Koji Ohno

Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition.

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.

Deletion of the Mouse Glycine Transporter 2 Results in a Hyperekplexia Phenotype and Postnatal Lethality

The glycine transporter subtype 2 (GlyT2) is localized in the axon terminals of glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.

The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system

Cation-chloride cotransporters have been considered to play pivotal roles in controlling intracellular and extracellular ionic environments of neurons and hence controlling neuronal function. We investigated the total distributions of K-Cl cotransporter 1 (KCC1), KCC2 (KCC2), and Na-K-2Cl cotransporter 1 (NKCC1) messenger RNAs in the adult rat nervous system using in situ hybridization histochemistry. KCC2 messenger RNA was abundantly expressed in most neurons throughout the nervous system. However, we could not detect KCC2 messenger RNA expression in the dorsal root ganglion and mesencephalic trigeminal nucleus, where primary sensory neurons show depolarizing responses to GABA, suggesting that the absence of KCC2 is necessary for this phenomenon. Furthermore, KCC2 messenger RNA was also not detected in the dorsolateral part of the paraventricular nucleus, dorsomedial part of the suprachiasmatic nucleus, and ventromedial part of the supraoptic nucleus where vasopressin neurons exist, and in the reticular thalamic nucleus. As vasopressin neurons in the suprachiasmatic nucleus and neurons in the reticular thalamic nucleus produce their intrinsic rhythmicity, the lack of KCC2 messenger RNA expression in these regions might be involved in the genesis of rhythmicity through the control of intracellular chloride concentration. The expression levels of KCC1 and NKCC1 messenger RNAs were relatively low, however, positive neurons were observed in several regions, including the olfactory bulb, hippocampus, and in the granular layer of the cerebellum. In addition, positive signals were seen in the non-neuronal cells, such as choroid plexus epithelial cells, glial cells, and ependymal cells, suggesting that KCC1 and NKCC1 messenger RNAs were widely expressed in both neuronal and non-neuronal cells in the nervous system. These results clearly indicate a wide area- and cell-specific variation of cation chloride cotransporters, emphasizing the central role of anionic homeostasis in neuronal function and communication.

Alteration of plasma glutamate and glutamine levels in children with high-functioning autism

Background It has recently been hypothesized that hyperglutamatergia in the brain is involved in the pathophysiology of autism. However, there is no conclusive evidence of the validity of this hypothesis. As peripheral glutamate/glutamine levels have been reported to be correlated with those of the central nervous system, the authors examined whether the levels of 25 amino acids, including glutamate and glutamine, in the platelet-poor plasma of drug-naïve, male children with high-functioning autism (HFA) would be altered compared with those of normal controls. Methodology/Principal Findings Plasma levels of 25 amino acids in male children (N = 23) with HFA and normally developed healthy male controls (N = 22) were determined using high-performance liquid chromatography. Multiple testing was allowed for in the analyses. Compared with the normal control group, the HFA group had higher levels of plasma glutamate and lower levels of plasma glutamine. No significant group difference was found in the remaining 23 amino acids. The effect size (Cohens d) for glutamate and glutamine was large: 1.13 and 1.36, respectively. Using discriminant analysis with logistic regression, the two values of plasma glutamate and glutamine were shown to well-differentiate the HFA group from the control group; the rate of correct classification was 91%. Conclusions/Significance The present study suggests that plasma glutamate and glutamine levels can serve as a diagnostic tool for the early detection of autism, especially normal IQ autism. These findings indicate that glutamatergic abnormalities in the brain may be associated with the pathobiology of autism.

The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1

The glycine transporter subtype 2 (GlyT2) is localized at glycinergic axon terminals where it mediates the re-uptake of glycine from the extracellular space. In this study, we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic carboxy terminal tail region of GlyT2. Screening of a rat brain cDNA library identified the PDZ domain protein syntenin-1 as an intracellular binding partner of GlyT2. In pull-down experiments, the interaction between GlyT2 and syntenin-1 was found to involve the C-terminal amino acid residues of GlyT2 and the PDZ2 domain of syntenin-1. Syntenin-1 is widely expressed in brain and co-localizes with GlyT2 in brainstem sections. Furthermore, syntenin-1 binds syntaxin 1A, which is known to regulate the plasma membrane insertion of GlyT2. Thus, syntenin-1 may be an in vivo binding partner of GlyT2 that regulates its trafficking and/or presynaptic localization in glycinergic neurons.

