Dynamic Interplay between Social Brain Development and Nutrient Intake in Young Children.

Myelination of the brain structures underlying social behavior in humans is a dynamic process that parallels the emergence of social-emotional development and social skills in early life. Of the many genetic and environmental factors regulating the myelination processes, nutrition is considered as a critical and modifiable early-life factor for establishing healthy social brain networks. However, the impact of nutrition on the longitudinal development of social brain myelination remains to be fully understood. This study examined the interplay between childhood nutrient intake and social brain development across the first 5 years of life. Myelin-sensitive neuroimaging and food-intake data were analyzed in 293 children, 0.5 to 5 years of age, and explored for dynamic patterns of nutrient-social brain myelin associations. We found three data-driven age windows with specific nutrient correlation patterns, 63 individual nutrient-myelin correlations, and six nutrient combinations with a statistically significant predictive value for social brain myelination. These results provide novel insights into the impact of specific nutrient intakes on early brain development, in particular social brain regions, and suggest a critical age-sensitive opportunity to impact these brain regions for potential longer-term improvements in socio-emotional development and related executive-function and critical-thinking skills.

Early Life to Adult Brain Lipidome Dynamic: A Temporospatial Study Investigating Dietary Polar Lipid Supplementation Efficacy.

The lipid composition of the brain is well regulated during development, and the specific temporospatial distribution of various lipid species is essential for the development of optimal neural functions. Dietary lipids are the main source of brain lipids and thus contribute to the brain lipidome. Human milk is the only source of a dietary lipids for exclusively breastfed infant. Notably, it contains milk fat globule membrane (MFGM) enriched in polar lipids (PL). While early life is a key for early brain development, the interplay between dietary intake of polar lipids and spatial dynamics of lipid distribution during brain development is poorly understood. Here, we carried out an exploratory study to assess the early postnatal temporal profiling of brain lipidome between postnatal day (PND) 7 and PND 50 using matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MSI) in an in vivo preclinical model. We also assessed the effect of chronic supplementation with PL extracted from alpha-lactalbumin-enriched whey protein concentrate (WPC) containing 10% lipids, including major lipid classes found in the brain (37% phospholipids and 15% sphingomyelin). MALDI-MSI of the spatial and temporal accretion of lipid species during brain development showed that the brain lipidome is changing heterogeneously along time during brain development. In addition, increases in 400+ PL supplement-dependent lipids were observed. PL supplementation had significant spatial and temporal effect on specific fatty esters, glycerophosphocholines, glycerophosphoethanolamines, and phosphosphingolipids. Interestingly, the average levels of these lipids per brain area tended to be constant in various brain structures across the age groups, paralleling the general brain growth. In contrast, other lipids, such as cytidine diphosphate diacylglycerol, diacylglycerophosphates, phosphocholines, specific ether-phosphoethanolamines, phosphosphingolipids, glycerophosphoinositols, and glycerophosphoserines showed clear age-dependent changes uncoupled from the general brain growth. These results suggest that the dietary PL supplementation may preferentially provide the building blocks for the general brain growth during development. Our findings add to the understanding of brain-nutrient relations, their temporospatial dynamics, and potential impact on neurodevelopment.

Effects of cigarette smoke exposure on a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory autoimmune disease associated with genetic and environmental factors. Cigarette smoking is harmful to health and may be one of the risk factors for MS. However, there have been no systematic investigations under controlled experimental conditions linking cigarette smoke (CS) and MS. The present study is the first inhalation study to correlate the pre-clinical and pathological manifestations affected by different doses of CS exposure in a mouse experimental autoimmune encephalomyelitis (EAE) model. Female C57BL/6 mice were whole-body exposed to either fresh air (sham) or three concentrations of CS from a reference cigarette (3R4F) for 2 weeks before and 4 weeks after EAE induction. The effects of exposure on body weight, clinical symptoms, spinal cord pathology, and serum biochemicals were then assessed. Exposure to low and medium concentrations of CS exacerbated the severity of symptoms and spinal cord pathology, while the high concentration had no effect relative to sham exposure in mice with EAE. Interestingly, the clinical chemistry parameters for metabolic profile as well as liver and renal function (e.g. triglycerides and creatinine levels, alkaline phosphatase activity) were lower in these mice than in naïve controls. Although the mouse EAE model does not fully recapitulate the pathology or symptoms of MS in humans, these findings largely corroborate previous epidemiological findings that exposure to CS can worsen the symptoms and pathology of MS. Furthermore, the study newly highlights the possible correlation of clinical chemistry findings such as metabolism and liver and renal function between MS patients and EAE mice.

