Axel Bernabeu

Anissa Bara

Michelle N Murphy Green

Antonia Manduca

Jim Wager-Miller

Milene Borsoi

Olivier Lassalle

Anne-Laure Pelissier-Alicot

Pascale Chavis

Ken Mackie

Olivier Jj Manzoni
email: olivier.manzoni@inserm.fr

Affiliations

Sexually dimorphic adolescent trajectories of prefrontal endocannabinoid synaptic plasticity equalize in adulthood, reflected by endocannabinoid system gene expression

Keywords: endocannabinoid, prefrontal cortex, postnatal development, sexual dimorphism Axel Bernabeu: Conceptualization, Data curation, Formal analysis, Validation, Writing-review and editing Conceptualization, Data curation, Formal analysis, Validation, Writingreview Conceptualization, Data curation, Formal analysis, Validation, Writing-original draft Data curation, Formal analysis, Validation Data curation, Formal analysis, Validation Data curation, Formal analysis, Validation, Methodology Conceptualization, Supervision, Funding acquisition, Conceptualization, Supervision, Project administration, Methodology

How sex influences PFC's synaptic development through adolescence remains unclear. Here we describe sex-specific cellular and synaptic trajectories in the rat PFC from adolescence to adulthood. The excitability of PFC layer 5 pyramidal neurons was lower in adult females compared with other developmental stages. The developmental course of endocannabinoid-mediated long-term depression (eCB-LTD) was sexually dimorphic, unlike long-term potentiation or mGluR3-LTD. eCB-LTD was expressed in juvenile females but appeared only at puberty in males. Endovanilloid TRPV1R or eCB receptors were engaged during LTD in a sequential and sexually dimorphic manner. Gene expression of the eCB/vanilloid systems was sequential and sex specific. LTDincompetent juvenile males had elevated expression levels of the CB1R-interacting inhibitory protein CRIP1a and of the 2AG-degrading enzyme ABHD6. Pharmacological inhibition of ABHD6 or MAGL, enabled LTD in young males, whereas inhibition of AEA's degradation was ineffective. These results reveal sex differences in the maturational trajectories of the rat PFC.

Introduction

The eCB system includes cannabinoid receptors (CB1R and CB2R), the vanilloid receptor TRPV1R, and proteins required for the metabolism, transport, and catabolism of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The eCB system contributes to CNS development beginning at ontogenesis 1 and continuing throughout the postnatal neurodevelopmental period. [START_REF] Harkany | The emerging functions of endocannabinoid signaling during CNS development[END_REF][START_REF] Harkany | Wiring and firing neuronal networks: endocannabinoids take center stage[END_REF][START_REF] Fride | Multiple roles for the endocannabinoid system during the earliest stages of life: pre-and postnatal development[END_REF][START_REF] Jutras-Aswad | Neurobiological consequences of maternal cannabis on human fetal development and its neuropsychiatric outcome[END_REF][START_REF] Galve-Roperh | Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation[END_REF][START_REF] Lu | Review of the Endocannabinoid System[END_REF][START_REF] Bossong | Adolescent brain maturation, the endogenous cannabinoid system and the neurobiology of cannabis-induced schizophrenia[END_REF] The eCB system is highly influenced by sex and hormonal regulation. [START_REF] Cooper | Sex-Dependent Effects of Cannabis and Cannabinoids: A Translational Perspective[END_REF][START_REF] Krebs-Kraft | Sex difference in cell proliferation in developing rat amygdala mediated by endocannabinoids has implications for social behavior[END_REF] For example, in the CNS, circulating levels eCBs and the expression of their major target receptor CB1R, vary with sex and during the phases of the estrous cycle. [START_REF] Rodriguez De Fonseca | Cannabinoid receptors in rat brain areas: sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement[END_REF][START_REF] Gonzalez | Sex steroid influence on cannabinoid CB(1) receptor mRNA and endocannabinoid levels in the anterior pituitary gland[END_REF][START_REF] Bradshaw | Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators[END_REF] Not surprisingly, human and rodent studies consistently indicate sex differences in the short and long-term effects of cannabis and its components, and the trajectory of cannabis use. [START_REF] Stinson | Cannabis use disorders in the USA: prevalence, correlates and co-morbidity[END_REF][START_REF] Schneider | Puberty as a highly vulnerable developmental period for the consequences of cannabis exposure[END_REF][START_REF] Schepis | Gender differences in adolescent marijuana use and associated psychosocial characteristics[END_REF][START_REF] Craft | Sex differences in cannabinoid pharmacology: a reflection of differences in the endocannabinoid system?[END_REF][START_REF] Lubman | Cannabis and adolescent brain development[END_REF][START_REF] Cuttler | Sex Differences in Cannabis Use and Effects: A Cross-Sectional Survey of Cannabis Users[END_REF] The prefrontal cortex (PFC) participates to multiple higher functions such as working memory, reasoning, cognitive flexibility, and emotionally guided behaviors, [START_REF] Goldman-Rakic | Cellular and circuit basis of working memory in prefrontal cortex of nonhuman primates[END_REF][START_REF] Seamans | Functional differences between the prelimbic and anterior cingulate regions of the rat prefrontal cortex[END_REF] and continues to mature until the end of adolescence. Emerging evidence pinpoints adolescence as a sensitive period of development, [START_REF] Fuhrmann | Adolescence as a Sensitive Period of Brain Development[END_REF] described as a postnatal "period of heightened malleability [START_REF] Steinberg | Adolescent development[END_REF] during which external stimuli shape changes in brain structure and function. Illuminating the maturational sequence of the PFC is of particular significance in understanding how sensitive periods contribute to physiological development and the pathological states that have their roots in early life adversity. During adolescence, the maturation of corticolimbic areas, notably the PFC, implicates the eCB system. [START_REF] Meyer | The Role of the Endocannabinoid System and Genetic Variation in Adolescent Brain Development[END_REF] The PFC is also a site of dense expression of CB1R [START_REF] Marsicano | Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain[END_REF] and is exquisitely sensitive to various synaptopathies which manifest as a variety of cognitive disease states. [START_REF] Scheyer | The endocannabinoid system in prefrontal synaptopathies[END_REF] Exogenous cannabinoid exposure during the prenatal or the perinatal period results in significant abnormalities in PFC synaptic function, which underlie longlasting behavioral alterations at adulthood in cannabinoid-exposed rats. [START_REF] Manduca | Sex-specific behavioural deficits induced at early life by prenatal exposure to the cannabinoid receptor agonist WIN55, 212-2 depend on mGlu5 receptor signalling[END_REF][START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Scheyer | Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors[END_REF][START_REF] Scheyer | Maternal Exposure to the Cannabinoid Agonist WIN 55,12,2 during Lactation Induces Lasting Behavioral and Synaptic Alterations in the Rat Adult Offspring of Both Sexes[END_REF] In spite of substantial evidence that cannabinoid exposure during adolescence affects males and/or females, [START_REF] Schneider | Puberty as a highly vulnerable developmental period for the consequences of cannabis exposure[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Cass | CB1 cannabinoid receptor stimulation during adolescence impairs the maturation of GABA function in the adult rat prefrontal cortex[END_REF][START_REF] Renard | What Can Rats Tell Us about Adolescent Cannabis Exposure? Insights from Preclinical Research[END_REF] the normal maturation profile of the eCB system in the PFC remains obscure. Here, we applied a cross-sectional strategy to explore the maturational sequence of PFC layer 5 (L5) pyramidal neurons and their excitatory inputs in male and female rats. We report period and sex specific differences in the development and the proteins involved in eCB-mediated long-term depression (LTD) in the rat PFC. In contrast, at the same PFC excitatory synapses, neither long-term potentiation (LTP) nor type II mGluR-LTD varied during adolescent maturation in either sex. These differences were not apparent in the nucleus accumbens, a cognate structure of the mesocorticolimbic system. The intrinsic properties of L5 pyramidal cells also displayed a degree of sexspecific maturation while basic synaptic properties were invariant. These data support the idea that the eCB system plays a key role in brain sex differences established in adolescence.

Material and Methods

Further information and requests for resources should be directed to the Lead Contact, Olivier J.J. Manzoni (olivier.manzoni@inserm.fr).