Amygdala kindling induces upregulation of mRNA for NKCC1, a Na+, K+–2Cl− cotransporter, in the rat piriform cortex

GABA, the main inhibitory neurotransmitter in the brain, elicits a hyperpolarizing response by activation of the GABA(A)-receptor/chloride-channel complex under conditions of normal Cl(-) homeostasis. Thus the pathogenesis of epilepsy could involve an impairment of GABA(A)-receptor-mediated inhibition due to a collapse of the Cl(-) gradient. We examined the expression patterns of Cl(-) transporters and a Cl(-) channel in a rat amygdala-kindling model. Activity-dependent increases were observed in the mRNA for NKCC1, an inwardly-directed Cl(-) transporter, in the piriform cortex. This suggests that an increase in [Cl(-)](i) and a resultant reduction in GABAergic inhibition may occur in the kindled piriform cortex.

Changes in chloride homeostasis-regulating gene expressions in the rat hippocampus following amygdala kindling

In a rat kindling model, we examined expression patterns of NKCC1, KCC1, KCC2, and CLC-2. In the dentate granule cell layer, there was an activity-dependent increase in NKCC1 mRNA but significant decreases in KCC1 and CLC-2 mRNAs. In addition, CLC-2 mRNA expression was markedly decreased in CA1 pyramidal layer. These results suggest that an increase in [Cl-]i and a resultant reduction in GABAergic inhibition may occur in hippocampus of epileptic rats.

Differential expression of KCC2 accounts for the differential GABA responses between relay and intrinsic neurons in the early postnatal rat olfactory bulb.

The rat olfactory bulb is anatomically immature at birth, and considerable neurogenesis and synaptogenesis are known to take place postnatally. In addition, significant physiological changes have also been reported in this period. For example, granule cell‐mediated inhibition following electrical stimulations to the lateral olfactory tract is robust during the first postnatal week, and then decreases abruptly after the second week. However, the mechanism underlying this enhanced inhibition remains to be elucidated. To know the cause of this phenomenon, we investigated the expression patterns of cation‐Cl– co‐transporters (KCC1, KCC2 and NKCC1) mRNAs, which are responsible for the regulation of [Cl–]i. In addition, responses to γ‐aminobutyric acid (GABA) were measured by gramicidin‐perforated patch‐clamp recordings and Ca2+ imaging using fura‐2. We found that in the early postnatal period, mitral cells expressing KCC2 mRNA were inhibited by GABA, while granule cells lacking KCC2 mRNA expression were depolarized or excited by GABA. These results indicate that transient GABA‐mediated excitation on granule cells might be the main cause of the enhanced inhibition on mitral cells, and suggest that these differential GABA responses between relay and intrinsic neurons play pivotal roles in the early postnatal rat olfactory bulb.

Differential expression patterns of three glutamate transporters (GLAST, GLT1 and EAAC1) in the rat main olfactory bulb

Glutamate is the main neurotransmitter in the olfactory bulb. Therefore, glutamate transporters, which regulate the concentration of extracellular glutamate, might play pivotal roles in odor processing. In this study, we examined expressions of three glutamate transporters (GLAST, GLT1 and EAAC1) in the olfactory bulb using in situ hybridization and immunohistochemistry. EAAC1 mRNA was expressed in neurons, such as periglomerular cells, tufted cells, mitral cells and granule cells as shown before in other brain areas. In contrast, GLAST and GLT1 were found in glial cells throughout the olfactory bulb, with intenser expressions in the glomerular layer, external plexiform layer and internal plexiform layer where glutamatergic synapses are concentrated. In addition, using double staining immunohistochemistry we clearly showed that GLAST and GLT1 were expressed in astrocytes. Furthermore, we found that GLAST was also intensely expressed in the subependymal layer where precursor cells exist. These results suggest each glutamate transporter plays its unique role not only in glutamatergic neurotransmission but also in cell differentiation and migration in the olfactory bulb.

Enzymes in the glutamate-glutamine cycle in the anterior cingulate cortex in postmortem brain of subjects with autism

BackgroundAccumulating evidence suggests that dysfunction in the glutamatergic system may underlie the pathophysiology of autism. The anterior cingulate cortex (ACC) has been implicated in autism as well as in glutamatergic neurotransmission. We hypothesized that alterations in the glutamate-glutamine cycle in the ACC might play a role in the pathophysiology of autism.MethodsWe performed Western blot analyses for the protein expression levels of enzymes in the glutamate-glutamine cycle, including glutamine synthetase, kidney-type glutaminase, liver-type glutaminase, and glutamate dehydrogenases 1 and 2, in the ACC of postmortem brain of individuals with autism (n = 7) and control subjects (n = 13).ResultsWe found that the protein levels of kidney-type glutaminase, but not those of the other enzymes measured, in the ACC were significantly lower in subjects with autism than in controls.ConclusionThe results suggest that reduced expression of kidney-type glutaminase may account for putative alterations in glutamatergic neurotransmission in the ACC in autism.