GABAergic Gene Expression Research Articles

Effects of gestational hypothyroidism on mouse brain development: Gabaergic systems and oxidative stress

Hormonal imbalance during pregnancy is a risk factor for neuropsychiatric impairment in the offspring. It has been suggested that hypothyroidism leads to dysfunction of cortical GABAergic interneurons and inhibitory system development that in turn underlies impairment of the central nervous system. Here we investigated how gestational hypothyroidism affected offspring GABAergic system development as well as redox regulation parameters, because of previous links identified between the two. Experimental Gestational Hypothyroidism (EGH) was induced in CD-1 mice with 0.02% methimazole (MMI) in drinking water from embryonic day 9 (E9) until tissue collection at embryonic day 14 (E14) or E18. We examined GABAergic cell distribution and inhibitory system development gene expression as well as redox relevant gene expression and direct measures across all embryos regardless of sex. Intrauterine restriction of maternal thyroid hormones significantly impacted both of these outcomes in brain, as well as altering redox regulation in the placenta. GAD67+ neuronal migration was reduced, accompanied by a disruption in gene expression influencing GABAergic cell migration and cortical inhibitory neural system development. EGH also altered embryonic brain gene expression of Gpx1, Nfe2l2, Cat levels in the dorsal E14 brains. Additionally, EGH resulted in elevated TBARS, Gpx1 and Nfe2l2 in the ventral E18 brains. Furthermore, EGH downregulated placental Gpx1 gene expression at E14 and increased protein oxidation at E18. These findings support the hypothesis that sufficient maternal thyroid hormone supply to the fetus influences central nervous system development, including processes of GABAergic system development and redox equilibrium.

Dbx1 is a dorsal midbrain-specific determinant of GABAergic neuron fate and regulates differentiation of the dorsal midbrain into the inferior and superior colliculi.

The mechanism underlying the differentiation of the dorsal midbrain into two morphologically and functionally distinct compartments, the inferior colliculus (IC) and superior colliculus (SC), which process auditory and visual information, respectively, remains largely unexplored. By using null and conditional alleles, we uncover the roles of a homeodomain transcription factor Dbx1 in the regulation of IC and SC differentiation. We show that Dbx1 regulates GABAergic neuron development in the dorsal midbrain. In the absence of Dbx1 function, the dorsal-most m1-m2 progenitor domains in the midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. These results identify Dbx1 as a dorsal midbrain-specific GABAergic determinant that regulates the selector genes, Helt, Gata2, and Tal2. Furthermore, we demonstrate that maturation of the dorsal midbrain into the IC and SC is dependent on Dbx1. Null mutation of Dbx1 impairs the identity and fate of IC and SC neurons. Surprisingly, Dbx1 is required for preventing IC into SC fate switch and thus Dbx1-deficient IC neurons undergo acquisition of SC identity. Conditional inactivation of Dbx1 at late developmental phase leads to alteration in the identity and fate of the IC, but not the SC. These results suggest that SC differentiation is dependent on the early function of Dbx1, and that the IC requires the prolonged action for its normal formation. Furthermore, we uncover that Tcf7l2 acts downstream of Dbx1 selectively to promote IC differentiation. Altogether, our study identifies a molecular mechanism underlying spatial and temporal control of dorsal midbrain development.

Fipronil disturbs the antigen-specific immune responses and GABAergic gene expression in the ovalbumin-immunized BALB/c mice

BackgroundFipronil (FPN) is a broad-spectrum pesticide and commonly known as low toxicity to vertebrates. However, increasing evidence suggests that exposure to FPN might induce unexpected adverse effects in the liver, reproductive, and nervous systems. Until now, the influence of FPN on immune responses, especially T-cell responses has not been well examined. Our study is designed to investigate the immunotoxicity of FPN in ovalbumin (OVA)-sensitized mice. The mice were administered with FPN by oral gavage and immunized with OVA. Primary splenocytes were prepared to examine the viability and functionality of antigen-specific T cells ex vivo. The expression of T cell cytokines, upstream transcription factors, and GABAergic signaling genes was detected by qPCR.ResultsIntragastric administration of FPN (1–10 mg/kg) for 11 doses did not show any significant clinical symptoms. The viability of antigen-stimulated splenocytes, the production of IL-2, IL-4, and IFN-γ by OVA-specific T cells, and the serum levels of OVA-specific IgG1 and IgG2a were significantly increased in FPN-treated groups. The expression of the GABAergic signaling genes was notably altered by FPN. The GAD67 gene was significantly decreased, while the GABAR β2 and GABAR δ were increased.ConclusionFPN disturbed antigen-specific immune responses by affecting GABAergic genes in vivo. We propose that the immunotoxic effects of FPN may enhance antigen-specific immunity by dysregulation of the negative regulation of GABAergic signaling on T cell immunity.