Differentiating the Neuropharmacological Properties of Nicotinic Acetylcholine Receptor-Activating Alkaloids.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids-nicotine, cotinine, and anatabine-in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release-the neurotransmitter systems relevant for anxiety-were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4ß2 and α6/3ß2ß3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4ß2 and α6/3ß2ß3 subtypes at high doses and no detectable activities against α3ß4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

Effects of nicotinic acetylcholine receptor-activating alkaloids on anxiety-like behavior in zebrafish.

Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4ß2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4ß2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood-brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.

Subchronic effects of plant alkaloids on anxiety-like behavior in zebrafish.

Zebrafish provide a valuable emerging complementary model for neurobehavioral research. They offer a powerful way to screen for the potential therapeutic effects of neuroactive drugs. A variety of behavioral tests for zebrafish have been developed and validated for assessing neurobehavioral function. The novel tank diving test is a straightforward, reproducible way of measuring anxiety-like behavior in zebrafish. When introduced into a novel tank, zebrafish normally dive to the bottom of the tank and then gradually explore the higher levels of the water column as time progresses. Buspirone is an effective anxiolytic drug in humans, which has been found, with acute administration, to reduce this anxiety-like response in zebrafish. The current study used the zebrafish model to evaluate the potential anxiolytic effects of alkaloids, commonly found in Solanaceae plants, with known neuropharmacology relevant to mood regulation. In line with previous findings, acute treatment with anxiolytic positive controls buspirone and the plant alkaloid nicotine reduced the anxiety-like diving response in the zebrafish novel tank diving test. Further, both buspirone and nicotine continued to produce anxiolytic-like effects in zebrafish after 5 days of exposure. In the same treatment paradigm, the effects of five other alkaloids-cotinine, anatabine, anabasine, harmane, and norharmane-were investigated. Cotinine, the major metabolite of nicotine, also caused anxiolytic-like effects, albeit at a dose higher than the effective dose of nicotine. Nicotine's anxiolytic-like effect was not shared by the other nicotinic alkaloids, anabasine and anatabine, or by the naturally present monoamine oxidase inhibitors harmane and norharmane. We conclude that nicotine uniquely induces anxiolytic-like effects after acute and subchronic treatment in zebrafish. The zebrafish model with the novel tank diving test could be a useful complement to rodent models for screening candidate compounds for anxiolytic effects in nonclinical studies.

Effects of Natural Monoamine Oxidase Inhibitors on Anxiety-Like Behavior in Zebrafish.

Monoamine oxidases (MAO) are a valuable class of mitochondrial enzymes with a critical role in neuromodulation. In this study, we investigated the effect of natural MAO inhibitors on novel environment-induced anxiety by using the zebrafish novel tank test (NTT). Because zebrafish spend more time at the bottom of the tank when they are anxious, anxiolytic compounds increase the time zebrafish spend at the top of the tank and vice versa. Using this paradigm, we found that harmane, norharmane, and 1,2,3,4-tetrahydroisoquinoline (TIQ) induce anxiolytic-like effects in zebrafish, causing them to spend more time at the top of the test tank and less time at the bottom. 2,3,6-trimethyl-1,4-naphtoquinone (TMN) induced an interesting mix of both anxiolytic- and anxiogenic-like effects during the first and second halves of the test, respectively. TIQ was unique in having no observable effect on general movement. Similarly, a reference MAO inhibitor clorgyline-but not pargyline-increased the time spent at the top in a concentration-dependent manner. We also demonstrated that the brain bioavailability of these compounds are high based on the ex vivo bioavailability assay and in silico prediction models, which support the notion that the observed effects on anxiety-like behavior in zebrafish were most likely due to the direct effect of these compounds in the brain. This study is the first investigation to demonstrate the anxiolytic-like effects of MAO inhibitors on novel environment-induced anxiety in zebrafish.

Differential effects of alkaloids on memory in rodents.

