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Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the "dematuration" of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities. [ABSTRACT FROM AUTHOR]

Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the “dematuration” of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities.

Abstract Background Lead (Pb2+) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca2+ signaling. How perinatal Pb2+ exposure affects Ca2+-related gene regulation remains unclear. However, Ca2+ activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission. Method A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam’s litters each ranged from N = 6–10 pups per litter (M = 8, SD = 2), irrespective of Pb2+ treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb2+− and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter’s obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb2+ N = 12) were used in the present study. Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Post-centrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 μL of DEPC treated H2O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb2+ exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABAAR-β3, NKCC1, KCC2, and GAD 80, 86, 65, and 67 isoforms. Results Perinatal Pb2+ exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb2+ exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb2+ competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb2+ at PND 2 and 7, and both PFC and HP were negatively impacted by Pb2+ at PND 22. Additionally, Pb2+ altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb2+ at PND 22. Conclusions Perinatal Pb2+ exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb2+ has on the GABAergic system preceding what is known regarding Pb2+ insults unto the glutamatergic system.

Background: Lead (Pb2+) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca2+ signaling. How perinatal Pb2+ exposure affects Ca2+-related gene regulation remains unclear. However, Ca2+ activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-to-inhibitory neurotransmission. Method: A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam's litters each ranged from N = 6–10 pups per litter (M = 8, SD = 2), irrespective of Pb2+ treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb2+− and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter's obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb2+N = 12) were used in the present study. Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Post-centrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 μL of DEPC treated H2O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb2+ exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABAAR-β3, NKCC1, KCC2, and GAD 80, 86, 65, and 67 isoforms. Results: Perinatal Pb2+ exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb2+ exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb2+ competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb2+ at PND 2 and 7, and both PFC and HP were negatively impacted by Pb2+ at PND 22. Additionally, Pb2+ altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb2+ at PND 22. Conclusions: Perinatal Pb2+ exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb2+ has on the GABAergic system preceding what is known regarding Pb2+ insults unto the glutamatergic system. [ABSTRACT FROM AUTHOR]