Chronic interleukin-6 mediated neuroinflammation decreases anxiety, and impaires spatial memory in aged female mice

IntroductionNeuroinflammation is a common feature of many psychiatric disorders as well as a common underlying mechanism of neurodegenerative diseases. Sex has been shown to strongly influence the development as well as the clinical expression of these pathologies. However, there is still a neglect regarding the consideration of sex effects in rodent experiments, and a substantial underrepresentation of females in studies. This work set out to expand our knowledge of neuroinflammatory mechanisms in female mice, at both a behavioral and molecular level.MethodsThis study used GFAP-IL6 mice, a model of chronic neuroinflammation, in which interleukin-6 (IL6) is overexpressed in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter. We evaluated aged (11-15-month-old) wild type-like (WT) and GFAP-IL6 female mice in behavioral tests assessing anxiety (elevated plus-maze, EPM, Light/dark box), and spatial learning and memory (Y-maze, YM and Barnes Maze, BM) and associative learning (fear conditioning, FC). We also examined gene expression of markers linked to neuroinflammation, neurodegeneration and neurotransmission via RT-qPCR in brain regions involved in motor control, anxiety, learning and memory.ResultsFemale GFAP-IL6 mice exhibited reduced anxiety-like behavior in the EPM, and hypolocomotion in the light-dark test and EPM. Short-term memory impairment was evident in the YM but associative learning in FC was intact in GFAP-IL6 mice, suggesting domain-specific cognitive deficits in female GFAP-IL6 mice. In the BM, all mice showed intact learning and memory, but GFAP-IL6 mice exhibited higher latencies to enter the escape hole than WT mice. We analyzed the search strategy and found differences in the way GFAP-IL6 mice searched for the escape hole compared to WTs. RT-qPCR showed increased mRNA levels for molecules involved in pro-inflammatory pathways in the cerebellum, motor cortex, hippocampus, and amygdala in GFAP-IL6 mice. Of the regions examined, the cerebellum and the hippocampus showed upregulation of neuroinflammatory makers as well as dysregulation of glutamatergic and GABAergic neurotransmission gene expression in GFAP-IL6 mice compared to WTs.ConclusionIn conclusion, we showed that chronic neuroinflammation via IL6 overexpression in aged female mice led to a less anxious-like phenotype, hypolocomotion and impaired intermediate-term spatial learning and memory in the YM.

16p11.2 deletion mice exhibit compromised fronto-temporal connectivity, GABAergic dysfunction, and enhanced attentional ability

Autism spectrum disorders are more common in males, and have a substantial genetic component. Chromosomal 16p11.2 deletions in particular carry strong genetic risk for autism, yet their neurobiological impact is poorly characterised, particularly at the integrated systems level. Here we show that mice reproducing this deletion (16p11.2 DEL mice) have reduced GABAergic interneuron gene expression (decreased parvalbumin mRNA in orbitofrontal cortex, and male-specific decreases in Gad67 mRNA in parietal and insular cortex and medial septum). Metabolic activity was increased in medial septum, and in its efferent targets: mammillary body and (males only) subiculum. Functional connectivity was altered between orbitofrontal, insular and auditory cortex, and between septum and hippocampus/subiculum. Consistent with this circuit dysfunction, 16p11.2 DEL mice showed reduced prepulse inhibition, but enhanced performance in the continuous performance test of attentional ability. Level 1 autistic individuals show similarly heightened performance in the equivalent human test, also associated with parietal, insular-orbitofrontal and septo-subicular dysfunction. The data implicate cortical and septal GABAergic dysfunction, and resulting connectivity changes, as the cause of pre-attentional and attentional changes in autism.

Interactive Effect of Dietary Gamma-Aminobutyric Acid (GABA) and Water Temperature on Growth Performance, Blood Plasma Indices, Heat Shock Proteins and GABAergic Gene Expression in Juvenile Olive Flounder Paralichthys olivaceus.