Animals

Animals were treated in compliance with the European Communities Council Directive (86/609/EEC) and the United States NIH Guide for the Care and Use of Laboratory Animals. The French Ethical committee authorized the project "Exposition Périnatale aux cannabimimétiques" (APAFIS#18476-2019022510121076 v3). All rats used in France were obtained from Janvier Labs and group-housed with 12h light/dark cycles with ad libitum access to food and water. All groups represent data from a minimum of 2 litters. Female data were collected blind to the estrous cycle. Female and male rats were classified based on the timing of their pubertal maturation. The pubertal period in female rats (approximately postnatal day (P) 28 to P 40) was determined by vaginal opening and first estrus. Balano preputial separation indicated pubertal onset in male rats (around P 40), and sexual maturity is indicated by the presence of mature spermatozoa in the vas deferens, which is achieved around P 60 (Schneider 2008). Thus, the female ages groups were Juvenile 21<P<28; Pubescent 30<P<50 and Adult at P >90. Male groups were: Juvenile 21<P<38; Pubescent 40<P<60 and Adult P >90.

Slice preparation

Adult male and female rats were anesthetized with isoflurane and killed as previously described. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Scheyer | Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors[END_REF][START_REF] Scheyer | Maternal Exposure to the Cannabinoid Agonist WIN 55,12,2 during Lactation Induces Lasting Behavioral and Synaptic Alterations in the Rat Adult Offspring of Both Sexes[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Scheyer | Perinatal THC exposure via lactation induces lasting alterations to social behavior and prefrontal cortex function in rats at adulthood[END_REF] The brain was sliced (300 µm) in the coronal plane with a vibratome (Integraslice, Campden Instruments) in a sucrose-based solution at 4°C (in mM as follows: 87 NaCl, 75 sucrose, 25 glucose, 2.5 KCl, 4 MgCl2, 0.5 CaCl2, 23 NaHCO3 and 1.25 NaH2PO4). Immediately after cutting, slices containing the medial prefrontal cortex (PFC) or the nucleus accumbens were stored for 1 hr at 32°C in a low-calcium ACSF that contained (in mm) as follows: 130 NaCl, 11 glucose, 2.5 KCl, 2.4 MgCl2, 1.2 CaCl2, 23 NaHCO3, 1.2 NaH2PO4, and were equilibrated with 95% O2/5% CO2 and then held at room temperature until the time of recording. During the recording, slices were submerged in the recording chamber and superfused at 2 ml/min with low Ca 2+ ACSF. All experiments were done at 32°C. The superfusion medium contained picrotoxin (100 µM) to block GABA-A receptors. All drugs were added at the final concentration to the superfusion medium.

Electrophysiology

Whole cell patch-clamp of visualized PFC layer five prelimbic pyramidal neurons and field potential recordings were made in coronal slices containing the PFC or the accumbens as previously described. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Scheyer | Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors[END_REF][START_REF] Scheyer | Maternal Exposure to the Cannabinoid Agonist WIN 55,12,2 during Lactation Induces Lasting Behavioral and Synaptic Alterations in the Rat Adult Offspring of Both Sexes[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Scheyer | Perinatal THC exposure via lactation induces lasting alterations to social behavior and prefrontal cortex function in rats at adulthood[END_REF] Neurons were visualized using an upright microscope with infrared illumination. The intracellular solution was based on K+ gluconate (in mM: 145 K+ gluconate, 3 NaCl, 1 MgCl2, 1 EGTA, 0.3 CaCl2, 2 Na 2+ ATP, and 0.3 Na + GTP, 0.2 cAMP, buffered with 10 HEPES). The pH was adjusted to 7.2 and osmolality to 290 -300 mOsm. Electrode resistance was 4 -6 MOhms. Whole cell patch-clamp recordings were performed with an Axopatch-200B amplifier as previously described. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Scheyer | Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors[END_REF][START_REF] Scheyer | Maternal Exposure to the Cannabinoid Agonist WIN 55,12,2 during Lactation Induces Lasting Behavioral and Synaptic Alterations in the Rat Adult Offspring of Both Sexes[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Scheyer | Perinatal THC exposure via lactation induces lasting alterations to social behavior and prefrontal cortex function in rats at adulthood[END_REF] Data were low pass filtered at 2kHz, digitized (10 kHz, DigiData 1440A, Axon Instruments), collected using Clampex 10.2 and analyzed using Clampfit 10.2 (all from Molecular Device, Sunnyvale, USA). Access resistance compensation was not used, and acceptable access resistance was <30 MOhms. The potential reference of the amplifier was adjusted to zero prior to breaking into the cell. Current-voltage (I-V) curves were made by a series of hyperpolarizing to depolarizing current steps immediately after breaking into the cell. Membrane resistance was estimated from the I-V curve around resting membrane potential (Bara et al. 2018; Borsoi et al. 2019; Scheyer et al. 2019, 2020a, 2020b). Field potential recordings were made in coronal slices containing the mPFC as previously described. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Scheyer | Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors[END_REF][START_REF] Scheyer | Maternal Exposure to the Cannabinoid Agonist WIN 55,12,2 during Lactation Induces Lasting Behavioral and Synaptic Alterations in the Rat Adult Offspring of Both Sexes[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Scheyer | Perinatal THC exposure via lactation induces lasting alterations to social behavior and prefrontal cortex function in rats at adulthood[END_REF] During the recording, slices were placed in the recording chamber and superfused at 2 ml/min with low Ca2+ ACSF. All experiments were done at 32 °C. The superfusion medium contained picrotoxin (100 µM) to block GABA Type A (GABA-A) receptors. All drugs were added at the final concentration to the superfusion medium. The glutamatergic nature of the field EPSP (fEPSP) was systematically confirmed at the end of the experiments using the ionotropic glutamate receptor antagonist CNQX (20 µM), which specifically blocked the synaptic component without altering the non-synaptic component. Both fEPSP area and amplitude were analyzed. Stimulation was performed with a glass electrode filled with ACSF and the stimulus intensity was adjusted ~60% of maximal intensity after performing an inputoutput curve. Stimulation frequency was set at 0.1 Hz.

Gene expression by qRT-PCR

Wistar rats (founders from Envigo (Indianapolis, IN, USA) and bred in house) were anesthetized, decapitated, and brains were harvested at postnatal days (PND) 25, 46, and 91 and flash frozen in chilled isopentane. The medial prefrontal cortex (mPFC) was isolated using the technique described. [START_REF] Wager-Miller | Collection of Frozen Rodent Brain Regions for Downstream Analyses[END_REF] RNA purification was conducted using Qiagen's RNeasy Plus Micro Kit (Ref. 74034) and reverse transcription was done using ThermoFisher Scientific's RevertAid First Strand cDNA Synthesis Kit (Ref. K1622) following the manufacturer's instructions. qRT-PCR was performed using Taqman Master Mix (Part No. 4369016) and a combination of probes and primers (Table 1). Samples were run in duplicate and normalized against PND 91 males. Outliers were identified and removed using a ROUT test and statistical significance was established with a one-way ANOVA comparing the means of each group of samples. All procedures were approved by the IU Bloomington Institutional Animal Care and Use Committee.

Data acquisition and analysis

The magnitude of plasticity was calculated at 0-10min and 30-40 min after induction (for TBS-LTP and eCB-LTD) or drug application (mGlu2/3-LTD) as percentage of baseline responses. Statistical analysis of data was performed with Prism (GraphPad Software, San Diego, CA, USA) using tests indicated in the main text after outlier subtraction (Grubb's test, alpha level 0.05). All values are given as mean ±SEM, and statistical significance was set at p<0.05.

Results

To establish the postnatal maturational trajectories layer 5 pyramidal PFC synapses, experiments were performed in rats of both sexes, at the juvenile (female 21<P<28; male 21<P<38), pubescent (female 30<P<50; male 40<P<60) and adult stages (P>90 for both sexes) [START_REF] Schneider | Puberty as a highly vulnerable developmental period for the consequences of cannabis exposure[END_REF] .

