Static Magnetic Field Stimulation Enhances Shunting Inhibition via a SLC26 Family Cl- Channel, Inducing Intrinsic Plasticity.

Magnetic fields are being used for detailed anatomical and functional examination of the human brain. In addition, evidence for their efficacy in treatment of brain dysfunctions is accumulating. Transcranial static magnetic field stimulation (tSMS) is a recently developed technique for noninvasively modifying brain functions. In tSMS, a strong and small magnet when placed over the skull can temporarily suppress brain functions. Its modulatory effects persist beyond the time of stimulation. However, the neurophysiological mechanisms underlying tSMS-induced plasticity remain unclear. Here, using acute motor cortical slice preparation obtained from male C57BL/6N mice, we show that tSMS alters the intrinsic electrical properties of neurons by altering the activity of chloride (Cl-) channels in neurons. Exposure of mouse pyramidal neurons to a static magnetic field (SMF) at a strength similar to human tSMS temporarily decreased their excitability and induced transient neuronal swelling. The effects of SMF were blocked by DIDS and GlyH-101, but not by NPPB, consistent with the pharmacological profile of SLC26A11, a transporter protein with Cl- channel activity. Whole-cell voltage-clamp recordings of the GlyH-101-sensitive Cl- current component showed significant enhancement of the component at both subthreshold and depolarized membrane potentials after SMF application, resulting in shunting inhibition and reduced repetitive action potential (AP) firing at the respective potentials. Thus, this study provides the first neurophysiological evidence for the inhibitory effect of tSMS on neuronal activity and advances our mechanistic understanding of noninvasive human neuromodulation.

Therapeutic effects of KCC2 chloride transporter activation on detrusor overactivity in mice with spinal cord injury.

This study aimed to clarify whether downregulation of K+-Cl- cotransporter 2 (KCC2) in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). SCI was produced by Th8-9 spinal cord transection in female C57BL/6 mice. At 4 wk after SCI, CLP290, a KCC2 activator, was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC2 expression in cholinergic preganglionic parasympathetic neurons in the SPN was examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in the spinal cord slice preparations. DO evident as nonvoiding contractions was significantly reduced by CLP290 treatment in SCI mice. The number of c-fos-positive cells and coexpression of c-fos in choline acetyltransferase-positive cells were decreased in the SPN region of the SCI CLP290-treated group versus the SCI vehicle-treated group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons and normalized fluorescence intensity of KCC2 in choline acetyltransferase-positive SPN neurons was decreased in the SCI vehicle-treated group versus the spinal intact vehicle-treated group but recovered in the SCI CLP290-treated group. Extracellular recordings showed that CLP290 suppressed the high-frequency firing activity of SPN neurons in SCI mice. These results indicated that SCI-induced DO is associated with downregulation of KCC2 in preganglionic parasympathetic neurons and that activation of KCC2 transporters can reduce DO, increase KCC2 expression in preganglionic parasympathetic neurons, and decrease neuronal firing of SPN neurons in SCI mice.NEW & NOTEWORTHY This study is the first report to suggest that activation of the Cl- transporter K+-Cl- cotransporter 2 may be a therapeutic modality for the treatment of spinal cord injury-induced detrusor overactivity by targeting bladder efferent pathways.

A novel de novo KCNB1 variant altering channel characteristics in a patient with periventricular heterotopia, abnormal corpus callosum, and mild seizure outcome.

KCNB1 encodes the α-subunit of Kv2.1, the main contributor to neuronal delayed rectifier potassium currents. The subunit consists of six transmembrane α helices (S1-S6), comprising the voltage-sensing domain (S1-S4) and the pore domain (S5-P-S6). Heterozygous KCNB1 pathogenic variants are associated with developmental and epileptic encephalopathy. Here we report an individual who shows the milder phenotype compared to the previously reported cases, including delayed language development, mild intellectual disability, attention deficit hyperactivity disorder, late-onset epilepsy responsive to an antiepileptic drug, elevation of serum creatine kinase, and peripheral axonal neuropathy. On the other hand, his brain MRI showed characteristic findings including periventricular heterotopia, polymicrogyria, and abnormal corpus callosum. Exome sequencing identified a novel de novo KCNB1 variant c.574G>A, p.(Ala192Thr) located in the S1 segment of the voltage-sensing domain. Functional analysis using the whole-cell patch-clamp technique in Neuro2a cells showed that the Ala192Thr mutant reduces both activation and inactivation of the channel at membrane voltages in the range of -50 to -30 mV. Our case could expand the phenotypic spectrum of patients with KCNB1 variants, and suggested that variants located in the S1 segment might be associated with a milder outcome of seizures.

