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Huntington's disease (HD) is an incurable inherited brain disorder characterized by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of newborn mice. Chimeric mice were subjected to behavioral testing and implanted human cells were examined by immunohistochemistry and electron microscopy. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connections within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed mHTT oligomers, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT oligomers, which were internalized by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT oligomers with key cellular organelles initially drives disease progression in HD patients, and that mHTT oligomers within extracellular vesicles contribute to spread the disease in a non-cell autonomous manner.

Human pluripotent stem cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a fast feeder-free neuronal differentiation protocol to direct hPSCs to mature forebrain neurons in 37 days in vitro (DIV). The protocol is based upon a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. A human-induced PSC line (Ctr-Q33) and a human embryonic stem cell line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSC-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitor cells (NPCs) with mostly a subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map 2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely “high”, “intermediate” and “low” firing neurons. Finally, transplantation experiments showed that the NPCs survived and differentiated within mouse striatum for at least 3 months. NPCs integrated host environmental cues and differentiated into striatal medium-sized spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without teratoma formation. Altogether, these findings demonstrate the potential of this robust human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, pharmacological studies and alternative in vitro toxicology. Electronic supplementary material The online version of this article (10.1007/s12035-020-01907-4) contains supplementary material, which is available to authorized users.

Huntington’s disease (HD) is a neurodegenerative disorder that primarily affects the medium spiny neurons (MSNs) of the striatum. Recent evidence indicates that there is a neurodevelopmental component to HD where MSN specification, maturation and cellular homeostasis may be affected. However, the dysregulated cellular mechanisms underlying these impairments remain to be established. To enhance understanding of how neurodevelopment is affected in HD, further study of striatal development in a healthy context is also required as it is a brain region whose development is relatively understudied. Our aim is to investigate human striatal development in both a healthy and HD context. To achieve this we differentiate control and HD human pluripotent stem cells in vitro towards an MSN fate. A range of analyses including bulk RNA-seq, single cell RNA-seq (scRNA-seq), and functional assays are performed during differentiation to evaluate the progression of striatal development and how it is altered in HD. Bulk RNA-seq analysis indicates that alternative splicing is affected in HD cells. A set of neurodevelopment-related genes display differential expression of specific isoforms that is likely to impact on neuronal development and function. scRNA-seq analysis has identified two main neuroblast populations. Using machine learning we have mapped the developmental trajectories that neural precursor cells follow to acquire these neuroblast identities. By plotting the expression of known striatal development genes onto these trajectories, we identify additional genes with a similar expression pattern that are also likely to have a role in striatal development. Analysis of this diverse range of data sets is ongoing as they are integrated to develop a predictive model of striatal development. Using this approach, we anticipate that we will identify genes, signaling pathways and developmental modules whose modification will revert MSNs in HD to a healthy state.

Progressive neuronal death in monoaminergic nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Given that α-synuclein may be an early mediator of the pathological cascade that ultimately leads to neurodegeneration, decreased α-synuclein synthesis will abate neurotoxicity if delivered to the key affected neurons.We used a non-viral gene therapy based on a new indatraline-conjugated antisense oligonucleotide (IND-ASO) to disrupt the α-synuclein mRNA transcription selectively in monoamine neurons of a PD-like mouse model and elderly nonhuman primates. Molecular, cell biology, histological, neurochemical and behavioral assays were performed.Intracerebroventricular and intranasal IND-ASO administration for four weeks in a mouse model with AAV-mediated wild-type human α-synuclein overexpression in dopamine neurons prevented the synthesis and accumulation of α-synuclein in the connected brain regions, improving dopamine neurotransmission. Likewise, the four-week IND-ASO treatment led to decreased levels of endogenous α-synuclein protein in the midbrain monoamine nuclei of nonhuman primates, which are affected early in PD.The inhibition of α-synuclein production in dopamine neurons and its accumulation in cortical/striatal projection areas may alleviate the early deficits of dopamine function, showing the high translational value of antisense oligonucleotides as a disease modifying therapy for PD and related synucleinopathies.Grants SAF2016-75797-R, RTC-2014-2812-1 and RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; Grant ID 9238, Michael J. Fox Foundation; and Centres for Networked Biomedical Research on Mental Health (CIBERSAM), and on Neurodegenerative Diseases (CIBERNED).