Sex-specific developmental sequence of retrograde eCB-LTD in the rat PFC Alterations in one or more categories of PFC synaptic plasticity have been associated with endophenotypes of neuropsychiatric disorders. [START_REF] Kasanetz | Prefrontal synaptic markers of cocaine addiction-like behavior in rats[END_REF][START_REF] Iafrati | Reelin, an extracellular matrix protein linked to early onset psychiatric diseases, drives postnatal development of the prefrontal cortex via GluN2B-NMDARs and the mTOR pathway[END_REF][START_REF] Thomazeau | Prefrontal deficits in a murine model overexpressing the down syndrome candidate gene dyrk1a[END_REF][START_REF] Labouesse | Hypervulnerability of the adolescent prefrontal cortex to nutritional stress via reelin deficiency[END_REF][START_REF] Manduca | Amplification of mGlu5-Endocannabinoid Signaling Rescues Behavioral and Synaptic Deficits in a Mouse Model of Adolescent and Adult Dietary Polyunsaturated Fatty Acid Imbalance[END_REF] However, most of the abovementioned studies were performed solely in adult males. A notable exception is the observation that in-utero cannabinoid exposure selectively ablates eCB-LTD in the adult male progeny, while females were spared. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF] Because the PFC and the eCB system both undergo rearrangements during adolescence, [START_REF] Bossong | Adolescent brain maturation, the endogenous cannabinoid system and the neurobiology of cannabis-induced schizophrenia[END_REF][START_REF] Schneider | Puberty as a highly vulnerable developmental period for the consequences of cannabis exposure[END_REF][START_REF] Fuhrmann | Adolescence as a Sensitive Period of Brain Development[END_REF] we elected to systematically investigate eCB-LTD in the PFC of juvenile, pubescent and adult rats of both sexes. First, we confirmed that a 10-minute, 10Hz stimulation of the superficiallayers of the PFC in slices obtained from the adults of both sexes, induces a robust eCB-LTD at layer 5 synapses (Figure 1 A-B). [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF][START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF] This identical protocol likewise elicited LTD in slices obtained from pubescent males and females (Figure 1 C-D). [START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF] To our surprise, eCB-LTD could not be induced in response to this protocol in the PFC of juvenile males, while juvenile females already expressed full-fledged LTD (Figure 1 E-F, Table 2 and3). Prefronto-accumbens glutamatergic circuits modulate reward-related behaviors [START_REF] Floresco | The nucleus accumbens: an interface between cognition, emotion, and action[END_REF][START_REF] Mateo | Endocannabinoid Actions on Cortical Terminals Orchestrate Local Modulation of Dopamine Release in the Nucleus Accumbens[END_REF] and the eCB-system of the accumbens is, much like that of the PFC, impeded following genetic deficits, [START_REF] Jung | Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome[END_REF] environmental insults, [START_REF] Lafourcade | Nutritional omega-3 deficiency abolishes endocannabinoid-mediated neuronal functions[END_REF][START_REF] Bosch-Bouju | Endocannabinoid-Mediated Plasticity in Nucleus Accumbens Controls Vulnerability to Anxiety after Social Defeat Stress[END_REF] and cannabinoid exposure. [START_REF] Mato | A single in-vivo exposure to delta 9THC blocks endocannabinoid-mediated synaptic plasticity[END_REF][START_REF] Mato | Presynaptic homeostatic plasticity rescues long-term depression after chronic Delta 9-tetrahydrocannabinol exposure[END_REF] As shown in Figure 2, accumbens eCB-LTD was readily induced in males (Figure 2 A-B) and females at all developmental stages (Figure 2 C-D, Table 4). None of the pairwise comparisons reached significance. Taken together, the results favor a degree of regional and sex specificity to the maturational profile of eCB-LTD. We next looked for functional properties that might correspond to the delayed expression of eCB-LTD in juvenile male rats.

mGluR-LTD and NMDAR-LTP do not follow a sex-specific maturational sequence in the rat PFC

We next determined if the sex-specific maturation of LTD in the PFC was global for all forms of LTD or limited to eCB-LTD. We examined a distinct form of LTD in the rat PFC mediated by type II mGluR (i.e. mGluR2/3). [START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Kasanetz | Prefrontal synaptic markers of cocaine addiction-like behavior in rats[END_REF][START_REF] Otani | Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex[END_REF][START_REF] Huang | Repeated cocaine administration impairs group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex[END_REF] mGlu2/3 LTD and eCB-LTD share common presynaptic mechanisms [START_REF] Mato | Presynaptic homeostatic plasticity rescues long-term depression after chronic Delta 9-tetrahydrocannabinol exposure[END_REF] and thus may display similar sex-specific maturational paths. PFC slices from our various groups were exposed for 10-minutes to the mGlu2/3 agonist, LY379268 (300nM) to induce an mGlu2/3-dependent LTD. As shown before, [START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF] this drug application elicited a significant LTD at layer 5 synapses in slices obtained from adult (Supplemental Figure 1 A-B) or pubescent (Supplemental Figure 1 C-D) rats of both sexes (Supplemental Figure 1 A-B). Similarly, the 10-minute application effectively elicited LTD in slices obtained from juvenile male and female rats (Supplemental Figure 1 E-F). Thus, despite shared downstream pathways, mGlu2/3-LTD does not follow the sex-specific maturational trajectory of eCB-LTD in the PFC of rats (Table 5 and6). Next, we elected to investigate a plasticity known to be altered in the PFC of rodent models of neuropsychiatric disorders, [START_REF] Thomazeau | Prefrontal deficits in a murine model overexpressing the down syndrome candidate gene dyrk1a[END_REF][START_REF] Labouesse | Hypervulnerability of the adolescent prefrontal cortex to nutritional stress via reelin deficiency[END_REF][START_REF] Manduca | Amplification of mGlu5-Endocannabinoid Signaling Rescues Behavioral and Synaptic Deficits in a Mouse Model of Adolescent and Adult Dietary Polyunsaturated Fatty Acid Imbalance[END_REF][START_REF] Iafrati | Multivariate synaptic and behavioral profiling reveals new developmental endophenotypes in the prefrontal cortex[END_REF] NMDAR-dependent theta-burst induced long-term potentiation (TBS-LTP). The TBS protocol effectively induced a lasting synaptic potentiation in slices obtained from rats of all age periods and both sexes (Supplemental Figure 2, Table 7 and8), thereby confirming and extending our previous study. [START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF] The data indicate that PFC TBS-LTP is already mature at the juvenile stage in rats of both sexes and consistently expressed in pubescents and adults of both sexes.

CB1R are functional across development and independently of sex

CB1R functionality is sensitive to several external factors. For example, nutritional imbalance [START_REF] Lafourcade | Nutritional omega-3 deficiency abolishes endocannabinoid-mediated neuronal functions[END_REF] and cannabinoid exposure [START_REF] Mato | A single in-vivo exposure to delta 9THC blocks endocannabinoid-mediated synaptic plasticity[END_REF][START_REF] Mato | Presynaptic homeostatic plasticity rescues long-term depression after chronic Delta 9-tetrahydrocannabinol exposure[END_REF][START_REF] Mikasova | Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization[END_REF] can desensitize CB1R and consequently ablate eCB-LTD. We tested if functional CB1R were present in both sexes at the three developmental stages by comparing full dose-response curves for the CB1 agonist CP55,940 in both sexes and across age groups. The dose response curves showed that the potency and efficacy of presynaptic CB1R were similar at all ages in both sexes (Figure 3). Thus, the lack of LTD in juvenile males cannot be attributed to a mere lack of presynaptic inhibitory CB1R. This pharmacological appraisal is supported by the qRT-PCR data showing similar levels of PFC CB1 mRNA in juveniles males and females (see Figure 8).

Sex-specific maturation of PFC pyramidal cell excitability and basic synaptic properties

In search of additional mechanistic insights, we compared intrinsic firing properties of pyramidal neurons in our various experimental groups (Table 9 to 12). Patch-clamp recordings of deep-layer PFC pyramidal neurons were performed in acute PFC slices obtained from juvenile, pubescent, and adult male and female rats. The membrane reaction profiles were measured in response to a series of somatic current steps. Independent of developmental stage or sex all recorded layer 5 PFC neurons showed similar and superimposable I-V plots (Figure 4 A-B). Similarly, the resting membrane potential (Figure 4 C-D) and the rheobase (Figure 4 E-F) were alike within and between age and sex groups. Closer examination of the depolarization-driven spikes showed less action potentials in response to somatic current steps in female adults compared to the other developmental stages (Figure 4 G-H). This trait, while suggestive of a lower excitability of PFC pyramidal neurons in adult females, does not provide a mechanism for the lack of LTD in juvenile male. Basic synaptic properties were also compared. Input-output profiles were measured in our 6 groups (Table 13 and14): fEPSPs evoked by electrical stimulation showed a consistent profile distribution in response to increasing stimulation intensity: "inputoutput" curves were similar across age and sex (Figure 5 A-C). Likewise, the pairedpulse ratio (PPR), a multifactorial parameter of the presynaptic probability of neurotransmitter release, remained unchanged in the experimental groups (Figure 5 D-E, Table 15 and16). When testing whether the PPR changed throughout development, we observed that paired stimuli elicited with intervals >150ms induced equivalent facilitation in juveniles, pubescent, and adults (Figure 5 D-E). Thus, the release probability of excitatory synapses to PFC layer 5 pyramidal neurons, as estimated from the PPR, was stable throughout developmental stages in both sexes.