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system of living organisms and has the ability to reduce the magnitude of stress in humans and animals. In this study, we evaluated the supplemental effects of GABA on normal and high water temperature based on growth, blood plasma composition as well as heat shock proteins and GABA-related gene expression in juvenile olive flounder. For this, a 2 × 2 factorial design of experiment was employed to investigate the dietary effects of GABA at 0 mg/kg of diet (GABA0 diet) and 200 mg/kg of diet (GABA200 diet) in water temperatures of 20 ± 1 °C (normal temperature) and 27 ± 1 °C (high temperature) for 28 days. A total of 180 fish with an average initial weight of 40.1 ± 0.4 g (mean ± SD) were distributed into 12 tanks, of which, each tank contained 15 fish based on the 4 dietary treatment groups in triplicate. At the end of the feeding trial, the results demonstrated that both temperature and GABA had significant effects on the growth performance of the fish. However, fish fed the GABA200 diet had a significantly higher final body weight, weight gain and specific growth rate as well as a significantly lower feed conversion ratio than the fish fed the GABA0 diet at the high water temperature. A significant interactive effect of water temperature and GABA was observed on the growth performance of olive flounder based on the two-way analysis of variance. The plasma GABA levels in fish were increased in a dose-dependent manner at normal or high water temperatures, whereas cortisol and glucose levels were decreased in fish fed GABA-supplemented diets under temperature stress. The GABA-related mRNA expression in the brains of the fish such as GABA type A receptor-associated protein (Gabarap), GABA type B receptor 1 (Gabbr1) and glutamate decarboxylase 1 (Gad1) were not significantly affected by GABA-supplemented diets under normal or temperature stressed conditions. On the other hand, the mRNA expression of heat shock proteins (hsp) in the livers of the fish, such as hsp70 and hsp90, were unchanged in fish fed the GABA diets compared to the control diet at the high water temperature. Collectively, the present study showed that dietary supplementation with GABA could enhance growth performance, and improve the feed utilization, plasma biochemical parameters and heat shock proteins and GABA-related gene expression under the stress of high water temperatures in juvenile olive flounder.

Comparative Investigation of Expression of Glutamatergic and GABAergic Genes in the Rat Hippocampus after Focal Brain Ischemia and Central LPS Administration

Among the responses in the early stages of stroke, activation of neurodegenerative and proinflammatory processes in the hippocampus is of key importance for the development of negative post-ischemic functional consequences. However, it remains unclear, what genes are involved in these processes. The aim of this work was a comparative study of the expression of genes encoding glutamate and GABA transporters and receptors, as well as inflammation markers in the hippocampus one day after two types of middle cerebral artery occlusion (according to Koizumi et al. method, MCAO-MK, and Longa et al. method, MCAO-ML), and direct pro-inflammatory activation by central administration of bacterial lipopolysaccharide (LPS). Differences and similarities in the effects of these challenges on gene expression were observed. Expression of a larger number of genes associated with activation of apoptosis and neuroinflammation, glutamate reception, and markers of the GABAergic system changed after the MCAO-ML and LPS administration than after the MCAO-MK. Compared with the MCAO-ML, the MCAO-MK and LPS challenges caused changes in the expression of more genes involved in glutamate transport. The most pronounced difference between the responses to different challenges was the changes in expression of calmodulin and calmodulin-dependent kinases genes observed after MCAO, especially MCAO-ML, but not after LPS. The revealed specific features of the hippocampal gene responses to the two types of ischemia and a pro-inflammatory stimulus could contribute to further understanding of the molecular mechanisms underlying diversity of the post-stroke consequences both in the model studies and in the clinic.

Developmental regulation of GABAergic gene expression in forebrain cholinergic neurons.