Nicotinic acetylcholine receptors (nAChRs) play a critical role in the neuropharmacology of learning and memory. As such, naturally occurring alkaloids that regulate nAChR activity have gained interest for understanding and potentially improving memory function. In this study, we tested the acute effects of three known nicotinic alkaloids, nicotine, cotinine, and anatabine, in suppressing scopolamine-induced memory deficit in rodents by using two classic memory paradigms, Y-maze and novel object recognition (NOR) in mice and rats, respectively. We found that all compounds were able to suppress scopolamine-induced spatial memory deficit in the Y-maze spontaneous alternation paradigm. However, only nicotine was able to suppress the short-term object memory deficit in NOR, despite the higher doses of cotinine and anatabine used to account for their potential differences in nAChR activity. These results indicate that cotinine and anatabine can uniquely regulate short-term spatial memory, while nicotine seems to have more robust and general role in memory regulation in rodents. Thus, nAChR-activating alkaloids may possess distinct procognitive properties in rodents, depending on the memory types examined.

In Vivo Profiling of a Natural Alkaloid, Anatabine, in Rodents: Pharmacokinetics and Anti-Inflammatory Efficacy.

Natural alkaloids, a large class of plant-derived substances, have attracted considerable interest because of their pharmacological activities. In this study, the in vivo pharmacokinetics and anti-inflammatory profile of anatabine, a naturally occurring alkaloid, were characterized in rodents. Anatabine was found to be bioavailable and brain-penetrant following systemic administration. Following intraperitoneal (i.p.) administration (1, 2, and 5 mg/kg), anatabine caused a dose-dependent reduction in carrageenan-induced paw edema in rats; in mice, it inhibited the production of pro-inflammatory cytokines and simultaneously elevated the levels of an anti-inflammatory cytokine in a dose-dependent manner 2 h after lipopolysaccharide challenge. Furthermore, anatabine (∼10 and ∼20 mg/kg/day for 4 weeks; inhalation exposure) had effects in a murine model of multiple sclerosis, reducing neurological deficits and bodyweight loss. Comparative studies of the pharmacokinetics and anti-inflammatory activity of anatabine demonstrated its bioequivalence in rats following i.p. administration and inhalation exposure. This study not only provides the first detailed profile of anatabine pharmacokinetics in rodents but also comprehensively characterizes the anti-inflammatory activities of anatabine in acute and chronic inflammatory models. These findings provide a basis for further characterizing and optimizing the anti-inflammatory properties of anatabine.

Neurobehavioral effects of selected tobacco constituents in rodents following subchronic administration.

Bidirectional correlations between cigarette smoking and affective disorders, such as depression, anxiety, and schizophrenia, are well documented. These findings have led to substantial investigations into the effects of the major tobacco alkaloid, nicotine, and to a lesser extent, of other tobacco constituents, on the central nervous system (CNS). However, systematic profiling of the neuropharmacological effects of tobacco constituents is limited. To elucidate the effects of selected tobacco constituents on the CNS, we used the SmartCube® system, which captures and classifies behavioral features of compound-treated mice, to profile the psychiatric drugs-like properties of previously reported neuroactive tobacco compounds in mice. Daily intraperitoneal injection of nicotine (0.5 and 1 mg/kg/day) and anatabine (5 mg/kg/day) for 7 days produced antidepressant-like behavioral SmartCube® signatures in mice, and these results were supported by the improved active coping responses in the forced swim tests. Conversely, ferulic acid did not show any identifiable class signatures in the SmartCube® tests, but rather displayed subclass signatures associated with acetylcholinesterase inhibitors. In novel object recognition memory test in rats, ferulic acid improved memory after 7 days of subcutaneous injection at 0.3 or 3 mg/kg/day. These results support previous findings showing the antidepressant drug-like effects of nicotine and the nootropic effects of ferulic acid. This is also the first report on the antidepressant drug-like effects of anatabine in rodents. This study provides a systemic behavioral evaluation of tobacco alkaloids and further insights into the association between affective disorders and smoking incidence.

Comparison of monoamine oxidase inhibition by cigarettes and modified risk tobacco products.