Sexually dimorphic mechanisms of LTD

In an earlier study, we revealed a previously unknown sexual difference in the mechanism of PFC eCB-LTD at adulthood: LTD is mediated solely by CB1R in males, in striking contrast to females where activation of TRPV1R (but not CB1R) is required to elicit LTD. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF] We reproduced and extended this observation to show that eCB-LTD engages distinct receptors in male and female depending on their developmental stages (Figure 6). In pubescent and adult males, the induction of LTD was blocked by the CB1R antagonist (SR141716A, Figure 6 A-B) but not by the TRPV1R antagonist (AMG 9810, Figure 6 C-D). Thus, regardless of the developmental stage, male eCB-LTD solely depends on CB1R. In marked contrast, SR141716A could prevent LTD induction in juvenile and pubescent females (Figure 6 E-H). As shown before, [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF] LTD was unaffected by the CB1R antagonist but blocked by AMG9810 in adult females (Figure 6 G-H) and we also now report that AMG9810 blocked LTD in juvenile females (Figure 8 G-H). Curiously, female pubescent LTD was not sensitive to AMG9810 (Figure 6 G-H). Thus, female rats require both CB1R and TRPV1R to make eCB-LTD as juveniles, only CB1R during the pubescent stage, and then solely TRPV1R at adulthood.

Stepwise and sex-specific expression of eCB-system genes in the PFC Throughout early neurodevelopment, specifically during the transition from the juvenile period to adulthood, significant changes occur in the prefrontal cortex. [START_REF] Arain | Maturation of the adolescent brain[END_REF][START_REF] Konrad | Brain development during adolescence: neuroscientific insights into this developmental period[END_REF] To investigate possible underlying mechanisms behind the sex differences in development of eCB-LTD that was discovered using electrophysiology, we used qRT-PCR to analyze several key molecules of the eCB system (Table 1 and19). There were several sex-specific and developmental differences identified across the molecules studied. Most relevant for the current study and the sexually dimorphic development of eCB-LTD, there was no difference in CB1 receptor mRNA expression levels between juvenile males and females. However, mRNAs for the cannabinoid receptor interacting protein 1a (CRIP1a) a protein that reduces CB1R mediated Gprotein-mediated signaling, or the 2-AG catabolic enzyme ABHD6 are expressed significantly more in males compared to females, only in the juveniles (Figure 7). Interestingly, this could suppress eCB-LTD in males relative to females and points to a potential mechanism. Additionally, males express more mRNA for the AEA catabolic enzyme FAAH, than females at both the juvenile and pubescent ages. The former may also contribute to the lack of eCB-LTD seen in juvenile males.

Augmentation of the circulating levels of 2-AG but not AEA uncovers LTD in juvenile male PFC

The current qRT-PCR profiling (Figure 8) and functional data showing that CB1R mediates LTD in pubescent and adult male rats (Figure 6), as well as the realization that inhibitory CB1R are fully functional in juvenile males (Figure 5), open the possibility that enhancing the circulating levels of 2-AG and/or AEA could allow for the induction of eCB-LTD in juvenile males. Thus, PFC slices were incubated (>45 min) in JZL184 (4 µM), a potent inhibitor of monoacyl-glycerol lipase, the main enzyme degrading 2-AG, to increase basal 2-AG levels prior to the 10-minute, 10Hz stimulation. Here, after JZL184 incubation slices obtained from juvenile males were found to exhibit robust, lasting LTD at layer 5 synapses (Figure 8 A-B, Table 20). Of importance, we verified that pre-treatment with SR141716A prevented the ameliorative actions of JZL184 (Figure 8 C), indicating that the "enabled LTD" was mediated by CB1R (Table 20). Additionally, guided by the qRT-PCR, we tested a specific inhibitor of ABHD6 (WWL70, >45 min 10 µM, Marrs et al. 2010, Jung et al. 2012) or an inhibitor of FAAH (URB597, >45 min 2µM, Bara et al. 2018). Inhibition of ABHD6-mediated 2-AG degradation with WWL70 permitted the full expression of LTD in prepubescent male rats (Figure 8 C), confirming that 2-AG is instrumental in eCB-LTD in immature male rats. In marked contrast, inhibiting the degradation of AEA in juvenile males did not uncover eCB-LTD (Figure 8 C), suggesting that AEA does not contribute to LTD in the immature PFC male system.

Discussion

We applied a cross-sectional strategy to show that in L5 synapses of the rat PFC, the maturation of excitatory input and eCB-mediated synaptic plasticity follows a sexspecific trajectory throughout the juvenile to adult stages. Specifically, we show that while eCB-mediated LTD is already apparent in juvenile females, it is expressed only after the onset of puberty in males. The age-related and sex-specific regulation of eCB plasticity contrasts with other forms of synaptic plasticity examined that were already mature by the initial time of experimental investigation in both sexes. The intrinsic properties of the major PFC neurons changed little from juvenile to adult age. Features such as resting membrane potentials and rheobase did not vary across age and sex, with the exception that the number of action potentials in response to depolarizing steps was reduced in adult females compared to all other groups, suggesting decreased excitability in the adult females. Like intrinsic membrane properties, two basic synaptic parameters, the paired-pulse ratio, and input-output relationships, were remarkably stable across maturation and sexes. These results suggest that for the most part, cellular and synaptic properties of PFC neurons are established early in life and that the inability to induce eCB-LTD in juvenile males (see below) cannot be attributed to differences in intrinsic properties. Early life and adolescence are well described periods of changes for the eCB system. [START_REF] Meyer | The Role of the Endocannabinoid System and Genetic Variation in Adolescent Brain Development[END_REF][START_REF] Ellgren | Dynamic changes of the endogenous cannabinoid and opioid mesocorticolimbic systems during adolescence: THC effects[END_REF][START_REF] Heng | Differential developmental trajectories for CB1 cannabinoid receptor expression in limbic/associative and sensorimotor cortical areas[END_REF][START_REF] Lee | Timing is everything: evidence for a role of corticolimbic endocannabinoids in modulating hypothalamic-pituitary-adrenal axis activity across developmental periods[END_REF][START_REF] Rubino | Sex-dependent vulnerability to cannabis abuse in adolescence[END_REF] The current data support and extend this concept by showing that a major form synaptic plasticity mediated by eCBs is also subject to developmental regulation and is the substrate of a previously unrecognized sex difference before puberty. Furthermore, the observation that eCB-LTD was consistently expressed in both sexes and at all developmental stages in the accumbens strongly suggests that this sexual dimorphism is specific to the PFC. The PFC achieves maturity late during adolescence and additional work will determine the role of eCB-LTD in the behavioral attributes of adolescence. Dysfunctional CB1R could underlie the lack of eCB-LTD in juvenile males. Sex differences in CB1R expression start early in life and peak around adolescence. [START_REF] Rodriguez De Fonseca | Cannabinoid receptors in rat brain areas: sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement[END_REF][START_REF] Rodriguez De Fonseca | Presence of cannabinoid binding sites in the brain from early postnatal ages[END_REF] Even though CB1R expression peaks with the onset of adolescence [START_REF] Meyer | The Role of the Endocannabinoid System and Genetic Variation in Adolescent Brain Development[END_REF] , our qRT-PCR data showed no difference in CB1R mRNA expression levels between juvenile males and females (Figure 7). The functionality of presynaptic CB1R was evaluated by building dose-response curves for the cannabinoid agonist CP55,940. Maximal inhibitory effects of CP55,940 and its EC50 were comparable in all groups (Figure 3) and like what we previously reported in adult male rats. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF] Thus, one can reasonably exclude the possibility that the lack of PFC eCB-LTD in juvenile males is due to low numbers of or non-functional presynaptic CB1R. Circulating levels of AEA and 2-AG fluctuate and peak around adolescence. [START_REF] Meyer | The Role of the Endocannabinoid System and Genetic Variation in Adolescent Brain Development[END_REF][START_REF] Ellgren | Dynamic changes of the endogenous cannabinoid and opioid mesocorticolimbic systems during adolescence: THC effects[END_REF][START_REF] Heng | Differential developmental trajectories for CB1 cannabinoid receptor expression in limbic/associative and sensorimotor cortical areas[END_REF][START_REF] Lee | Timing is everything: evidence for a role of corticolimbic endocannabinoids in modulating hypothalamic-pituitary-adrenal axis activity across developmental periods[END_REF][START_REF] Rubino | Sex-dependent vulnerability to cannabis abuse in adolescence[END_REF] Here, we used qRT-PCR to show sex-specific and developmental differences in the expression of key molecules of the eCB system. We found that CRIP1a, ABHD6 and FAAH mRNA are expressed significantly more in juvenile males compared to females. It is tempting to hypothesize that this expression profile could hinder the induction of eCB-LTD in males relative to females. Indeed, LTD was fully expressed in when PFC slices from prepubescent male rats were incubated with WWL70 to inhibit ABHD6. In keeping with the idea that 2-AG is instrumental to eCB-LTD in juvenile male rats, we found that inhibiting MAGL uncover LTD in juvenile males. In marked contrast, pharmacological inhibition of FAAH in juvenile males did enable eCB-LTD, showing that AEA does not contribute to LTD at this developmental stage.