Astrocytic NKCC1 inhibits seizures by buffering Cl- and antagonizing neuronal NKCC1 at GABAergic synapses.

OBJECTIVE: A pathological excitatory action of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) has been observed in epilepsy. Blocking the Cl- importer NKCC1 with bumetanide is expected to reduce the neuronal intracellular Cl- concentration ([Cl- ]i ) and thereby attenuate the excitatory GABA response. Accordingly, several clinical trials of bumetanide for epilepsy were conducted. Although NKCC1 is expressed in both neurons and glial cells, an involvement of glial NKCC1 in seizures has not yet been reported. Astrocytes maintain high [Cl- ]i with NKCC1, and this gradient promotes Cl- efflux via the astrocytic GABAA receptor (GABAA R). This Cl- efflux buffers the synaptic cleft Cl- concentration to maintain the postsynaptic Cl- gradient during intense firing of GABAergic neurons, thereby sustaining its inhibitory action during seizure. In this study, we investigated the function of astrocytic NKCC1 in modulating the postsynaptic action of GABA in acute seizure models. METHODS: We used the astrocyte-specific conditional NKCC1 knockout (AstroNKCC1KO) mice. The seizurelike events (SLEs) in CA1 pyramidal neurons were triggered by tetanic stimulation of stratum radiatum in acute hippocampus slices. The SLE underlying GABAA R-mediated depolarization was evaluated by applying the GABAA R antagonist bicuculline. The pilocarpine-induced seizure in vivo was monitored in adult mice by the Racine scale. The SLE duration and tetanus stimulation intensity threshold and seizure behavior in AstroNKCC1KO mice and wild-type (WT) mice were compared. RESULTS: The AstroNKCC1KO mice were prone to seizures with lower threshold and longer duration of SLEs and larger GABAA R-mediated depolarization underlying the SLEs, accompanied by higher Racine-scored seizures. Bumetanide reduced these indicators of seizure in AstroNKCC1KO mice (which still express neuronal NKCC1), but not in the WT, both in vitro and in vivo. SIGNIFICANCE: Astrocytic NKCC1 inhibits GABA-mediated excitatory action during seizures, whereas neuronal NKCC1 has the converse effect, suggesting opposing actions of bumetanide on these cells.

Elucidation of pathological mechanism caused by human disease mutation in CaMKIIß.

Recently, we have identified CaMKIIα and CaMKIIß mutations in patients with neurodevelopmental disorders by whole exome sequencing study. Most CaMKII mutants have increased phosphorylation of Thr286/287, which induces autonomous activity of CaMKII, using cell culture experiments. In this study, we explored the pathological mechanism of motor dysfunction observed exclusively in a patient with Pro213Leu mutation in CaMKIIß using a mouse model of the human disease. The homozygous CaMKIIß Pro213Leu knockin mice showed age-dependent motor dysfunction and growth failure from 2 weeks after birth. In the cerebellum, the mutation did not alter the mRNA transcript level, but the CaMKIIß protein level was dramatically decreased. Furthermore, in contrast to previous result from cell culture, Thr287 phosphorylation of CaMKIIß was also reduced. CaMKIIß Pro213Leu knockin mice showed similar motor dysfunction as CaMKIIß knockout mice, newly providing evidence for a loss of function rather than a gain of function. Our disease model mouse showed similar phenotypes of the patient, except for epileptic seizures. We clearly demonstrated that the pathological mechanism is a reduction of mutant CaMKIIß in the brain, and the physiological aspects of mutation were greatly different between in vivo and cell culture.

GABAA Receptors and Maternally Derived Taurine Regulate the Temporal Specification of Progenitors of Excitatory Glutamatergic Neurons in the Mouse Developing Cortex.

