Environmental Enrichment Rescues Social Behavioral Deficits and Synaptic Abnormalities in Pten Haploinsufficient Mice.

Pten germline haploinsufficient (Pten+/-) mice, which model macrocephaly/autism syndrome, show social and repetitive behavior deficits, early brain overgrowth, and cortical-subcortical hyperconnectivity. Previous work indicated that altered neuronal connectivity may be a substrate for behavioral deficits. We hypothesized that exposing Pten+/- mice to environmental enrichment after brain overgrowth has occurred may facilitate adaptation to abnormal "hard-wired" connectivity through enhancing synaptic plasticity. Thus, we reared Pten+/- mice and their wild-type littermates from weaning under either standard (4-5 mice per standard-sized cage, containing only bedding and nestlet) or enriched (9-10 mice per large-sized cage, containing objects for exploration and a running wheel, plus bedding and nestlet) conditions. Adult mice were tested on social and non-social assays in which Pten+/- mice display deficits. Environmental enrichment rescued sex-specific deficits in social behavior in Pten+/- mice and partially rescued increased repetitive behavior in Pten+/- males. We found that Pten+/- mice show increased excitatory and decreased inhibitory pre-synaptic proteins; this phenotype was also rescued by environmental enrichment. Together, our results indicate that environmental enrichment can rescue social behavioral deficits in Pten+/- mice, possibly through normalizing the excitatory synaptic protein abundance.

10 K: a large-scale prospective longitudinal study in Israel.

The 10 K is a large-scale prospective longitudinal cohort and biobank that was established in Israel. The primary aims of the study include development of prediction models for disease onset and progression and identification of novel molecular markers with a diagnostic, prognostic and therapeutic value. The recruitment was initiated in 2018 and is expected to complete in 2021. Between 28/01/2019 and 13/12/2020, 4,629 from the expected 10,000 participants were recruited (46 %). Follow-up visits are scheduled every year for a total of 25 years. The cohort includes individuals between the ages of 40 and 70 years. Predefined medical conditions were determined as exclusions. Information collected at baseline includes medical history, lifestyle and nutritional habits, vital signs, anthropometrics, blood tests results, Electrocardiography, Ankle-brachial pressure index (ABI), liver US and Dual-energy X-ray absorptiometry (DXA) tests. Molecular profiling includes transcriptome, proteome, gut and oral microbiome, metabolome and immune system profiling. Continuous measurements include glucose levels using a continuous glucose monitoring device for 2 weeks and sleep monitoring by a home sleep apnea test device for 3 nights. Blood and stool samples are collected and stored at - 80 °C in a storage facility for future research. Linkage is being established with national disease registries.

A Prediction Model to Prioritize Individuals for a SARS-CoV-2 Test Built from National Symptom Surveys.

BACKGROUND: The gold standard for COVID-19 diagnosis is detection of viral RNA through PCR. Due to global limitations in testing capacity, effective prioritization of individuals for testing is essential. METHODS: We devised a model estimating the probability of an individual to test positive for COVID-19 based on answers to 9 simple questions that have been associated with SARS-CoV-2 infection. Our model was devised from a subsample of a national symptom survey that was answered over 2 million times in Israel in its first 2 months and a targeted survey distributed to all residents of several cities in Israel. Overall, 43,752 adults were included, from which 498 self-reported as being COVID-19 positive. FINDINGS: Our model was validated on a held-out set of individuals from Israel where it achieved an auROC of 0.737 (CI: 0.712-0.759) and auPR of 0.144 (CI: 0.119-0.177) and demonstrated its applicability outside of Israel in an independently collected symptom survey dataset from the US, UK, and Sweden. Our analyses revealed interactions between several symptoms and age, suggesting variation in the clinical manifestation of the disease in different age groups. CONCLUSIONS: Our tool can be used online and without exposure to suspected patients, thus suggesting worldwide utility in combating COVID-19 by better directing the limited testing resources through prioritization of individuals for testing, thereby increasing the rate at which positive individuals can be identified. Moreover, individuals at high risk for a positive test result can be isolated prior to testing. FUNDING: E.S. is supported by the Crown Human Genome Center, Larson Charitable Foundation New Scientist Fund, Else Kroener Fresenius Foundation, White Rose International Foundation, Ben B. and Joyce E. Eisenberg Foundation, Nissenbaum Family, Marcos Pinheiro de Andrade and Vanessa Buchheim, Lady Michelle Michels, and Aliza Moussaieff and grants funded by the Minerva foundation with funding from the Federal German Ministry for Education and Research and by the European Research Council and the Israel Science Foundation. H.R. is supported by the Israeli Council for Higher Education (CHE) via the Weizmann Data Science Research Center and by a research grant from Madame Olga Klein - Astrachan.