Cannabis exposure during adolescence produces multiple long-lasting and sexspecific molecular, synaptic and behavioral alterations. [START_REF] Borsoi | Sex Differences in the Behavioral and Synaptic Consequences of a Single in vivo Exposure to the Synthetic Cannabimimetic WIN55,212-2 at Puberty and Adulthood[END_REF][START_REF] Cass | CB1 cannabinoid receptor stimulation during adolescence impairs the maturation of GABA function in the adult rat prefrontal cortex[END_REF][START_REF] Renard | What Can Rats Tell Us about Adolescent Cannabis Exposure? Insights from Preclinical Research[END_REF][START_REF] Rubino | Sex-dependent vulnerability to cannabis abuse in adolescence[END_REF] Based on the current findings it is tempting to attribute at least partially, such differences to underlying sex and state-dependent differences in the maturational window of development during which cannabinoid exposure takes place. Additional behavioral and molecular investigations as well as extended characterizations of plasticity and synaptic functions in animals of both sexes in other brain regions are necessary to provide a more thorough picture of the extent to which adolescent maturation shapes brain functions in adulthood. Changes in plasticity in response to cannabis in-utero or during adolescence were selective to mPFC eCB-LTD, as mPFC NMDAR-LTP and mGlu3-LTD were consistently expressed and eCB-LTD was not developmentally regulated in the accumbens. The recent finding of increased hippocampal LTP in prepubertal female rats [START_REF] Le | Prepubescent female rodents have enhanced hippocampal LTP and learning relative to males, reversing in adulthood as inhibition increases[END_REF] may seem inconsistent with these results, but the presence (current study) or absence [START_REF] Le | Prepubescent female rodents have enhanced hippocampal LTP and learning relative to males, reversing in adulthood as inhibition increases[END_REF] of a GABA blocker during LTP induction and perhaps more importantly, the different brain areas (PFC vs. Hippocampus) likely explain this apparent discrepancy. In addition, we also recently found opposite changes in LTD and LTP in the basolateral rat amygdala in males and females during puberty and adulthood. [START_REF] Guily | Sex-specific divergent maturational trajectories in the postnatal rat basolateral amygdala[END_REF] Collectively, these data highlight that sex is a major determinant of prolonged central synapse maturation. Finally, we previously showed that the consequences of in-utero cannabinoids are expressed differently in the offspring: LTD and social behaviors were impeded in adult male but spared in female. [START_REF] Bara | Sex-dependent effects of in utero cannabinoid exposure on cortical function[END_REF] The current findings advance the interpretation of the in-utero data. Taken together, these two studies support the hypothesis that in-utero exposure to THC from cannabis blocks the gain of function in eCB-LTD normally occurring at puberty in the male PFC. A. A 10-minute, 10Hz field stimulation (arrow) of layer 2/3 cells in the PFC elicited a robust eCB-LTD at deep layer synapses in adult rat of both sexes. Average timecourses of mean fEPSPs in PFC slices prepared from male (white circles, n=19) or female rats (grey circle, n=8). B. fEPSP magnitude at baseline (-10 to 0 minutes) and LTD (30-40 minutes post-tetanus) values corresponding to the normalized values in A. Individual experiments (light circles) and group average (bold circles), before and after LTD induction show similar eCB-LTD in the PFC of adult of both sexes. C. Average time-courses of mean fEPSPs showing that a 10-minute, 10Hz field stimulation (arrow) of layer 2/3 cells in the PFC elicited a robust eCB-LTD at deep layer synapses in pubescent rats of both sexes (male, white circles, n=10; female, grey circle, n=16). D. Individual experiments (light circles) and group average (bold circles), before and after LTD induction show similar PFC eCB-LTD in pubescent rats of both sexes. E. Average time-courses of mean fEPSPs showing the induction of a robust eCB-LTD at deep layer synapses of pubescent female but not male pubescent rats (male, white circles, n=10; female, grey circle, n=14). F. Individual experiments (light circles) and group average (bold circles), before and after LTD induction in the juvenile male (left) and female (right) PFC. Data represent mean ± SEM. Two tailed paired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05. A. In male rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=14), Pubescents (orange circles, n=5) and Adults (grey circle, n=5). B. Individual experiments (light circles) and group average (bold circles), before (baseline) and after (40min) LTD induction showing similar NAC eCB-LTD in male juvenile, pubescent, and adult stages. C. In female rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=5), Pubescents (orange circles, n=4) and Adults (grey circle, n=5). A. Dose-response curve for the cannabimimetic CP55,940 in juvenile male (orange symbols, n = 3-4 animals, EC50 = 0.079 μM, top value 42.17%, 95% CI for EC50 = 0.026 -0.238) and female rats (black symbols, n = 5-6, EC50 = 0.316 μM, top value 33.63%, 95% CI for EC50 = 0.08 -1.245). B. Dose-response curve for the CP55,940 in pubescent male (orange symbols, n = 4-7 animals, EC50 = 0.026 μM, top value 34.89%, 95% CI for EC50 = 0.006 -0.204) and female rats (black symbols, n = 4-6, EC50 = 0.082 μM, top value 36.85%, 95% CI for EC50 = 0.027 -0.255). C. Doseresponse curve for the CP55,940 in adult male (orange symbols, n = 3-5 animals, EC50 = 0.253 μM, top value 41.21%, 95% CI for EC50 = 0.0813 -0.7853) and female rats (black symbols, n = 5-6, EC50 = 0.122 μM, top value 30.5%, 95% CI for EC50 = 0.0296 -0.504). fEPSP amplitudes were measured 30 min after application of CP55,940. Each point is expressed as the percentage of inhibition of its basal value. Error bars indicate SEM. A-B. Comparison of current injection steps of 50pA from -400pA to 100pA revealed no differences in the I-V relationship in layer 5 pyramidal neurons of the PFC of female and male groups at the juvenile (white symbols), pubescent (orange symbols), and adult stages (black symbols). C-D. Similarly, no difference was found in resting membrane potentials and (E-F) rheobases (i.e., the minimum current injection required to elicit an action potential with 10pA progressive current injections steps from 0-200pA). G. In male, the number of evoked action potentials in response to increasing depolarizing current steps from 0-600pA did not differ in PFC pyramidal neurons of male rats during the juvenile to adulthood maturation. H. In female however, the number of evoked action potentials in response to increasing depolarizing current steps was lower in adults versus juvenile and pubescent. Data represent mean ± SEM. Scatter dot plot represents one animal. Males (Juvenile n=16; Pubescent n=9; Adult n=9); Females (Juvenile n=9; Pubescent n=6; Adult n=7). n= individual animal. Sidak's multiple comparisons test, *P<0.05. In males' PFC, LTD is always mediated by CB1R. A. In pubescent and adult males, a > 45 min pre-incubation with the CB1R antagonist (SR141716A) prevents the induction of 10Hz LTD (arrow). Average time-courses of mean fEPSPs in PFC slices prepared from SR141716A-treated pubescent (orange circles, n=5) and adult (grey circle, n=5) males. B. fEPSP magnitude at baseline (-10 to 0 min) and LTD (30-40 min post-tetanus) values. Individual experiments (light circles) and group average (bold circles) in the PFC of male pubescent and adult rats. C. In pubescent and adult males, a > 45 min pre-incubation with the TRPV1R antagonist (AMG9810) had no effect on LTD induction (arrow). Average time-courses of mean fEPSPs in PFC slices prepared from AMG9810 pubescent (orange circles, n=5) and adult (grey circle, n=11) males. D. fEPSP magnitude at baseline and LTD values. Individual experiments (light circles) and group average (bold circles) in the PFC of pubescent and adult male rats. E-H. In females' PFC, back-and-forth CB1R and TRPV1R are required for LTD E. In juvenile and pubescent female, the CB1R antagonist prevents LTD induction, while the same protocol elicited a robust eCB-LTD in adult females. Average time-courses of mean fEPSPs in PFC slices prepared from SR141716A-treated juvenile (white circles, n=5) pubescent (orange circles, n=9) and adult (grey circle, n=6) females. F. fEPSP magnitude at baseline and LTD. Individual experiments (light circles) and group average (bold circles) after SR141716A in the PFC of pubescent and adult females. G. In juvenile and adult but not pubescent females, the TRPV1R antagonist prevented eCB-LTD. Average time-courses of mean fEPSPs in PFC slices prepared from AMG9810-tretaed juvenile (white circles, n=13) pubescent (orange circles, n=7) and adult (grey circle, n=5) females H. fEPSP magnitude at baseline and LTD. Individual experiments (light circles) and group average (bold circles) show eCB-LTD after AMG9810 incubation in the PFC of female juvenile, pubescent and adult rats. Data represent mean ± SEM. Two tailed paired T-test, Two tailed unpaired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05. Individual experiments (light circles) and group average (bold circles) show eCB-LTD after JZL 184-incubation in the PFC of male juvenile rats. C. Bar histograms summarizing the effects of inhibiting eCB degradation in juvenile male PFC. As shown in detail in A, JZL184 (light grey bar, n=4) restores LTD in otherwise incompetent juvenile (white bar, n=10) male PFC slices. CB1R were necessary for the permissive effect of JZL184 effect on LTD: SR141716A preincubation blocked LTD induction in the presence of JZL184 (dark grey bar, n=5). Blocking ABHD6-mediated 2-AG degradation with WWL70 (10µM, > 45 min pre-incubation) allowed the previously ineffective low frequency protocol to induce a robust LTD in male juvenile rats (stiped bar, n=8). In contrast, a > 45 min pre-incubation with the FAAH inhibitor, URB597, 2µM,) was not permissive for LTD in the PFC of male juvenile rats (black bar, n=9). Data represent mean ± SEM. Mann-Whitney test, *P<0.05 Tables Table 1: Taqman probe and primers. Catalog numbers for the Taqman probe and primer sets from ThermoFisher Scientific (Waltham, MA, USA).