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson’s disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%–40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD., This work was supported by grant SAF2016-75797-R (to A.B.), INNPACTO Subprogram IPT-2012- 1208-300000 (to A.B.), and Retos-Colaboración Subprogram RTC-2014-2812-1 (to M.V. and A.B.); Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; grants PI13/01897 (to M.V.) and PI13/01390 (to A.B.), Fondo de Investigación Sanitaria-Instituto de Salud Carlos III (Spain), co-financed by ERDF; and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED). CERCA Programme/Generalitat de Catalunya is also acknowledged.

Poster presentado en: ACNP (American College of Neuropsychopharmacology) 52nd Annual Conference, celebrada del 8 al 12 de diciembre de 2013 en Hollywood, Florida (Estados Unidos)

Pesticide exposure has been associated with neuropsychological and psychiatric impairments and neurodegenerative disorders. Pesticide exposure commonly causes a deficit in inhibitory control behaviours. In the present study, we investigated whether acute exposure to organophosphate (OP) chlorpyrifos (CPF) is related to long-term lack of inhibitory control; we also examined the possible neurochemical basis of this association. Lister Hooded rats were exposed to an acute dose of CPF (250mg/kg). Seven months later, we tested inhibitory control with the 5-choice serial reaction time task (5-CSRTT). We manipulated the baseline conditions of this task and also systemically pre-administered d-amphetamine, quinpirole, dizocilpine (MK-801) or ketanserin. We also analysed the post-mortem baseline levels of monoamines and amino acids in different brain regions. On the 5-CSRT task, CPF-exposed rats showed elevated perseverative responses that persisted across manipulation of baseline conditions of the task and under most of the pharmacological challenges tested. Only d-amphetamine induced a dose-dependent amelioration of the increased perseverative responses in the CPF group. The CPF group also exhibited increased levels of dopamine metabolism in the hippocampus and decreased levels of gamma-aminobutyric acid (GABA) and glutamate in the striatum compared to the vehicle group. These findings suggest that CPF induced a long-term compulsivity that was apparent in the 5-CSRT task and associated with changes in monoaminergic and amino acid brain systems of inhibitory control function. Exposure to high doses of OP should be taken into account in studies of environmental causes for neurodegenerative, neuropsychological and neuropsychiatric disorders., This work was supported by Grants from the Ministerio de Ciencia e Innovacion of Spain (PSI2009-08626), Ministerio de Sanidad y Consumo from Spain (FIS, PI09-01163) and FEDER funds.

Although deep brain stimulation (DBS) has been used with success in treatment-resistant depression, little is known about its mechanism of action. We examined the antidepressant-like activity of short (1 h) DBS applied to the infralimbic prefrontal cortex in the forced swim test (FST) and the novelty-suppressed feeding test (NSFT). We also used in vivo microdialysis to evaluate the release of glutamate, γ-aminobutyric acid, serotonin, dopamine, and noradrenaline in the prefrontal cortex and c-Fos immunohistochemistry to determine the brain regions activated by DBS. One hour of DBS of the infralimbic prefrontal cortex has antidepressant-like effects in FST and NSFT, and increases prefrontal efflux of glutamate, which would activate AMPA receptors (AMPARs). This effect is specific of the infralimbic area since it is not observed after DBS of the prelimbic subregion. The activation of prefrontal AMPARs would result in a stimulation of prefrontal output to the brainstem, thus increasing serotonin, dopamine, and noradrenaline in the prefrontal cortex. Further, the activation of prefrontal AMPARs is necessary and sufficient condition for the antidepressant response of 1 h DBS., This work was supported by the Junta de Andalucía (CTS-510, CTS-7748 to E.B.) and by the Instituto de Salud Carlos III, Subdirección General de Evaluación y Fomento de la Investigación (FIS grants PI10-01103 and PI13-00038 to A.A. and PI12-00915 to E.B.) that were co-funded by the European Regional Development Fund (A way to build Europe). Funding from the Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM, Intramural Project Grant P91G), and the Generalitat de Catalunya (2009SGR220) is also acknowledged. L.J.-S. was recipient of a predoctoral fellowship from the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS).

Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl(-) channels. Here we explore the effects of thymol, and propofol as positive control, on t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABA(A) receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [(35)S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [(35)S]TBPS to its recognition site at the GABA(A) receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABA(A) receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABA(A) receptor. Furthermore, we corroborate [(35)S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABA(A) receptor.

A Ferrés-Coy et al., Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, similar to 80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive -like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant -like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies., This work was supported by grants from CDTI—Spanish Ministry of Science and Innovation—DENDRIA contribution, 'nLife all rights reserved' (to AB and FA); Instituto de Salud Carlos III PI10/00290 and PI13/01390 (to AB), PI/10/0123 (to JCL) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM); NARSAD Independent Investigator Grant from the Brain & Behavior Research Foundation Grant 20003 (to AB); Ministry of Economy and Competitiveness SAF2012-35183 (to FA) and SAF2011-25020 (to AP); and Generalitat de Catalunya, Secretaria d’Universitat i Recerca del Departament d’Economia i Coneixement (SGR2014) Catalan Government Grant 2009SGR220 (to FA). Some of these grants are co-financed by the European Regional Development Fund 'A way to build Europe'. AF-C is a recipient of a fellowship from Spanish Ministry of Education, Culture and Sport.

Trabajo presentado en el 29th ECNP Congress, celebrado en Viena, Austria, del 17 al 20 de septiembre de 2016

Dieldrin was previously used as a pesticide. Although its use has been discontinued, humans are still exposed to it due to its high environmental persistence and because it accumulates in the adipose tissue of animals. Acute exposure to dieldrin provokes convulsions due to its antagonism on the gamma-aminobutyric acid-A (GABAA) receptor. However, little is known about the effects of low chronic exposure to this pollutant. In the present work, we use primary cultures of cortical neurons to study the mechanisms involved in the toxic action of dieldrin. We found that 2 and 6 days in vitro (DIV) exposure to a subcytotoxic concentration (60nM) of dieldrin reduced the increase in intracellular calcium concentration ([Ca2+]i) and the excitotoxicity caused by glutamate. Exposure to dieldrin for 6 DIV induced N-methyl-D-aspartate receptor (NMDAR) internalization and reduced metabotropic glutamate receptor 5 (mGLUR5) levels. Double immunostaining for NMDAR and mGLUR5 showed that these receptors lose colocalization on the cell membrane in neurons treated with dieldrin. No changes were observed in receptor functionalities or receptor levels after 2 DIV of exposure to dieldrin. However, the increase in [Ca2+]i induced by coactivation of NMDAR and mGLUR5 was significantly reduced. Thus, a functional interaction between the two receptors seems to play an important role in glutamate-induced excitotoxicity. We confirm that permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptive neuronal changes consisting of a reduction of glutamatergic neurotransmission. This might explain the cognitive and learning deficits observed in animals after chronic treatment with dieldrin. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology., This work was funded by the Instituto de Salud Carlos III, Ministry of Health, Spain (grant number PI 061212); Generalitat de Catalunya (grant number 2005/SGR/00826). V.B. is recipient of a predoctoral fellowship from Institut d’Investigacions Biomèdiques August Pi i Sunyer.

Trabajo presentado en el 45th Congress of the European Societies of Toxicology. EUROTOX 2008., celebrado en Rhodes (Grecia) del 05 al 08 de octubre de 2008., Validated alternative test methods are required for safety toxicology of drugs and chemicals. Studies have demonstrated that there is ∼70% correlation between in vitro cytotoxic concentrations and in vivo LD50 or human lethal concentrations. We are developing a testing strategy that could identify both factors to improve the correlation between in vitro data and human acute toxicity and alerts for the identification of outliers. A set of pharmaceutical, industrial, biocide and drug abuse chemicals have been tested against GABAA receptor activity, GABA and glutamate transport, acetylcholinaesterase activity and membrane potential in primary neuronal cultures, in the frame of the European project AcuteTox. GABAA receptor-mediated chloride influx was inhibited by compounds for which seizures have been observed after severe human poisoning. Abuse drugs inhibit [3H]GABA uptake but not [3H]aspartate uptake. Exposure to organophosphorus compounds for 48 h resulted in higher inhibition of acetylcholinaesterase activity than shorter exposure for 30–60 min, suggesting that primary cortical neurons have metabolic capacity.Most neurotoxic compounds alteredmembrane potential. Mercuric chloride, pentachlorophenol and sodium lauryl sulfate decreased cell viability, however they were more potent at neuronal endpoints. Acetaminophen and acetylsalicylic acid did not modify any of the endpoints assayed; they do not produce acute neurotoxicity in humans. These results show that in vitro evaluation of neural endpoints may identify compounds that produce acute neurotoxicity in humans, provided that relevant in vitro models for acute neural dysfunctions are used., Financed by EU contract LSHB-CT-2004-512051 and Spanish FIS IP 06/1212.

Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl(-) channels. Here we explore the effects of thymol, and propofol as positive control, on t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABA(A) receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [(35)S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [(35)S]TBPS to its recognition site at the GABA(A) receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABA(A) receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABA(A) receptor. Furthermore, we corroborate [(35)S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABA(A) receptor.

El pdf del artículo es la versión post-print., Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity., This work was supported by the Grant FIS PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain) and the EU Integrated Project LSHB-CT-2004-512051. .Z. Babot, E. Fonfría and I. Vendrell were recipients of postgraduate fellowships (FPU from the Ministry of Education, FI from the Generalitat de Catalunya and CSIC - I3P program cofinanced with European Social funds, respectively). D. García was recipient of a postdoctoral fellowship from the Fundación Carolina (Spain).

Poster presentado en el 26th ECNP (European College of Neuropsychopharmacology) Congress, celebrado del 5 al 9 de octubre de 2013, en Barcelona (Espana)

Poster presentado en el 2013 ECNP Workshop on Neuropsychopharmacology for Young Scientists in Euope, celebrado del 7 al 10 de marzo de 2013 en Nice (Francia) Abstract publicado en: European Neuropsychopharmacology 23(Suppl. 1): S88 (2013). ISSN: 0924-977X. e-ISSN: 1873-7862. DOI: 10.1016/S0924-977X(13)70100-7

Trabajo presentado en el XII International Congress of Toxicology, celebrado en Barcelona, España, del 19 al 23 de julio de 2010, Dieldrin is a previously used pesticide that accumulates in the adipose tissue as well as in the brain of mammals. This organochlorine has convulsant activity because it blocks the GABAA receptor. However, little is known about the effects of a chronic exposure to this pollutant. We have previously reported that long-term exposure to dieldrin reduces the number of functional NMDA receptors (NMDARs) in neuronal cultures. On the other hand, estradiol increases synaptogenesis and promotes the delivery of NMDAR to the cell membrane. The aims of this study are: (i) elucidate which subunits of NMDAR are affected by dieldrin exposure and (ii) evaluate the effects of estradiol against dieldrin-induced NMDAR internalization. Long-term (6DIV) exposure to a subcitotoxic concentration (60 nM) of dieldrin caused the internalization of NR1 and NR2B, but not NR2A, subunits of NMDAR. Treatment with 1 nM (but not 10 nM) 17β-estradiol (E2) for 2DIV rescued dieldrin-induced decrease of NR1 and NR2B on the cell membrane but not their internalization, suggesting different mechanisms of action between the two compounds regarding NMDAR trafficking. E2 also restored NMDAR function, as measured by NMDA-induced [Ca2+]i increase. Furthermore, prolonged exposure to a higher concentration (200 nM) of dieldrin also reduced cell surface expression of NR2A, but this effect was not prevented by E2. In addition, we showed here by immunoprecipitation on membrane extraction that PSD-95 is highly physically associated with NR2A-containing (but not NR2B-containing) NMDARs in cortical neurons and moreover that this association is lost after prolonged exposure to 200 nM dieldrin. In the present study, we confirmed that the permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptive neuronal changes consisting on NMDAR internalization, that might explain the learning and cognitive deficits observed in animals after chronic treatment with dieldrin. In addition, estradiol may exert beneficial effects against dieldrin-induced neurotoxicity., Supported by FIS PI061212.