Publisher Correction: Building an international consortium for tracking coronavirus health status.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pten haploinsufficiency disrupts scaling across brain areas during development in mice.

Haploinsufficiency for PTEN is a cause of autism spectrum disorder and brain overgrowth; however, it is not known if PTEN mutations disrupt scaling across brain areas during development. To address this question, we used magnetic resonance imaging to analyze brains of male Pten haploinsufficient (Pten+/-) mice and wild-type littermates during early postnatal development and adulthood. Adult Pten+/- mice display a consistent pattern of abnormal scaling across brain areas, with white matter (WM) areas being particularly affected. This regional and WM enlargement recapitulates structural abnormalities found in individuals with PTEN haploinsufficiency and autism. Early postnatal Pten+/- mice do not display the same pattern, instead exhibiting greater variability across mice and brain regions than controls. This suggests that Pten haploinsufficiency may desynchronize growth across brain regions during early development before stabilizing by maturity. Pten+/- cortical cultures display increased proliferation of glial cell populations, indicating a potential substrate of WM enlargement, and provide a platform for testing candidate therapeutics. Pten haploinsufficiency dysregulates coordinated growth across brain regions during development. This results in abnormally scaled brain areas and associated behavioral deficits, potentially explaining the relationship between PTEN mutations and neurodevelopmental disorders.

Association of the combination of time-weighted variability of tacrolimus blood level and exposure to low drug levels with graft survival after kidney transplantation.

Background: The variability of tacrolimus blood levels has been shown to be associated with inferior graft survival. However, the effect of variability during the early post-transplantation period has not been evaluated. We sought to evaluate the association between time-weighted variability in the early post-transplantation period and graft survival. We also explored the interaction between drug level variability and exposure to inadequate drug levels. Methods: This retrospective cohort study included all patients who underwent kidney transplantation in the Rabin Medical Center and were treated with tacrolimus. Time-weighted coefficient of variability (TWCV) was defined as time-weighted standard deviation divided by the mean drug level. Univariate and multivariate Cox proportional hazard model was used with the primary outcome of patients and graft survival. Results: The study population included 803 patients who underwent kidney transplantation between 1 January 2000 and 29 September 2013. The high tertile of TWCV of tacrolimus blood levels was associated with reduced graft survival by univariate and multivariate analyses [hazard ratio (HR) 1.69, 95% confidence interval (CI) 1.14-2.53, P = 0.01 and HR 1.74, 95% CI 1.14-2.63, P = 0.01, respectively]. The interaction between high TWCV and exposure to inadequately low drug levels was significantly associated with reduced survival (P = 0.004), while the interaction between TWCV and high drug blood levels was not. One hundred and thirty patients (16.2%) had the combination of high TWCV and exposure to low drug values (<5 ng/mL). These patients had reduced graft survival by univariate and multivariate analyses (HR 2.42, 95% CI 1.57-3.74, P < 0.001 and HR 2.6, 95% CI 1.65-4.11, P < 0.001, respectively). Conclusions: The combination of high TWCV and exposure to low drug levels might identify high-risk patients in the early post-transplantation period.

RNA sequencing of transformed lymphoblastoid cells from siblings discordant for autism spectrum disorders reveals transcriptomic and functional alterations: Evidence for sex-specific effects.

Genome-wide expression studies of samples derived from individuals with autism spectrum disorder (ASD) and their unaffected siblings have been widely used to shed light on transcriptomic differences associated with this condition. Females have historically been under-represented in ASD genomic studies. Emerging evidence from studies of structural genetic variants and peripheral biomarkers suggest that sex-differences may exist in the biological correlates of ASD. Relatively few studies have explicitly examined whether sex-differences exist in the transcriptomic signature of ASD. The present study quantified genome-wide expression values by performing RNA sequencing on transformed lymphoblastoid cell lines and identified transcripts differentially expressed between same-sex, proximal-aged sibling pairs. We found that performing separate analyses for each sex improved our ability to detect ASD-related transcriptomic differences; we observed a larger number of dysregulated genes within our smaller set of female samples (n = 12 sibling pairs), as compared with the set of male samples (n = 24 sibling pairs), with small, but statistically significant overlap between the sexes. Permutation-based gene-set analyses and weighted gene co-expression network analyses also supported the idea that the transcriptomic signature of ASD may differ between males and females. We discuss our findings in the context of the relevant literature, underscoring the need for future ASD studies to explicitly account for differences between the sexes. Autism Res 2017, 10: 439-455. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Cytomegalovirus-negative kidney transplant recipients are at an increased risk for malignancy after kidney transplantation.

