultaneous blockade of histamine h3 receptors and inhibition of acetylcholine esterase alleviate autistic-like behaviors in btbr t+ tf/j mouse model of autism. Biomolecules 10, 1–20 (2020).
Nadeem, A. et al. Nrf2 activator, sulforaphane ameliorates autism-like symptoms through suppression of Th17 related signaling and rectification of oxidant-antioxidant imbalance in periphery and brain of BTBR T+tf/J mice. Behav. Brain Res. 364, 213–224 (2019).
Cai, Y. et al. Autism-like behavior in the BTBR mouse model of autism is improved by propofol. Neuropharmacology 118, 175–187 (2017).
Silverman, J. L., Oliver, C. F., Karras, M. N., Gastrell, P. T. & Crawley, J. N. Neuropharmacology AMPAKINE enhancement of social interaction in the BTBR mouse model of autism. 64, 268–282 (2013).
Cristiano, C. et al. Palmitoylethanolamide counteracts autistic-like behaviours in BTBR T+tf/J mice: Contribution of central and peripheral mechanisms. Brain. Behav. Immun. 74, 166–175 (2018).
Wu, H. et al. Selenium inhibits ferroptosis and ameliorates autistic-like behaviors of BTBR mice by regulating the Nrf2/GPx4 pathway. Brain Res. Bull. 183, 38–48 (2022).
Perets, N., Hertz, S., London, M. & Offen, D. Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice. Mol. Autism 9, 1–12 (2018).
Hagen, E. et al. Autism-Like Behavior in BTBR Mice Is Improved by Electroconvulsive Therapy. Neurotherapeutics 12, 657–666 (2015).
Bales, K. L. et al. Long-term exposure to intranasal oxytocin in a mouse autism model. Transl. Psychiatry 4, 1–10 (2014).
Queen, N. J. et al. Environmental enrichment improves metabolic and behavioral health in the BTBR mouse model of autism. Psychoneuroendocrinology 111, (2020).
PARK, H. Y. et al. Humulus japonicus rescues autistic-like behaviours in the BTBR T+ Itpr3tf/J mouse model of autism. Mol. Med. Rep. 23, 1–8 (2021).
Cui, J. et al. General Anesthesia During Neurodevelopment Reduces Autistic Behavior in Adult BTBR Mice, a Murine Model of Autism. Front. Cell. Neurosci. 15, 1–10 (2021).
Wang, L., Cai, Y. & Fan, X. Metformin administration during early postnatal life rescues autistic-like behaviors in the BTBR T+ Itpr3tf/J mouse model of autism. Front. Behav. Neurosci. 12, 1–10 (2018).
Zhong, H. et al. Neonatal curcumin treatment restores hippocampal neurogenesis and improves autism-related behaviors in a mouse model of autism. Psychopharmacology (Berl). 237, 3539–3552 (2020).
Cai, Y. et al. The liver X receptor agonist TO901317 ameliorates behavioral deficits in two mouse models of autism. Front. Cell. Neurosci. 13, 1–12 (2019).
Nettleton, J. E. et al. Prebiotic, probiotic, and synbiotic consumption alter behavioral variables and intestinal permeability and microbiota in btbr mice. Microorganisms 9, (2021).
Chao, O. Y. et al. Targeting inhibitory cerebellar circuitry to alleviate behavioral deficits in a mouse model for studying idiopathic autism. Neuropsychopharmacology 45, 1159–1170 (2020).
Wang, X. et al. Overexpression of mGluR7 in the Prefrontal Cortex Attenuates Autistic Behaviors in Mice. Front. Cell. Neurosci. 15, 1–11 (2021).
Branchi, I., Santucci, D. & Alleva, E. Ultrasonic vocalisation emitted by infant rodents: A tool for assessment of neurobehavioural development. Behav. Brain Res. 125, 49–56 (2001).
Nitschke, W., Bell, R. W. & Zachman, T. Distress vocalizations of young in three inbred strains of mice. Dev. Psychobiol. 5, 363–370 (1972).
Wolff, R. J. Solitary and social play in wild Mus musculus (Mammalia). J. Zool. 195, 405–412 (1981).
Panksepp, J. The ontogeny of play in rats. Dev. Psychobiol. 14, 327–32 (1981).
Ferri, S. L. et al. Sociability development in mice with cell-specific deletion of the NMDA receptor NR1 subunit gene. Genes, Brain Behav. 19, 1–12 (2020).
Deitchman, R., Lavine, A. & Burkholder, J. Litter size, emotionality, learning, and social interaction in C57BL/6j mice. Prycbological Reports 39, 27–31 (1976).
Bicks, L. K. et al. Prefrontal parvalbumin interneurons require juvenile social experience to establish adult social behavior. Nat. Commun. 11, (2020).
Branchi, I., D’Andrea, I., Santarelli, S., Bonsignore, L. T. & Alleva, E. The richness of social stimuli shapes developmental trajectories: Are laboratory mouse pups impoverished? Prog. Neuro-Psychopharmacology Biol. Psychiatry 35, 1452–1460 (2011).
Walker, D. M., Cunningham, A. M., Gregory, J. K. & Nestler, E. J. Long-term behavioral effects of post-weaning social isolation in males and females. Front. Behav. Neurosci. 13, 1–20 (2019).
Li, D. C., Hinton, E. A. & Gourley, S. L. Persistent behavioral and neurobiological consequences of social isolation during adolescence. Semin. Cell Dev. Biol. 118, 73–82 (2021).

AdditionalFile1.docx

Download PDF

Journal Publication

published 24 May, 2023

Read the published version in Molecular Brain →

Editorial decision: Major revision
17 Mar, 2023
Reviewer #2 agreed at journal
25 Feb, 2023
Reviewers agreed at journal
19 Feb, 2023
Reviewers invited by journal
18 Feb, 2023
Reviewer #1 agreed at journal
18 Feb, 2023
Editor invited by journal
16 Feb, 2023
Editor assigned by journal
15 Feb, 2023
First submitted to journal
15 Feb, 2023
Submission checks completed at journal
14 Feb, 2023

You are reading this latest preprint version

Maturation of Nucleus Accumbens Synaptic Transmission Signals a Critical Period for the Rescue of Social Deficits in a Mouse Model of Autism Spectrum Disorder

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1