Temporal specification of the neural progenitors (NPs) producing excitatory glutamatergic neurons is essential for histogenesis of the cerebral cortex. Neuroepithelial cells, the primary NPs, transit to radial glia (RG). To coincide with the transition, NPs start to differentiate into neurons, undergoing a switch from symmetric to asymmetric cell division. After the onset of neurogenesis, NPs produce layer-specific neurons in a defined order with precise timing. Here, we show that GABAA receptors (GABAARs) and taurine are involved in this regulatory mechanism. Foetal exposure to GABAAR-antagonists suppressed the transition to RG, switch to asymmetric division, and differentiation into upper-layer neurons. Foetal exposure to GABAAR-agonists caused the opposite effects. Mammalian foetuses are dependent on taurine derived from the mothers. GABA and taurine function as endogenous ligands for GABAARs. Ca2+ imaging showed that NPs principally responded to taurine but not GABA before E13. The histological phenotypes of the taurine transporter knockout mice resembled those of the mice foetally exposed to GABAAR-antagonists. Foetal exposure to GABAAR-modulators resulted in considerable alterations in offspring behavior like core symptoms of autism. These results show that taurine regulates the temporal specification of NPs and that disrupting the taurine-receptor interaction possibly leads to neurodevelopmental disorders.

Biallelic Variants in CNPY3, Encoding an Endoplasmic Reticulum Chaperone, Cause Early-Onset Epileptic Encephalopathy.

Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses.

Functional MHCI deficiency induces ADHD-like symptoms with increased dopamine D1 receptor expression.

Inappropriate synaptic development has been proposed as a potential mechanism of neurodevelopmental disorders, including attention-deficit hyperactivity disorder (ADHD). Major histocompatibility complex class I (MHCI), an immunity-associated molecule expressed by neurons in the brain, regulates synaptic development; however, the involvement of MHCI in these disorders remains elusive. We evaluated whether functional MHCI deficiency induced by ß2m-/-Tap1-/- double-knockout in mice leads to abnormalities akin to those seen in neurodevelopmental disorders. We found that functional MHCI deficiency induced locomotor hyperactivity, motor impulsivity, and attention deficits, three major symptoms of ADHD. In contrast, these mice showed normal spatial learning, behavioral flexibility, social behavior, and sensorimotor integration. In the analysis of the dopamine system, upregulation of dopamine D1 receptor (D1R) expression in the nucleus accumbens and a greater locomotor response to D1R agonist SKF 81297 were found in the functional MHCI-deficient mice. Low-dose methylphenidate, used for the treatment of ADHD patients, alleviated the three behavioral symptoms and suppressed c-Fos expression in the D1R-expressing medium spiny neurons of the mice. These findings reveal an unexpected role of MHCI in three major symptoms of ADHD and may provide a novel landmark in the pathogenesis of ADHD.

Intracellular Cl- dysregulation causing and caused by pathogenic neuronal activity.

The intracellular Cl- concentration ([Cl-]i) is tightly regulated in brain neurons for stabilizing brain performance. The [Cl-]i in mature neurons is determined by the balance between the rate of Cl- extrusion mainly mediated by the neuron-specific type 2 K+-Cl- cotransporter (KCC2) and the rate of Cl- entry through various Cl- channels including GABAA receptors during neuronal activity. Disturbance of the balance causes instability of brain circuit performance and may lead to epileptic seizures. In the first part of this review, we discuss how genetic alterations in KCC2 in humans cause infantile migrating focal seizures, based on our previous report and others. Depolarization of the membrane potential increases the driving force for Cl- entry into neurons. Thus, the duration of action potential spike generation and the frequency of excitatory synaptic inputs are the crucial factors for determining the total amount of Cl- entry and the equilibrium [Cl-]i in neurons. Moreover, there is also a significant interdependence between the neuronal activity and the KCC2 expression. In the second part, we discuss plausible mechanisms by which excessive neuronal activity due to excitotoxic brain insults or other epilepsy-associated gene mutations may cause the Cl- imbalance in neurons and lead to epileptic discharges over the brain, using the schematic "unifying foci" model based on literature.

Can we understand human brain development from experimental studies in rodents?