Acetylcholine and GABA are often co-released, including from VIP-expressing neurons of the cortex, cortically-projecting neurons of the globus pallidus externus and basal forebrain, and hippocampal-projecting neurons of the medial septum. The co-release of the functionally antagonistic neurotransmitters GABA and acetylcholine (ACh) greatly expands the possible functional effects of cholinergic neurons and provides an additional exogenous source of inhibition to the cortex. Transgene expression suggests that nearly all forebrain cholinergic neurons in mice at some point in development express Slc32a1, which encodes the vesicular GABA transporter (VGAT). To determine the degree of co-expression of GABA and Ach handling proteins, we measured expression in adult mice of Slc32a1, Gad1 and Gad2 (which encode GAD67 and GAD65, respectively, the GABA synthetic enzymes) in cholinergic neurons using fluorescent in situ hybridization. We found that only a subset of cholinergic neurons express the necessary machinery for GABA release at a single time in adult mice. This suggests that GABA co-release from cholinergic neurons is dynamic and potentially developmentally regulated. By measuring expression of Slc32a1, Gad1, Gad2, and Chat in the basal forebrain and medial septum in mice from post-natal day 0 to 28, we noted abundant yet variable expressions of GABAergic markers across early development, which are subsequently downregulated in adulthood. This is in contrast with the forebrain-projecting pedunculopontine nucleus, which showed no evidence of co-expression of GABAergic genes. These results suggest that expression of GABA signaling machinery in the cortically-projecting cholinergic system peaks during early development before settling at a non-zero level that is maintained through adulthood.

Maternal behaviours and adult offspring behavioural deficits are predicted by maternal TNFα concentration in a rat model of neurodevelopmental disorders

Exposure to inflammatory stressors during fetal development is a major risk factor for neurodevelopmental disorders (NDDs) in adult offspring. Maternal immune activation (MIA), induced by infection, causes an acute increase in pro-inflammatory cytokines which can increase the risk for NDDs directly by inducing placental and fetal brain inflammation, or indirectly through affecting maternal care behaviours thereby affecting postnatal brain development. Which of these two potential mechanisms dominates in increasing offspring risk for NDDs remains unclear. Here, we show that acute systemic maternal inflammation induced by the viral mimetic polyinosinic:polycytidylic acid (poly I:C) on gestational day 15 of rat pregnancy affects offspring and maternal behaviour, offspring cognition, and expression of NDD-relevant genes in the offspring brain. Dams exposed to poly I:C elicited an acute increase in the pro-inflammatory cytokine tumour necrosis factor (TNF; referred to here as TNFα), which predicted disruption of key maternal care behaviours. Offspring of poly I:C-treated dams showed early behavioural and adult cognitive deficits correlated to the maternal TNFα response, but, importantly, not with altered maternal care. We also found interacting effects of sex and treatment on GABAergic gene expression and DNA methylation in these offspring in a brain region-specific manner, including increased parvalbumin expression in the female adolescent frontal cortex. We conclude that the MIA-induced elevation of TNFα in the maternal compartment affects fetal neurodevelopment leading to altered offspring behaviour and cognition. Our results suggest that a focus on prenatal pathways affecting fetal neurodevelopment would provide greater insights into the mechanisms underpinning the TNFα-mediated genesis of altered offspring behaviour and cognition following maternal inflammation.

Altered Expression of Genes Associated with Major Neurotransmitter Systems in the Reward-Related Brain Regions of Mice with Positive Fighting Experience.

The main neurotransmitters in the brain-dopamine, γ-aminobutyric acid (GABA), glutamate, and opioids-are recognized to be the most important for the regulation of aggression and addiction. The aim of this work was to study differentially expressed genes (DEGs) in the main reward-related brain regions, including the ventral tegmental area (VTA), dorsal striatum (STR), ventral striatum (nucleus accumbens, NAcc), prefrontal cortex (PFC), and midbrain raphe nuclei (MRNs), in male mice with 20-day positive fighting experience in daily agonistic interactions. Expression of opioidergic, catecholaminergic, glutamatergic, and GABAergic genes was analyzed to confirm or refute the influence of repeated positive fighting experience on the development of "addiction-like" signs shown in our previous studies. High-throughput RNA sequencing was performed to identify differentially expressed genes in the brain regions of chronically aggressive mice. In the aggressive mice, upregulation of opioidergic genes was shown (Oprk1 in VTA, Pdyn in NAcc, Penk in PFC, and Oprd1 in MRNs and PFC), as was downregulation of genes Opcml and Oprk1 in STR and Pomc in VTA and NAcc. Upregulation of catecholaminergic genes in VTA (Ddc and Slc6a2) and in NAcc (Th and Drd2) and downregulation of some differentially expressed genes in MRNs (Th, Ddc, Dbh, Drd2, Slc18a2, and Sncg) and in VTA (Adra2c, Sncg, and Sncb) were also documented. The expression of GABAergic and glutamatergic genes that participate in drug addiction changed in all brain regions. According to literature data, the proteins encoded by genes Drd2, Oprk1, Oprd1, Pdyn, Penk, and Pomc are directly involved in drug addiction in humans. Thus, our results confirm our earlier claim about the formation of addiction-like signs following repeated positive fighting experience in mice, as shown previously in our biobehavioral studies.