Figure 1 :

 1 Figure 1: Sex-specific maturational trajectory of eCB-LTD in the rat PFC.A. A 10-minute, 10Hz field stimulation (arrow) of layer 2/3 cells in the PFC elicited a robust eCB-LTD at deep layer synapses in adult rat of both sexes. Average timecourses of mean fEPSPs in PFC slices prepared from male (white circles, n=19) or female rats (grey circle, n=8). B. fEPSP magnitude at baseline (-10 to 0 minutes) and LTD (30-40 minutes post-tetanus) values corresponding to the normalized values in A. Individual experiments (light circles) and group average (bold circles), before and after LTD induction show similar eCB-LTD in the PFC of adult of both sexes. C. Average time-courses of mean fEPSPs showing that a 10-minute, 10Hz field stimulation (arrow) of layer 2/3 cells in the PFC elicited a robust eCB-LTD at deep layer synapses in pubescent rats of both sexes (male, white circles, n=10; female, grey circle, n=16). D. Individual experiments (light circles) and group average (bold circles), before and after LTD induction show similar PFC eCB-LTD in pubescent rats of both sexes. E. Average time-courses of mean fEPSPs showing the induction of a robust eCB-LTD at deep layer synapses of pubescent female but not male pubescent rats (male, white circles, n=10; female, grey circle, n=14). F. Individual experiments (light circles) and group average (bold circles), before and after LTD induction in the juvenile male (left) and female (right) PFC. Data represent mean ± SEM. Two tailed paired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05.

Figure 2 :

 2 Figure 2: Similar eCB-LTD in the nucleus accumbens core is expressed similarly at all age groups in both sexes.A. In male rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=14), Pubescents (orange circles, n=5) and Adults (grey circle, n=5). B. Individual experiments (light circles) and group average (bold circles), before (baseline) and after (40min) LTD induction showing similar NAC eCB-LTD in male juvenile, pubescent, and adult stages. C. In female rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=5), Pubescents (orange circles, n=4) and Adults (grey circle, n=5). D. Individual experiments (light circles) and group average (bold circles), before (baseline) and after (40min) LTD induction showing similar NAC eCB-LTD in female juvenile, pubescent, and adult stages. Data represent mean ± SEM. Two tailed paired T-test, Two tailed unpaired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05.

  Figure 2: Similar eCB-LTD in the nucleus accumbens core is expressed similarly at all age groups in both sexes.A. In male rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=14), Pubescents (orange circles, n=5) and Adults (grey circle, n=5). B. Individual experiments (light circles) and group average (bold circles), before (baseline) and after (40min) LTD induction showing similar NAC eCB-LTD in male juvenile, pubescent, and adult stages. C. In female rats, eCB-LTD is induced similarly at accumbens synapses at all age groups in response to the 10-minute, 10Hz field stimulation (arrow) the juvenile to adulthood maturation. Average time-courses of mean fEPSPs in Juveniles (white circles, n=5), Pubescents (orange circles, n=4) and Adults (grey circle, n=5). D. Individual experiments (light circles) and group average (bold circles), before (baseline) and after (40min) LTD induction showing similar NAC eCB-LTD in female juvenile, pubescent, and adult stages. Data represent mean ± SEM. Two tailed paired T-test, Two tailed unpaired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05.

Figure 3 :

 3 Figure 3: Inhibitory CB1R are similarly functional at all developmental stages in both sexes.A. Dose-response curve for the cannabimimetic CP55,940 in juvenile male (orange symbols, n = 3-4 animals, EC50 = 0.079 μM, top value 42.17%, 95% CI for EC50 = 0.026 -0.238) and female rats (black symbols, n = 5-6, EC50 = 0.316 μM, top value 33.63%, 95% CI for EC50 = 0.08 -1.245). B. Dose-response curve for the CP55,940 in pubescent male (orange symbols, n = 4-7 animals, EC50 = 0.026 μM, top value 34.89%, 95% CI for EC50 = 0.006 -0.204) and female rats (black symbols, n = 4-6,

Figure 4 :

 4 Figure 4: Sex-specific maturation of the intrinsic properties of layer 5 PFC pyramidal neurons. A-B.Comparison of current injection steps of 50pA from -400pA to 100pA revealed no differences in the I-V relationship in layer 5 pyramidal neurons of the PFC of female and male groups at the juvenile (white symbols), pubescent (orange symbols), and adult stages (black symbols). C-D. Similarly, no difference was found in resting membrane potentials and (E-F) rheobases (i.e., the minimum current injection required to elicit an action potential with 10pA progressive current injections steps from 0-200pA). G. In male, the number of evoked action potentials in response to increasing depolarizing current steps from 0-600pA did not differ in PFC pyramidal neurons of male rats during the juvenile to adulthood maturation. H. In female however, the number of evoked action potentials in response to increasing depolarizing current steps was lower in adults versus juvenile and pubescent. Data represent mean ± SEM. Scatter dot plot represents one animal. Males (Juvenile n=16; Pubescent n=9; Adult n=9); Females (Juvenile n=9; Pubescent n=6; Adult n=7). n= individual animal. Sidak's multiple comparisons test, *P<0.05.

Figure 5 :

 5 Figure 5: The basic synaptic properties of mPFC pyramidal are identical at all age groups in both sexes. A-C. Input-output profile from juvenile (A), pubescent (B) and adult mPFC layer 5 fEPSPs. Averaged fEPSP area measured as a factor of stimulus intensity. D-E: Shortterm plasticity of fEPSPs estimated by the ratio of paired stimulus-induced fEPSPs normalized to the amplitude of the first response in juvenile (white symbols), pubescent (orange symbols), and adult (grey symbols) male (D) or female (E) rats showed no modification in PPR ratio curves along late postnatal development. n= individual animal. Sidak's multiple comparisons test.