The adverse effects of cigarette smoking are well documented, and the two main strategies for reducing smoking prevalence are prevention of smoking initiation and promotion of smoking cessation. More recently, a third and complementary avenue, tobacco harm reduction has emerged, which is aimed to reduce the burden of smoking-related diseases. This has been enabled by the development of novel products such as electronic cigarettes (e-cigarettes) and heated tobacco products, designed to deliver nicotine with significantly reduced levels of the toxicants that are emitted by cigarettes. Several potential modified risk tobacco products (pMRTP) have been reported to emit significantly less toxicants than cigarettes and significantly reduce toxicant exposure in smokers who switch completely to such products. These are two prerequisites for pMRTPs to reduce harm and the risk of smoking-related disease. However, concerns remain regarding the addictive nature of these products. Smoking addiction is a complex phenomenon involving multiple pharmacological and non-pharmacological factors. Although the main pharmacological substance associated with smoking addiction is nicotine, accumulating evidence suggests that nicotine mostly acts as a primary reinforcer and that other factors are involved in establishing smoking addiction. Inhibition of monoamine oxidases (MAO)-mammalian flavoenzymes with a central role in neurotransmitter metabolism-has also been suggested to be involved in this process. Therefore, we aimed to comparatively investigate the ability of several types of pMRTPs and cigarette smoke (3R4F) to inhibit MAO activity. The results showed that the heated tobacco product Tobacco Heating System (THS) 2.2 and the MESH 1.1 e-cigarette possessed no MAO inhibitory activity while 3R4F significantly inhibits the levels of MAO activity (3R4F MAO-A and B; > 2 µM nicotine). Snus products have similar inhibition profiles as 3R4F but for larger nicotine concentrations (snus MAO-A; ∼68-fold, snus MAO-B; ∼23-fold higher compared to 3R4F). These observations were confirmed by analytical datasets of potential MAO inhibitors emitted by these products. In conclusion, we have demonstrated that specific pMRTPs, namely THS 2.2 and MESH 1.1, have a significantly lower MAO-inhibitory activity than 3R4F. These findings provide a basis for further investigation of the role of MAO inhibitors in cigarette addiction as well as the implications of the findings for abuse liability of pMRTPs in comparison with cigarettes.

Alternative to LRRK2-IN-1 for Pharmacological Studies of Parkinson's Disease.

BACKGROUND/AIMS: LRRK2 (leucine-rich repeat protein kinase 2) is one of the most commonly accepted genes associated with Parkinson's disease (PD). The overexpression of disease-associated mutations in LRRK2 is toxic to the cells, while reduction or elimination of LRRK2 expression promotes cell health and growth. Thus, the identification of an LRRK2 inhibitor with good physiochemical and pharmacokinetic properties is of great interest for the treatment of PD. METHODS: In this study, we have investigated LRRK2 compounds, LRRK2-IN-1 and Compound 1, in vitro and in vivo to determine how suitable they are as a selective LRRK2 tool compound. RESULTS: We report that Compound 1, patented by GSK, is a potent and selective LRRK2 inhibitor with good blood-brain barrier permeability as reflected by its high brain to plasma ratio in rats. In addition, Compound 1 can significantly promote neurite outgrowth in a primary cortical culture, indicating an optimistic cellular function of this compound in a biological system. In contrast, LRRK2-IN-1 is a less selective LRRK2 inhibitor and has low brain penetration. Furthermore, LRRK2-IN-1 is cyto- and genotoxic, while Compound 1 does not exhibit any toxicity. CONCLUSIONS: These results suggest that Compound 1 may be a superior tool compound than LRRK2-IN-1 to advance future pharmacological research on LRRK2.

Procognitive Compounds Promote Neurite Outgrowth.

BACKGROUND/AIMS: To this date, the only available drugs for treating Alzheimer's disease are cognitive enhancers, which may improve the cognitive function of patients for a few years while the disease continues to progress. As such, there are intense investigations to develop disease-modifying drugs to suppress progressive neurodegeneration. METHODS: In this study, a range of procognitive compounds are tested in a primary neuronal culture to determine their relative potential for promoting neuritogenesis. RESULTS: We report that donepezil, memantine, dimebon, Pre-084 and 4-IBP are neuritogenic while tacrine, rosemarinic acid, memoquin and a BACE1 inhibitor suppress neurite outgrowth of neurons. CONCLUSIONS: The results of this study indicate that some procognitive compounds may possess a disease-modifying potential.

Protein phosphatase 1-dependent transcriptional programs for long-term memory and plasticity.

Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and phosphatases (PPs) are important players in these mechanisms. Protein serine/threonine phosphatase 1 (PP1), in particular, was recently shown to be important for transcription-dependent memory by regulating chromatin remodeling. However, the impact of PP1 on gene transcription in adult neurons remains not fully delineated. Here, we demonstrate that the nuclear pool of PP1 is associated with transcriptional events involving molecular components of signaling cascades acting as positive and negative regulators of memory and brain plasticity. The data show that inhibiting this pool selectively in forebrain neurons improves memory performance, enhances long-term potentiation (LTP), and modulates gene transcription. These findings highlight an important role for PP1 in the regulation of gene transcription in LTM and synaptic plasticity in the adult brain.

Protein phosphatase 1 regulates the histone code for long-term memory.