Predator odor (TMT) exposure potentiates interoceptive sensitivity to alcohol and increases GABAergic gene expression in the anterior insular cortex and nucleus accumbens in male rats.

Post-traumatic stress disorder (PTSD) confers enhanced vulnerability to developing comorbid alcohol use disorder (AUD). Exposure to the scent of a predator, such as the fox odor TMT, has been used to model a traumatic stressor with relevance to PTSD symptomatology. Alcohol produces distinct interoceptive (subjective) effects that may influence vulnerability to problem drinking and AUD. As such, understanding the lasting impact of stressors on sensitivity to the interoceptive effects of alcohol is clinically relevant. The present study used a 2-lever, operant drug discrimination procedure to train male Long-Evans rats to discriminate the interoceptive effects of alcohol (2g/kg, i.g. [intragastrically]) from water. Upon stable performance, rats underwent a 15-min exposure to TMT. Two weeks later, an alcohol dose-response curve was conducted to evaluate the lasting effects of the TMT stressor on the interoceptive effects of alcohol. The TMT group showed a leftward shift in the effective dose (ED50) of the dose-response curve compared to controls, reflecting potentiated interoceptive sensitivity to alcohol. TMT exposure did not affect response rate. GABAergic signaling in both the anterior insular cortex (aIC) and the nucleus accumbens (Acb) is involved in the interoceptive effects of alcohol and stressor-induced adaptations. As such, follow-up experiments in alcohol-naïve rats examined neuronal activation (as measured by c-Fos immunoreactivity) following TMT and showed that TMT exposure increased c-Fos expression in the aIC and the nucleus accumbens core (AcbC). Two weeks after TMT exposure, Gad-1 gene expression was elevated in the aIC and Gat-1 was increased in the Acb, compared to controls. Lastly, the alcohol discrimination and alcohol-naïve groups displayed dramatic differences in stress reactive behaviors during the TMT exposure, suggesting that alcohol exposure may alter the behavioral response to predator odor. Together, these data suggest that predator odor stressor results in potentiated sensitivity to alcohol, possibly through GABAergic adaptations in the aIC and Acb, which may be relevant to understanding PTSD-AUD comorbidity.

EZH2-Mediated H3K27me3 Targets Transcriptional Circuits of Neuronal Differentiation.

The Polycomb Repressive Complex 2 (PRC2) plays important roles in the epigenetic regulation of cellular development and differentiation through H3K27me3-dependent transcriptional repression. Aberrant PRC2 activity has been associated with cancer and neurodevelopmental disorders, particularly with respect to the malfunction of sits catalytic subunit EZH2. Here, we investigated the role of the EZH2-mediated H3K27me3 apposition in neuronal differentiation. We made use of a transgenic mouse model harboring Ezh2 conditional KO alleles to derive embryonic stem cells and differentiate them into glutamatergic neurons. Time course transcriptomics and epigenomic analyses of H3K27me3 in absence of EZH2 revealed a significant dysregulation of molecular networks affecting the glutamatergic differentiation trajectory that resulted in: (i) the deregulation of transcriptional circuitries related to neuronal differentiation and synaptic plasticity, in particular LTD, as a direct effect of EZH2 loss and (ii) the appearance of a GABAergic gene expression signature during glutamatergic neuron differentiation. These results expand the knowledge about the molecular pathways targeted by Polycomb during glutamatergic neuron differentiation.