Figure 6 :

 6 Figure 6: Sex specific mechanisms of LTD across postnatal development A-D. In males' PFC, LTD is always mediated by CB1R. A. In pubescent and adult males, a > 45 min pre-incubation with the CB1R antagonist (SR141716A) prevents the induction of 10Hz LTD (arrow). Average time-courses of mean fEPSPs in PFC slices prepared from SR141716A-treated pubescent (orange circles, n=5) and adult (grey circle, n=5) males. B. fEPSP magnitude at baseline (-10 to 0 min) and LTD (30-40 min post-tetanus) values.Individual experiments (light circles) and group average (bold circles) in the PFC of male pubescent and adult rats. C. In pubescent and adult males, a > 45 min pre-incubation with the TRPV1R antagonist (AMG9810) had no effect on LTD induction (arrow). Average time-courses of mean fEPSPs in PFC slices prepared from AMG9810 pubescent (orange circles, n=5) and adult (grey circle, n=11) males. D. fEPSP magnitude at baseline and LTD values. Individual experiments (light circles) and group average (bold circles) in the PFC of pubescent and adult male rats. E-H. In females' PFC, back-and-forth CB1R and TRPV1R are required for LTD E. In juvenile and pubescent female, the CB1R antagonist prevents LTD induction, while the

Figure 7 :

 7 Figure 7: Developmental & sex-specific changes in mRNA levels of key components of the eCB system. A-E. qRT-PCR analysis of eCB system receptors; F-H synthesizing enzymes; I-K degrading enzymes, and L other relevant molecules. One-way ANOVA. Error bars represent SD. **p<0.01, ***p<0.001, ****p<0.0001

Figure 8 :

 8 Figure 8: Enhancing 2-AG but not AEA effectively uncovers eCB-LTD in the juvenile male PFC.A. Averaged time courses of 10Hz field stimulations (arrow) of layer 2/3 cells in the PFC of male juvenile rats. In control medium, this protocol failed to induce eCB-LTD at deep layer synapses. However, following a > 45 min pre-incubation with the MAGL inhibitor, JZL 184 (1µM), the previously ineffective low frequency protocol now induced a robust LTD in male juvenile rats. Average time-courses of mean fEPSPs in PFC slices prepared from JZL 184 treated (grey circles, n=8) or untreated (white circle, n=11) juvenile males. B. fEPSP magnitude at baseline (-10 to 0 minutes) and LTD (30-40 minutes post-tetanus) values corresponding to the normalized values in A. Individual experiments (light circles) and group average (bold circles) show eCB-LTD after JZL 184-incubation in the PFC of male juvenile rats. C. Bar histograms summarizing the effects of inhibiting eCB degradation in juvenile male PFC. As shown in detail in A, JZL184 (light grey bar, n=4) restores LTD in otherwise incompetent juvenile (white bar, n=10) male PFC slices. CB1R were necessary for the permissive effect of JZL184 effect on LTD: SR141716A preincubation blocked LTD induction in the presence of JZL184 (dark grey bar, n=5). Blocking ABHD6-mediated 2-AG degradation with WWL70 (10µM, > 45 min pre-incubation) allowed the previously ineffective low frequency protocol to induce a robust LTD in male juvenile rats (stiped bar, n=8). In contrast, a > 45 min pre-incubation with the FAAH inhibitor, URB597, 2µM,) was not permissive for LTD in the PFC of male juvenile rats (black bar, n=9). Data represent mean ± SEM. Mann-Whitney test, *P<0.05

Table 2 . eCB-LTD in the PFC data; comparisons by age

 2 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test	-	Measure	Condition Value (mean, SEM,	Two tailed paired
	(unit)				n)	T-test
	eCB-LTD		M J	102,1 ± 3,069 N=10	P = 0,512
	Normalized fEPSP	P	75,91 ± 5,153 N=10	P = 0,001
	0-10min baseline	A	76,41 ± 2,741 N=19	P < 0,0001
	vs			F J	81,25 ± 3,263 N=14	P < 0,0001
	30-40min	post-	P	83,27 ± 3,609 N=16	P = 0,0002
	tetanus			A	83,51 ± 3,603 N=8	P = 0,0026
	(%)
						Ordinary one-way
						ANOVA
						Tukey	multiple
						comparisons
	eCB-LTD		M J	102,1 ± 3,069 N=10	F (2, 36) = 15,43 ;
	Normalized fEPSP	P	75,91 ± 5,153 N=10	P<0,0001
	30-40min	post-	A	76,41 ± 2,741 N=19	Juvenile	vs
	tetanus					Pubescent	;
	(%)					P<0,0001
						Juvenile vs Adult;
						P=0,001
				F J	81,25 ± 3,263 N=14	F (2, 32) = 0,5402;
				P	83,27 ± 3,609 N=16	P=0,5878
				A	83,51 ± 3,603 N=8

Table 3 . eCB-LTD in the PFC data; comparisons by sex

 3

	Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold
	characters represent statistics with significant p values.

Test -Measure (unit) Condition Value (mean, SEM, n) Two tailed unpaired T- test

	eCB-LTD	P M	75,91 ± 5,153	P = 0,3007
	Normalized fEPSP		N=10
	30-40min post-tetanus	F	83,27 ± 3,609
	(%)		N=16
		A M	76,41 ± 2,741	P = 0,1546
			N=19
		F	83,51 ± 3,603
			N=8

Table 4 . eCB-LTD in the NAc data; comparisons by age

 4 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test	-	Measure	Condition Value (mean, SEM,	Two tailed paired
	(unit)				n)	T-test
	eCB-LTD		M J	83.81 ± 3.826 N=14	P = 0.002
	Normalized fEPSP	P	83.75 ± 3.428 N=5	P = 0.001
	0-10min baseline	A	73.68 ± 3.605 N=5	P < 0.0001
	vs			F J	90.14 ± 2.544 N=5	P = 0.014
	30-40min	post-	P	84.17 ± 5.411 N=5	P = 0.022
	tetanus			A	80.09 ± 8.451 N=5	P = 0.046
	(%)

Table 5 . mGlu2/3-LTD data, by age

 5 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test	-	Measure	Condition	Value	Two tailed paired T-
	(unit)					(mean,	test
						SEM, n)
	LY379268-LTD	M	J	34,77	±	P = 0,0001
	Normalized fEPSP			4,410 N=5
	0-10min baseline		P	36,02	±	P = 0,0001
	vs					5,429 N=8
	30-40min	post-		A	51 ± 4,395	P = 0,0001
	tetanus					N=6
	(%)			F	J	43,33	±	P = 0,0005
						3,698 N=4
					P	41,9	±	P = 0,0008
						3,609 N=5
					A	54,63	±	P = 0,0140
						8,768 N=4
							Ordinary	one-way
							ANOVA Tukey multiple
							comparisons
	LY379268-LTD	M	J	34,77	±	F (2, 16) = 3,014;
	Normalized fEPSP			4,410 N=5	P=0,0775
	30-40min	post-		P	36,02	±
	tetanus					5,429 N=8
	(%)				A	51 ± 4,395
						N=6
				F	J	43,33	±	F (2, 10) = 1,088;
						3,698 N=4	P=0,3737
					P	41,9	±
						3,609 N=5
					A	54,63	±
						8,768 N=4

Table 6 . mGlu2/3-LTD data, by sex

 6 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure	Condition		Value		Two tailed unpaired T-test
	(unit)			(mean,
				SEM, n)
	LY379268-LTD	J	M	34,77 ±	P = 0,1943
	Normalized			4,410
	fEPSP			N=5
	30-40min post-		F	43,33 ±
	tetanus			3,698
	(%)			N=4
		P	M	36,02 ±	P = 0,5032
				5,429
				N=8
			F	41,9	±
				3,609
				N=5
		A	M	51	±	P = 0,6923
				4,395
				N=6
			F	54,63 ±
				8,768
				N=4

Table 7 . LTP-TBS data, by age

 7 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test	-	Measure	Conditio	Value (mean, SEM,	Two tailed paired T-
	(unit)			n	n)	test
	LTP-TBS		M	J 129,5 ± 9,073 N=10	P = 0,0102
	Normalized fEPSP		P 131,3 ± 5,748 N=7	P = 0,0016
	0-10min baseline		A 128,2 ± 3,487 N=11	P = 0,0001
	vs			F	J 148,9 ± 10,13 N=9	P = 0,0013
	30-40min	post-		P 146,8 ± 7,542 N=7	P = 0,0008
	tetanus				A 125,7 ± 6,286 N=5	P = 0,015
	(%)
						Ordinary	one-way
						ANOVA
						Tukey	multiple
						comparisons
	LTP-TBS		M	J 129,5 ± 9,073 N=10	F (2, 25) = 0,048;
	Normalized fEPSP		P 131,3 ± 5,748 N=7	P=0,9527
	30-40min	post-		A 128,2 ± 3,487 N=11
	tetanus			F	J 148,9 ± 10,13 N=9	F (2, 18) = 1,631;
	(%)				P 146,8 ± 7,542 N=7	P=0,2234
					A 125,7 ± 6,286 N=5

Table 8 . LTP-TBS data, by sex

 8 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure	Condition Value (mean, SEM,	Two tailed unpaired T-test
	(unit)				n)
	LTP-TBS		J		129,5 ± 9,073 N=10 P = 0,1704
	Normalized			F	148,9 ± 10,13 N=9
	fEPSP		P	M	131,3 ± 5,748 N=7	P = 0,1272
	30-40min	post-		F	146,8 ± 7,542 N=7
	tetanus		A	M	128,2 ± 3,487 N=11 P = 0,7077
	(%)			F	125,7 ± 6,286 N=5

Table 9 .