BACKGROUND: The effect of cytomegalovirus (CMV) serology status on malignancy risk in kidney transplanted patients is not clear yet. METHODS: In a nested case-control study, CMV serology status was compared between patients with a malignancy and 2:1 matched control patients without a malignancy. In a cohort study, the hazard of malignancy was compared between patients that were CMV-negative but had a CMV-positive donor and other patients, using Cox analysis. RESULTS: Fifty-two of 599 patients transplanted in our center between 2001 and 2014 developed a malignancy. Nine (17.3%) of the 52 patients that developed cancer were CMV-negative but had a-CMV-positive donor compared with 6 (5.8%) of the 104 matched control patients (odd ratio 3.42, 95% confidence interval [CI] 1.15-10.2, P=.021). By univariate Cox model, there was a trend toward increased cancer risk in CMV-negative patients with a positive donor (hazard ratio [HR] 1.95, 95% CI 0.95-4.0, P=.07), but after adjusting for multiple covariates, CMV-negative status was significantly associated with increased risk of cancer (HR 2.55, 95% CI 1.23-5.26; P=.012). CONCLUSIONS: CMV-negative patients that had a CMV-positive donor were found to have a higher risk of malignancy after kidney transplantation.

Characterization of a Novel Mutation in SLC1A1 Associated with Schizophrenia.

We have recently described a hemi-deletion on chromosome 9p24.2 at the SLC1A1 gene locus and its co-segregation with schizophrenia in an extended Palauan pedigree. This finding represents a point of convergence for several pathophysiological models of schizophrenia. The present report sought to characterize the biological consequences of this hemi-deletion. Dual luciferase assays demonstrated that the partially deleted allele (lacking exon 1 and the native promoter) can drive expression of a 5'-truncated SLC1A1 using sequence upstream of exon 2 as a surrogate promoter. However, confocal microscopy and electrophysiological recordings demonstrate that the 5'-truncated SLC1A1 lacks normal membrane localization and glutamate transport ability. To identify downstream consequences of the hemi-deletion, we first used a themed qRT-PCR array to compare expression of 84 GABA and glutamate genes in RNA from peripheral blood leukocytes in deletion carriers (n = 11) versus noncarriers (n = 8) as well as deletion carriers with psychosis (n = 5) versus those without (n = 3). Then, targeted RNA-Seq (TREx) was used to quantify expression of 375 genes associated with neuropsychiatric disorders in HEK293 cells subjected to either knockdown of SLC1A1 or overexpression of full-length or 5'-truncated SLC1A1. Expression changes of several genes strongly implicated in schizophrenia pathophysiology were detected (e.g. SLC1A2, SLC1A3, SLC1A6, SLC7A11, GRIN2A, GRIA1 and DLX1).

Controlling an avatar by thought using real-time fMRI.

OBJECTIVE: We have developed a brain-computer interface (BCI) system based on real-time functional magnetic resonance imaging (fMRI) with virtual reality feedback. The advantage of fMRI is the relatively high spatial resolution and the coverage of the whole brain; thus we expect that it may be used to explore novel BCI strategies, based on new types of mental activities. However, fMRI suffers from a low temporal resolution and an inherent delay, since it is based on a hemodynamic response rather than electrical signals. Thus, our objective in this paper was to explore whether subjects could perform a BCI task in a virtual environment using our system, and how their performance was affected by the delay. APPROACH: The subjects controlled an avatar by left-hand, right-hand and leg motion or imagery. The BCI classification is based on locating the regions of interest (ROIs) related with each of the motor classes, and selecting the ROI with maximum average values online. The subjects performed a cue-based task and a free-choice task, and the analysis includes evaluation of the performance as well as subjective reports. MAIN RESULTS: Six subjects performed the task with high accuracy when allowed to move their fingers and toes, and three subjects achieved high accuracy using imagery alone. In the cue-based task the accuracy was highest 8-12 s after the trigger, whereas in the free-choice task the subjects performed best when the feedback was provided 6 s after the trigger. SIGNIFICANCE: We show that subjects are able to perform a navigation task in a virtual environment using an fMRI-based BCI, despite the hemodynamic delay. The same approach can be extended to other mental tasks and other brain areas.