Enzymatic Degradation of Cortical Perineuronal Nets Reverses GABAergic Interneuron Maturation

Perineuronal nets (PNNs) are specialised extracellular matrix structures which preferentially enwrap fast-spiking (FS) parvalbumin interneurons and have diverse roles in the cortex. PNN maturation coincides with closure of the critical period of cortical plasticity. We have previously demonstrated that BDNF accelerates interneuron development in a c-Jun-NH2-terminal kinase (JNK)–dependent manner, which may involve upstream thousand-and-one amino acid kinase 2 (TAOK2). Chondroitinase-ABC (ChABC) enzymatic digestion of PNNs reportedly reactivates ‘juvenile-like’ plasticity in the adult CNS. However, the mechanisms involved are unclear. We show that ChABC produces an immature molecular phenotype in cultured cortical neurons, corresponding to the phenotype prior to critical period closure. ChABC produced different patterns of PNN-related, GABAergic and immediate early (IE) gene expression than well-characterised modulators of mature plasticity and network activity (GABAA-R antagonist, bicuculline, and sodium-channel blocker, tetrodotoxin (TTX)). ChABC downregulated JNK activity, while this was upregulated by bicuculline. Bicuculline, but not ChABC, upregulated Bdnf expression and ERK activity. Furthermore, we found that BDNF upregulation of semaphorin-3A and IE genes was TAOK mediated. Our data suggest that ChABC heightens structural flexibility and network disinhibition, potentially contributing to ‘juvenile-like’ plasticity. The molecular phenotype appears to be distinct from heightened mature synaptic plasticity and could relate to JNK signalling. Finally, we highlight that BDNF regulation of plasticity and PNNs involves TAOK signalling.

Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors.

It is essential to generate isolated populations of human neuronal subtypes in order to understand cell-type-specific roles in brain function and susceptibility to disease pathology. Here we describe a protocol for in-parallel generation of cortical glutamatergic (excitatory) and GABAergic (inhibitory) neurons from human pluripotent stem cells (hPSCs) by using the neurogenic transcription factors Ngn2 and a combination of Ascl1 and Dlx2, respectively. In contrast to the majority of neural transdifferentiation protocols that use transient lentiviral infection, the use of stable hPSC lines carrying doxycycline-inducible transcription factors allows neuronal differentiation to be initiated by addition of doxycycline and neural medium. This article presents a method to generate lentivirus from cultured mammalian cells and establish stable transcription factor-expressing cell lines (Basic Protocol 1), followed by a method for monolayer excitatory and inhibitory neuronal differentiation from the established lines (Basic Protocol 2). The resulting neurons reproducibly exhibit properties consistent with human cortical neurons, including the expected morphologies, expression of glutamatergic and GABAergic genes, and functional properties. Our approach enables the scalable and rapid production of human neurons suitable for modeling human brain diseases in a subtype-specific manner and examination of differential cellular vulnerability. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and generation of stable hPSC lines Support Protocol 1: Expansion and maintenance of hPSCs Basic Protocol 2: Differentiation of EX- and IN-neurons Support Protocol 2: Experimental methods for validation of EX- and IN-neurons.

Age‐associated Downregulation of GABAergic and Glutamatergic Synapse‐related Gene Expression in the Rostral Ventrolateral Medulla of Male Rats

The rostral ventrolateral medulla (RVLM), a part of the medullary reticular formation, plays a major role in several physiological responses, including cardiovascular and sympathetic nervous system functions. Although aging causes disturbances in the responses of these physiological systems, RVLM involvement in these age-related changes is not clear. Previous work on high-throughput gene expression analysis of the RVLM in aged animals suggested that neurotransmission- and synapse-related genes might be downregulated in these animals (Balivada S et al., 2017. Physiol Genomics. 49, 400). To identify the synapse-related genes, we hypothesized that aging leads to downregulation of GABAergic and glutamatergic synapse-related gene expression in the RVLM of aged rats. To test this hypothesis, we micropunched an RVLM-containing area from young (3 months), middle-aged (12 months), and aged (24 months) F344 male rats. Sampled sites were mapped onto the Brain Maps 4.0 rat brain reference atlas (Swanson LW, 2018. J. Comp. Neurol. 526, 935) to localize and validate the spatial location of the RVLM-containing micropunch. RNA purified from these micropunches was analyzed using GABA and Glutamate RT2 Profiler PCR arrays (n= 8–10). In addition, the expression of selected genes was analyzed at the protein level using western blotting (n=10). Genes that displayed significant differential expression (1.5-fold, p < .05, FDR < .05) were identified to be downregulated in the RVLM of aged and middle-aged rats. Compared to GABAergic synapse-related genes, more glutamatergic synapse-related genes were observed to be downregulated in the aged (76.6%) and middle-aged (77.7%) RVLM. Slc1a6 showed the highest fold downregulation (—1.8) at the RNA level in the RVLM of aged as compared to young rats, and its protein product, Excitatory Amino Acid Transporter-4, showed a downregulatory trend in the RVLM of aged rats. These results suggest that certain molecular constituents of both GABAergic and glutamatergic synapses might be altered in the RVLM of aged and middle-aged rats, and that the changes in glutamatergic synapse composition might be more prominent. Investigating the anatomical and functional changes associated with these glutamatergic synapses might help understand the effects of aging on the RVLM.