Animal models are needed to gain an understanding of the genetic, molecular, cellular, and network mechanisms of human brain development. In rodents, a large spectrum of in vitro and in vivo approaches allows detailed analyses and specific experimental manipulations for studying the sequence of developmental steps in corticogenesis. Neurogenesis, neuronal migration, cellular differentiation, programmed cell death, synaptogenesis, and myelination are surprisingly similar in the rodent cortex and the human cortex. Spontaneous EEG activity in the pre- and early postnatal human cortex resembles the activity patterns recorded with intracortical multi-electrode arrays in newborn rodents. This early activity is generated by thalamic activation of a subplate-driven local network coupled via gap junctions, which controls the development of cortical columns and the spatio-temporal pattern of apoptosis. Disturbances of this activity may induce disturbances in cortical structure and function leading to neurological and psychiatric disorders.

Diverse Actions of Astrocytes in GABAergic Signaling.

An imbalance of excitatory and inhibitory neurotransmission leading to over excitation plays a crucial role in generating seizures, while enhancing GABAergic mechanisms are critical in terminating seizures. In recent years, it has been reported in many studies that astrocytes are deeply involved in synaptic transmission. Astrocytes form a critical component of the "tripartite" synapses by wrapping around the pre- and post-synaptic elements. From this location, astrocytes are known to greatly influence the dynamics of ions and transmitters in the synaptic cleft. Despite recent extensive research on excitatory tripartite synapses, inhibitory tripartite synapses have received less attention, even though they influence inhibitory synaptic transmission by affecting chloride and GABA concentration dynamics. In this review, we will discuss the diverse actions of astrocytic chloride and GABA homeostasis at GABAergic tripartite synapses. We will then consider the pathophysiological impacts of disturbed GABA homeostasis at the tripartite synapse.

De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy.

Heterotrimeric G proteins, composed of α, ß, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.

Accumulation of GABAergic neurons, causing a focal ambient GABA gradient, and downregulation of KCC2 are induced during microgyrus formation in a mouse model of polymicrogyria.

Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase-green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl(-) transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl(-) concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca(2+) oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca(2+) oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.

Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus.

While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 µM taurine or 10 µM glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 µM 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 µM strychnine or after incubation with the loop diuretic bumetanide (10 µM), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (≥ 1 mM) or glycine (100 µM) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 µM) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus.

Early establishment of chloride homeostasis in CRH neurons is altered by prenatal stress leading to fetal HPA axis dysregulation.

Corticotropin-releasing hormone (CRH) neurons play an important role in the regulation of neuroendocrine responses to stress. The excitability of CRH neurons is regulated by inhibitory GABAergic inputs. However, it is unclear when GABAergic regulation of CRH neurons is established during fetal brain development. Furthermore, the exact progression of the developmental shift of GABA action from depolarization to hyperpolarization remains unelucidated. Considering the importance of CRH neuron function in subsequent hypothalamic-pituitary-adrenal (HPA) axis regulation during this critical phase of development, we investigated the ontogeny of GABAergic inputs to CRH neurons and consequent development of chloride homeostasis. Both CRH neuron soma in the paraventricular nucleus (PVN) and axons projecting to the median eminence could be identified at embryonic day 15 (E15). Using acute slices containing the PVN of CRF-VenusΔNeo mice, gramicidin perforated-patch clamp-recordings of CRH neurons at E15, postnatal day 0 (P0), and P7 were performed to evaluate the developmental shift of GABA action. The equilibrium potential of GABA (EGABA) was similar between E15 and P0 and showed a further hyperpolarizing shift between P0 and P7 that was comparable to EGABA values in adult CRH neurons. GABA primarily acted as an inhibitory signal at E15 and KCC2 expression was detected in CRH neurons at this age. Activation of the HPA axis has been proposed as the primary mechanism through which prenatal maternal stress shapes fetal development and subsequent long-term disease risk. We therefore examined the impact of maternal food restriction stress on the development of chloride homeostasis in CRH neurons. We observed a depolarization shift of EGABA in CRH neurons of pups exposed to maternal food restriction stress. These results suggest that Cl- homeostasis in early developmental CRH neurons attains mature intracellular Cl- levels, GABA acts primarily as inhibitory, and CRH neurons mature and function early compared with neurons in other brain regions, such as the cortex and hippocampus. Maternal food restriction stress alters chloride homeostasis in CRH neurons of pups, reducing their inhibitory control by GABA. This may contribute to increased CRH neuron activity and cause activation of the HPA axis in pups.

Cl⁻ homeodynamics in gap junction-coupled astrocytic networks on activation of GABAergic synapses.