Acute prenatal exposure to a moderate dose of valproic acid increases social behavior and alters gene expression in rats.

Prenatal exposure to moderate doses of valproic acid (VPA) produces brainstem abnormalities, while higher doses of this teratogen elicit social deficits in the rat. In this pilot study, we examined effects of prenatal exposure to a moderate dose of VPA on behavior and on transcriptomic expression in three brain regions that mediate social behavior. Pregnant Long Evans rats were injected with 350 mg/kg VPA or saline on gestational day 13. A modified social interaction test was used to assess social behavior and social preference/avoidance during early and late adolescence and in adulthood. VPA-exposed animals demonstrated more social investigation and play fighting than control animals. Social investigation, play fighting, and contact behavior also differed as a function of age; the frequency of these behaviors increased in late adolescence. Social preference and locomotor activity under social circumstances were unaffected by treatment or age. Thus, a moderate prenatal dose of VPA produces behavioral alterations that are substantially different from the outcomes that occur following exposure to a higher dose. At adulthood, VPA-exposed subjects exhibited transcriptomic abnormalities in three brain regions: anterior amygdala, cerebellar vermis, and orbitofrontal cortex. A common feature among the proteins encoded by the dysregulated genes was their ability to be modulated by acetylation. Analysis of the expression of individual exons also revealed that genes involved in post-translational modification and epigenetic regulation had particular isoforms that were ubiquitously dysregulated across brain regions. The vulnerability of these genes to the epigenetic effects of VPA may highlight potential mechanisms by which prenatal VPA exposure alters the development of social behavior.

Transcriptomic analysis of postmortem brain identifies dysregulated splicing events in novel candidate genes for schizophrenia.

The diverse spatial and temporal expression of alternatively spliced transcript isoforms shapes neurodevelopment and plays a major role in neuronal adaptability. Although alternative splicing is extremely common in the brain, its role in mental illnesses such as schizophrenia has received little attention. To examine this relationship, postmortem brain tissue was obtained from 20 individuals with schizophrenia (SZ) and 20 neuropsychiatrically normal comparison subjects. Gray matter samples were extracted from two brain regions implicated in the disorder: Brodmann Area 10 and caudate. Affymetrix Human Gene 1.0 ST arrays were used on four subjects per group to attain an initial profile of differential expression of transcribed elements within and across brain regions in SZ. Numerous genes of interest with altered mRNA transcripts were identified by microarray through the differential expression of particular exons and 3' untranslated regions (UTRs) between diagnostic groups. Select microarray results--including dysregulation of ENAH exon 11a and CPNE3 3'UTR--were verified by qRTPCR and replicated in the remaining independent sample of 16 SZ patients and 16 normal comparison subjects. These results, if further replicated, clearly illustrate the importance of Identifying transcriptomic variants in expression studies, and implicate novel candidate genes in the disorder.

A re-review of the association between the NOTCH4 locus and schizophrenia.

NOTCH4 has long been identified as a candidate susceptibility gene for schizophrenia, but the collective body of genetic association studies of this gene has been less than conclusive. Recently a variant in NOTCH4 was implicated as one of the most reliably associated polymorphisms observed in a genome-wide association scan of the disorder, and the collective evidence for this polymorphism now surpasses criteria for genome-wide significance. To place these developments in context, we now summarize the initial work identifying NOTCH4 as a candidate gene for schizophrenia. The results of the genome-wide association studies that have confirmed this as a risk gene, and novel bioinformatics analyses that reveal potential functional profiles of the most likely risk-conferring polymorphisms. These analyses suggest that the NOTCH4 polymorphisms most strongly associated with schizophrenia exert their effects on susceptibility by altering the efficiency and/or alternative splicing of Notch4 transcripts. Further experimental evidence should be pursued to clarify the NOTCH4-regulated molecular and cellular phenotypes of relevance to the disorder, and the functional consequences of the implicated polymorphisms in the gene.