A Mediation Analysis to Identify Links between Gut Bacteria and Memory in Context of Human Milk Oligosaccharides

Elucidating relationships between the gut and brain is of intense research focus. Multiple studies have demonstrated that modulation of the intestinal environment via prebiotics or probiotics can induce cognitively beneficial effects, such as improved memory or reduced anxiety. However, the mechanisms by which either act remain largely unknown. We previously demonstrated that different types of oligosaccharides affected short- and long-term memory in distinct ways. Given that the oligosaccharide content of human milk is highly variable, and that formula-fed infants typically do not consume similar amounts or types of oligosaccharides, their potential effects on brain development warrant investigation. Herein, a mediation analysis was performed on existing datasets, including relative abundance of bacterial genera, gene expression, brain volume, and cognition in young pigs. Analyses revealed that numerous bacterial genera in both the colon and feces were related to short- and/or long-term memory. Relationships between genera and memory appeared to differ between diets. Mediating variables frequently included GABAergic and glutamatergic hippocampal gene expression. Other mediating variables included genes related to myelination, transcription factors, brain volume, and exploratory behavior. Overall, this analysis identified multiple pathways between the gut and brain, with a focus on genes related to excitatory/inhibitory neurotransmission.

A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders

We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson’s disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer’s disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk.

Neonatal Tactile Stimulation Alters Behaviors in Heterozygous Serotonin Transporter Male Rats: Role of the Amygdala.

The serotonin transporter (SERT) gene, especially the short allele of the human serotonin transporter linked polymorphic region (5-HTTLPR), has been associated with the development of stress-related neuropsychiatric disorders. In line, exposure to early life stress in SERT knockout animals contributes to anxiety- and depression-like behavior. However, there is a lack of investigation of how early-life exposure to beneficial stimuli, such as tactile stimulation (TS), affects later life behavior in these animals. In this study, we investigated the effect of TS on social, anxiety, and anhedonic behavior in heterozygous SERT knockouts rats and wild-type controls and its impact on gene expression in the basolateral amygdala. Heterozygous SERT+/– rats were submitted to TS during postnatal days 8–14, for 10 min per day. In adulthood, rats were assessed for social and affective behavior. Besides, brain-derived neurotrophic factor (Bdnf) gene expression and its isoforms, components of glutamatergic and GABAergic systems as well as glucocorticoid-responsive genes were measured in the basolateral amygdala. We found that exposure to neonatal TS improved social and affective behavior in SERT+/– animals compared to naïve SERT+/– animals and was normalized to the level of naïve SERT+/+ animals. At the molecular level, we observed that TS per se affected Bdnf, the glucocorticoid-responsive genes Nr4a1, Gadd45β, the co-chaperone Fkbp5 as well as glutamatergic and GABAergic gene expression markers including the enzyme Gad67, the vesicular GABA transporter, and the vesicular glutamate transporter genes. Our results suggest that exposure of SERT+/– rats to neonatal TS can normalize their phenotype in adulthood and that TS per se alters the expression of plasticity and stress-related genes in the basolateral amygdala. These findings demonstrate the potential effect of a supportive stimulus in SERT rodents, which are more susceptible to develop psychiatric disorders.