Chromatin remodeling through histone posttranslational modifications (PTMs) and DNA methylation has recently been implicated in cognitive functions, but the mechanisms involved in such epigenetic regulation remain poorly understood. Here, we show that protein phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Our data show that PP1 is present at the chromatin in brain cells and interacts with enzymes of the epigenetic machinery including HDAC1 (histone deacetylase 1) and histone demethylase JMJD2A (jumonji domain-containing protein 2A). The selective inhibition of the nuclear pool of PP1 in forebrain neurons in transgenic mice is shown to induce several histone PTMs that include not only phosphorylation but also acetylation and methylation. These PTMs are residue-specific and occur at the promoter of genes important for memory formation like CREB (cAMP response element-binding protein) and NF-kappaB (nuclear factor-kappaB). These histone PTMs further co-occur with selective binding of RNA polymerase II and altered gene transcription, and are associated with improved long-term memory for objects and space. Together, these findings reveal a novel mechanism for the epigenetic control of gene transcription and long-term memory in the adult brain that depends on PP1.

Control of the establishment of aversive memory by calcineurin and Zif268.

Emotional memory is a rapidly acquired and persistent form of memory, and its robustness is in part determined by the initial strength of the memory. Here, we provide new evidence that the protein phosphatase calcineurin (CaN), a potent negative regulator of neuronal signaling that is known to constrain learning and memory, critically regulates the establishment of emotional memory through mechanisms involving the immediate early gene Zif268 (also known as Egr1). We found that CaN is inhibited in the amygdala during the establishment of aversive memory, but Zif268 is activated. Using inducible transgenesis in mice, we further saw that CaN inhibition and Zif268 overexpression during memory establishment strengthen the memory trace and enhance its resistance to extinction. We found that CaN inhibition correlates with increased Zif268 expression and that a common pool of proteins is regulated in the amygdala after CaN inhibition and Zif268 overexpression. Together, these findings reveal a previously unknown mechanism for the control of emotional memory that depends on CaN and Zif268.

Protein phosphatase 1-dependent bidirectional synaptic plasticity controls ischemic recovery in the adult brain.

Protein kinases and phosphatases can alter the impact of excitotoxicity resulting from ischemia by concurrently modulating apoptotic/survival pathways. Here, we show that protein phosphatase 1 (PP1), known to constrain neuronal signaling and synaptic strength (Mansuy et al., 1998; Morishita et al., 2001), critically regulates neuroprotective pathways in the adult brain. When PP1 is inhibited pharmacologically or genetically, recovery from oxygen/glucose deprivation (OGD) in vitro, or ischemia in vivo is impaired. Furthermore, in vitro, inducing LTP shortly before OGD similarly impairs recovery, an effect that correlates with strong PP1 inhibition. Conversely, inducing LTD before OGD elicits full recovery by preserving PP1 activity, an effect that is abolished by PP1 inhibition. The mechanisms of action of PP1 appear to be coupled with several components of apoptotic pathways, in particular ERK1/2 (extracellular signal-regulated kinase 1/2) whose activation is increased by PP1 inhibition both in vitro and in vivo. Together, these results reveal that the mechanisms of recovery in the adult brain critically involve PP1, and highlight a novel physiological function for long-term potentiation and long-term depression in the control of brain damage and repair.

Gene x environment effects: stress and memory dysfunctions caused by stress and gonadal factor irregularities during puberty in control and TGF-alpha hypomorphic mice.

The maturation of many neural functions occurs during puberty. An abnormal development of these processes, in the context of genetic vulnerability, may result in sex- and age-dependent penetrance of neuropsychiatric disorders. Reduced transforming growth factors-alpha (TGF-alpha) expression in Waved-1 (Wa-1) mice impairs the stress response and fear memory in adult males, but are absent or far less prominent in adult females and in pubertal males. Gonadectomy around the onset of puberty, when the mutant anatomical and behavioral phenotypes are undetectable, results in significant gene x environment effects. Adult control males show reduced physiological stress response as a result of gonadectomy, but not adult Wa-1 males. In females, pubertal gonadectomy elevates specific anxiety parameters only in adult control mice. There also are general sex-specific effects of pubertal gonadectomy on adult stress and fear memory. Surgical stress alone also induces sex- and genotype-dependent effects, albeit in different behavioral parameters than those affected by gonadectomy. We conclude that normal development of stress and memory processes is reliant on the levels of stress and gonadal factors during puberty, the effects of which are modulated by genetic factors and sex.