 9 Intrinsic properties data, by ageValues are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure (unit)	Condition	Value	Ordinary one-way ANOVA
				Tukey	multiple
				comparisons
	Vm charges (mV)	M J	N=16	F (2, 30) = 0,3808; P=0,6866
		P	N=9
		A	N=8
		F J	N=9	F (2, 30) = 0,2674; P=0,7672
		P	N=6
		A	N=7
	Number of spikes	M J	N=16	F (2, 36) = 0,1771; P=0,8385
		P	N=9
		A	N=8
		F J	N=9	F (2, 36) = 1,619; P=0,2122
		P	N=6
		A	N=7

Table 10 .

 10 Intrinsic properties data, by ageValues are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure	Condition	Value	Ordinary	one-way
	(unit)				ANOVA
					Tukey	multiple
					comparisons
	Resting membrane	M	J	-69,25 ± 1,010	F (2, 31) = 2,441;
	potential (mV)			N=16	P=0,1037
			P	-70,53 ± 1,263
				N=9
			A	-73,21 ± 1,625
				N=8
		F	J	-69,81 ± 2,077	F (2, 19) = 1,330;
				N=9	P=0,2880
			P	-67,13 ± 0,9310
				N=6
			A	-71,68 ± 1,843
				N=7
	Rheobase (pA)	M	J	111,4 ± 8,881	F (2, 30) = 0,2810;
				N=16	P=0,7570
			P	113,1 ± 19,95
				N=9
			A	125±14,94
				N=8
		F	J	134,4±1,752	F (2, 18) = 0,2178;
				N=9	P=0,8063
			P	144,4±23,45
				N=6
			A	128,8±19,83
				N=7

Table 11 .

 11 Intrinsic properties data, by sexValues are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test	-	Condition	Value	Two-way RM ANOVA; Sidak
	Measure					multiple comparisons
	(unit)
	Vm charges	J	Male	N=16	F (10, 264) = 0,09421; P=0,9999
	(mV)			Female N=9
			P	Male	N=9	F (10, 143) = 0,1164; P=0,9996
				Female N=6
			A	Male	N=8	F (10, 143) = 0,3991; P=0,9453
				Female N=7
	Number	of	J	Male	N=16	F (12, 313) = 0,1069; P>0,9999
	spikes			Female N=9
			P	Male	N=9	F (12, 169) = 0,2945; P=0,9896
				Female N=6
			A	Male	N=8	F (12, 169) = 0,1679; P=0,9993
				Female N=7

Table 12 . Intrinsic properties data, by sex

 12 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure (unit) Condition	Value	Two tailed unpaired T-
					test
	Resting membrane	J	M	-69,25 ± 1,010	P = 0,7871
	potential (mV)			N=16
			F	-69,81 ± 2,077
				N=9
		P	M	-70,53 ± 1,263	P = 0,0720
				N=9
			F	-67,13 ± 0,9310
				N=8
		A	M	-73,21 ± 1,625	P = 0,5442
				N=8
			F	-71,68 ± 1,843
				N=7
	Rheobase (pA)	J	M	111,4 ± 8,881	P = 0,0933
				N=16
			F	134,4±1,752
				N=9
		P	M	113,1 ± 19,95	P = 0,3279
				N=9
			F	144,4±23,45
				N=8
		A	M	125±14,94	P = 0,8786
				N=8
			F	128,8±19,83
				N=7

Table 13 .

 13 Input-output data, by sexValues are mean ± SEM. F: Female, M: Male, P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

	Test -Measure	Condition	Value	Two Way RM ANOVA; Sidak
	(unit)				multiple comparisons
	input (µA)	J	M	N=9	F (8, 168) = 0,07051; P=0,9998
			F	N=12
		P	M	N=12	F (8, 204) = 0,5453; P=0,8214
			F	N=13
		A	M	N=7	F (8, 189) = 0,08236; P=0,9996
			F	N=16

Table 19 . Changes in mRNA levels of key components of the eCB system data, by age and sex

 19 Values are mean ± SEM. F: Female, M: Male, J: Juvenile; P: Pubescent, A: Adult. Bold characters represent statistics with significant p values.

			J	P	A
	Targets	Sex	n p value	n p value	n p value
	CB1	F M	8 0,999 8	8 <0.0001 7	8 0,0002 8
	CB2	F M	8 0,0005 8	8 <0.0001 8	8 0,5045 8
	mGluR1	F M	8 0,2747 8	8 0,5494 8	8 0,9768 8
	mGluR5	F M	8 0,0006 8	8 <0.0001 8	8 <0.0001 8
	TRPV1	F M	8 0,2580 8	8 0,0032 8	8 <0.0001 8
	DAGLα	F M	8 0,0048 8	8 0,1056 8	8 0,9868 8
	DAGLβ	F M	8 0,8287 8	8 >0.9999 8	8 0,7092 8
	NAPE-PLD	F M	8 <0.0001 8	8 <0.0001 8	8 <0.0001 8
	MAGL	F M	8 <0.0001 8	8 0,0866 8	8 0,0073 8
	FAAH	F M	8 <0.0001 8	8 0,0006 8	8 0,0940 8
	ABHD6	F M	8 <0.0001 8	8 0,3445 8	8 >0.9999 8
	CRIP1a	F M	8 <0.0001 8	8 0,8622 8	8 0,1951 8

Table 20 . Effects of inhibitors of eCB catabolic enzymes on the induction of LTD in juvenile males, PFC data

 20 Values are mean ± SEM. Bold characters represent statistics with significant p values.

	Test -Measure	Condition	Value	Mann-Whitney
	(unit)			test
	eCB-LTD	JZL184	78,64 ± 3,842 N=10 P = 0,004
	Normalized	JZL184 + SR141716 98,92 ± 9,178 N=4	P = 0,111
	fEPSP	WWL70	85,00 ± 5,894 N=8	P = 0,012
	30-40min post-
	tetanus
	(%)
		URB597	93,64 ± 3,350 N=9	P = 0,156

Acknowledgements:

The authors are grateful to the Chavis-Manzoni team members for helpful discussions and to Dr. A.F. Scheyer for critical reading and help with writing the manuscript.

Funding and Disclosures: This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM); Fondation pour la Recherche Médicale (Equipe FRM 2015 to O.M.) and the NIH (R01DA043982 to K.M and O.M.).

Declarations of interest:

The authors declare no competing interests.

Supplemental Figure 1: mGluR2/3-LTD does not follow a sex-specific maturational sequence in the rat PFC. A. mGluR-LTD, induced by a 10-min application of 30nM LY379268, produced a significant LTD at deep layer synapses in adult rat of both sexes. Average time-courses of mean fEPSPs in PFC slices prepared from male (white circles, n=6) or female rats (grey circle, n=4). B. fEPSP magnitude at baseline (-10 to 0 minutes) and LTD (30-40 minutes post-drug washout) values corresponding to the normalized values in A. Individual experiments (light circles) and group average (bold circles), show similar mGluR2/3-LTD in the PFC of adult of both sexes. C. Average time-courses showing mGluR2/3-LTD in the PFC of pubescent rats of both sexes (male, white circles, n=8; female, grey circle, n=5). D. Individual experiments (light circles) and group average (bold circles), before and after mGluR2/3-LTD induction in pubescent rats of both sexes. E. Average time-courses showing the induction of a robust mGluR2/3-LTD in pubescent male and female rats (male, white circles, n=5; female, grey circle, n=4). F. Individual experiments (light circles) and group average (bold circles), before and after mGluR2/3-LTD induction in the juvenile male (left) and female (right) PFC. Data represent mean ± SEM. Two tailed paired T-test, Two tailed unpaired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05.

Supplemental Figure 2: LTP does not follow a sex-specific maturational sequence in the rat PFC.

A. In male PFC, LTP (arrow, the theta-burst stimulation protocol consisted of five trains of burst with four pulses at 100 Hz, at 200 ms interval, repeated four times at intervals of 10 s) of layer 2/3 cells in elicited a robust LTP at deep-layer synapses at all age groups. Average time-courses of mean fEPSPs in PFC slices prepared from juvenile (white circles, n=10), pubescent (orange circle, n=7) and adult (grey circle, n=11) male rats. B. Individual experiments (light circles) and group average (bold circles), before and after LTP induction show similar LTP at all stages. C. In female rats, LTP is induced similarly in juvenile, pubescent, and adult groups. Average time-courses of mean fEPSPs in Juveniles (white circles, n=9), Pubescents (orange circles, n=7) and Adults (grey circle, n=5). D. Individual experiments (light circles) and group average (bold circles) show similar LTP in female at all stages. Data represent mean ± SEM. Two tailed paired T-test, Two tailed unpaired T-test, Ordinary one-way ANOVA, Tukey multiple comparisons test, *P<0.05.