M9. RATS REARED IN SOCIAL ISOLATION INDUCES EPIGENETIC MODIFICATIONS IN THE NMDA RECEPTOR SUBUNITS

BackgroundEarly-life stress is a key risk for psychiatric disorders that may produce changes in the neurodevelopment. N-methyl-d-aspartate receptor (NMDAR) have been associated with the pathophysiology of schizophrenia and evidence supports that epigenetic changes in NMDAR imply deficiencies in excitatory neurotransmission suggest its role in the neurobiology of psychoses (Uno and Coyle, 2019; Fachim et al., 2019; Gulchina et al., 2017). Aims: Although previous studies have shown abnormalities in the glutamatergic system in animal model of schizophrenia, it is not known if there are equivalent mRNA/protein alterations and DNA methylation changes in the brains of rats reared in isolation. Thus, in order to improve the knowledge of glutamatergic system role in psychosis, we investigated the NR1 and NR2 mRNA/protein and the DNA methylation levels of Grin1, Grin2a and Grin2b promoter region in the prefrontal cortex (PFC) and hippocampus (HIPPO) of male Wistar rats after isolation rearing. Furthermore, because the Parvalbumin (PV) deficit is the most consistent finding across animal models and schizophrenia itself, we also evaluated the expression of PV and other related GABAergic genes (REL and GAD1) in the brain of rats undergoing social isolation rearing as a validation of this animal model. We hypothesized that isolation rearing reduces mRNA and protein expressions of NMDAR subunits and cause DNA methylation changes.MethodsWistar rats were kept isolated or grouped (n=10/group) from weaning (21 days after birth) to 10 weeks and then exposed to the Open Field Test to assess locomotion. Afterwards the behavioural tests, the tissues were dissected for RNA/DNA extraction and NMDAR subunits were analysed using qRT-PCR, ELISA and pyrosequencing. Data were analysed by parametric tests.ResultsIsolated-reared animals presented: (i) decreased mRNA levels of Grin1 (p=0.011), Grin2a (p=0.039) and Grin2b (p=0.037) in the PFC followed by reduction in the GABAergic markers; (ii) increased NR1 protein levels in the HIPPO (p=0.001); (iii) hypermethylation of Grin1 at CpG5 in the PFC (p=0.047) and Grin2b CpG4 in the HIPPO when compared to grouped (p=0.024). Moreover, isolated and grouped animals presented a negative correlation between Grin1 mRNA and Grin1 methylation levels at CpG5 in the PFC (r: -0.577; p=0.010) and isolated rats presented a negative correlation between Grin2b methylation at CpG4 and NR2 protein levels in the HIPPO (r: -0.753; p=0.012).DiscussionThis study supports the hypothesis that the NMDAR methylation changes found in the brain tissues may underlie the NMDAR mRNA/protein expression alterations caused by the isolation period. These results highlighted the importance of the environmental influence during the development that may lead to cognitive impairments in adulthood. Moreover, we demonstrated that the social isolation rearing during 10 weeks causes long-lasting behavioral changes that may be more associated with late stages of schizophrenia. Our study contributes to the identification of the epigenetic mechanisms involved in the neuropathophysiology of schizophrenia, which can bring new pharmacotherapeutic strategies and to identify biomarkers that can improve the early interventions in schizophrenia patients. Finally, our data thus reinforce the validity of rats reared in social isolation after weaning in modelling aspects of schizophrenia, highlighting the glutamatergic and GABAergic features involved principally in the cognitive impairments related to prefrontal cortex.

Altered expression of glutamatergic and GABAergic genes in the valproic acid-induced rat model of autism: A screening test

Autism spectrum disorders (ASD) include neurodevelopmental disorders in which behavioral deficits can result from neuronal imbalance of excitation to inhibition (E/I) in the brain. Here we used RT-qPCR to screen for the expression of 99 genes associated with excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission in the cerebral cortex, hippocampus and cerebellum of rats in an established VPA model of ASD. The largest changes in the expression of glutamatergic genes were found in the cerebral cortex, where 12 genes including these encoding some of the subunits of the ionotropic glutamate receptors, were upregulated, while 2 genes were downregulated. The expression of genes encoding the presynaptic glutamatergic proteins vGluT1 and mGluR7 and PKA, involved in downstream glutamatergic signaling, was elevated more than 100-fold. Changes in GABAergic gene expression were found in the cortex, cerebellum and hippocampus; 3 genes were upregulated, and 3 were downregulated. In conclusion, these results revealed that, in the ASD model, several glutamatergic genes in the rat cerebral cortex were upregulated, which contrasts with small and balanced changes in the expression of GABAergic genes. The VPA rat model, useful in studying the molecular basis of ASD, may be suitable for testing experimental therapies in these disabilities.