The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction-coupled astrocytic network are still unclear. GABA-induced astrocytic Cl⁻ flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl⁻]o. Thus, revealing the properties of GABA-mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl⁻ dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl⁻ imaging during GABA application, and whole cell recordings from interneuron-astrocyte pairs in the stratum lacunosum-moleculare. Astrocytic [Cl⁻]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl⁻ flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor-mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing-induced currents and induced bicuculline-insensitive, mGAT4 inhibitor-sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl⁻ efflux because GABAA receptors are localized near the synaptic clefts. This GABA-induced Cl⁻ efflux was accompanied by Cl⁻ siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing-induced currents. Thus, Cl⁻ efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity-dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl⁻ conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl⁻]o.

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway.

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.

Dominant-negative mutations in alpha-II spectrin cause West syndrome with severe cerebral hypomyelination, spastic quadriplegia, and developmental delay.

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

A perturbation of multimodal GABA functions underlying the formation of focal cortical malformations: assessments by using animal models.

In order to study how the formation of focal cortical malformations is attributed to perturbation of developmentally multimodal γ-aminobutyric acid (GABA) functions, we made a focal cortical freeze-lesion on a rodent cerebral cortex at P0 (postnatal day 0). The microgyrus was formed at P7. GABA neurons were accumulated in the region surrounding necrosis at P4. Cortical plate cells born at E17.5 gathered, surrounding the GABA neurons, forming the cell dense portions in layer 2 of the microgyrus. Ambient GABA level was increased in the area corresponding to populated GABA neurons at P4. A KCC2 expression was downregulated, whereas an NKCC1 expression was upregulated in both the gathering GABA and cortical plate neurons, suggesting these cells had high intracellular Cl(-) concentration rendering GABA action depolarizing. GABAA receptor activation was involved in Ca(2+) oscillation in these gathering cells. In vivo blockade of GABAA receptor prevented the above characteristic pattern of cell accumulation and hence microgyrus formation. Thus, neonatal freeze-lesion causes characteristic accumulation of differential populations of neurons preceded by characteristic release of GABA at an early stage, which induces GABAA receptor-mediated depolarization and Ca(2+) oscillation. This paracrine/autocrine GABA may underlie the formation of neocortical malformations such as polymicrogyria.

Maternal taurine as a modulator of Cl- homeostasis as well as of glycine/GABAA receptors for neocortical development.

During brain and spinal cord development, GABA and glycine, the inhibitory neurotransmitters, cause depolarization instead of hyperpolarization in adults. Since glycine and GABAA receptors (GABAARs) are chloride (Cl-) ion channel receptor, the conversion of GABA/glycine actions during development is influenced by changes in the transmembrane Cl- gradient, which is regulated by Cl- transporters, NKCC1 (absorption) and KCC2 (expulsion). In immature neurons, inhibitory neurotransmitters are released in a non-vesicular/non-synaptic manner, transitioning to vesicular/synaptic release as the neuron matures. In other word, in immature neurons, neurotransmitters generally act tonically. Thus, the glycine/GABA system is a developmentally multimodal system that is required for neurogenesis, differentiation, migration, and synaptogenesis. The endogenous agonists for these receptors are not fully understood, we address taurine. In this review, we will discuss about the properties and function of taurine during development of neocortex. Taurine cannot be synthesized by fetuses or neonates, and is transferred from maternal blood through the placenta or maternal milk ingestion. In developing neocortex, taurine level is higher than GABA level, and taurine tonically activates GABAARs to control radial migration as a stop signal. In the marginal zone (MZ) of the developing neocortex, endogenous taurine modulates the spread of excitatory synaptic transmission, activating glycine receptors (GlyRs) as an endogenous agonist. Thus, taurine affects information processing and crucial developmental processes such as axonal growth, cell migration, and lamination in the developing cerebral cortex. Additionally, we also refer to the possible mechanism of taurine-regulating Cl- homeostasis. External taurine is uptake by taurine transporter (TauT) and regulates NKCC1 and KCC2 mediated by intracellular signaling pathway, with-no-lysine kinase 1 (WNK1) and its subsequent kinases STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress response kinase-1 (OSR1). Through the regulation of NKCC1 and KCC2, mediated by the WNK-SPAK/OSR1 signaling pathway, taurine plays a role in maintaining Cl- homeostasis during normal brain development.