Dysfunctional gene splicing as a potential contributor to neuropsychiatric disorders.

Alternative pre-mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here, we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of "gene-focused" epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders.

Stimulation of AQP2 membrane insertion in renal epithelial cells in vitro and in vivo by the cGMP phosphodiesterase inhibitor sildenafil citrate (Viagra).

Vasopressin-stimulated insertion of the aquaporin 2 (AQP2) water channel into the plasma membrane of kidney collecting duct principal cells is a key event in the urinary concentrating mechanism. The paradigm for vasopressin-receptor signaling involves cAMP-mediated protein kinase A activation, which results in the functionally critical phosphorylation of AQP2 on amino acid serine 256. We previously showed that a parallel cGMP-mediated signaling pathway also leads to AQP2 membrane insertion in AQP2-transfected LLC-PK1 (LLC-AQP2) cells and in outer medullary collecting duct principal cells in situ (Bouley R, Breton S, Sun T, McLaughlin M, Nsumu NN, Lin HY, Ausiello DA, and Brown D. J Clin Invest 106: 1115-1126, 2000). In the present report, we show by immunofluorescence microscopy, and Western blotting of plasma membrane fractions, that 45-min exposure of LLC-AQP2 cells to the cGMP phosphodiesterase type 5 (PDE5) inhibitors sildenafil citrate (Viagra) or 4-{[3',4'-methylene-dioxybenzyl]amino}-6-methoxyquinazoline elevates intracellular cGMP levels and results in the plasma membrane accumulation of AQP2; i.e., they mimic the vasopressin effect. Importantly, our data also show that acute exposure to PDE5 inhibitors for 60 min induces apical accumulation of AQP2 in kidney medullary collecting duct principal cells both in tissue slices incubated in vitro as well as in vivo after intravenous injection of Viagra into rats. These data suggest that AQP2 membrane insertion can be induced independently of vasopressin-receptor activation by activating a parallel cGMP-mediated signal transduction pathway with cGMP PDE inhibitors. These results provide proof-of-principle that pharmacological activation of vasopressin-independent, cGMP signaling pathways could aid in the treatment of those forms of nephrogenic diabetes insipidus that are due to vasopressin-2 receptor dysfunction.

Prenatal cocaine exposure decreases nigrostriatal dopamine release in vitro: effects of age and sex.

The present study examined the effects of prenatal cocaine (PCOC) exposure, age, sex, and estrous phase on the functional development of nigrostriatal dopamine (DA) neurons. Striatal tissue was obtained from prepubescent and adult rats of both sexes after bidaily exposure to saline (1 ml/kg) or cocaine (20 mg/kg/ml saline) from embryonic days 15-21. Tissue levels, basal release, and electrically evoked (1 or 8 Hz) overflow of endogenous DA and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as their efflux in response to superfusion with the DA transport blocker, nomifensine (10 microM), were measured from superfused striatal slices. Generally, these measures were highest in tissue from males and adults. Tissue DA and DOPAC levels and the rate of DA turnover were unaffected by PCOC exposure. Slices from PCOC-exposed juvenile and adult male rats exhibited significantly reduced basal and electrically evoked DA release at both stimulation intensities, in conjunction with higher levels of presynaptic DA reuptake. Female rats were largely spared from the effects of PCOC exposure, and measures did not vary with estrous phase. These findings demonstrate that the effects of PCOC exposure on various parameters of nigrostriatal DA neuronal function are not uniform across age, sex, or phases of the estrous cycle. These novel alterations in nigrostriatal DA transmission are in need of independent replication, but they may have profound implications for behavioral activities regulated by these neurons and, thus, may provide a basis for sex-selective effects of PCOC in exposed humans. Possible mechanisms of deleterious effects of PCOC exposure in select groups are discussed.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation.

Smad4, the common Smad, is central for transforming growth factor (TGF)-beta superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-beta and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-beta-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats.

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT3 agonist-induced inhibition of electrically evoked [3H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT2, 5-HT3 and D2 receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT3 agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 microM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 microM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 microM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT2 receptor antagonist. ICS-205,930, a 5-HT3 receptor antagonist or sulpiride, a D2 receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 microM) and was completely reversed by sulpiride (1 microM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT2, 5-HT3 and D2 receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D2 receptors and high concentrations of the 5-HT3 receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.