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The delta 2 subunit of the glutamate receptor (GLuR) channel is localized selectively in cerebellar Purkinje cells. Analysis by gene targeting has shown that it is important for motor coordination, as well as the formation of synapses between parallel fibers and Purkinje cells and between climbing fibers and Purkinje cells. Loss of the delta 2 subunit results in changes in the long-term depression of synaptic transmission between parallel fiber and Purkinje cells. Result suggests that synaptic plasticity is closely related to synapse formation in the cerebellum.

Gamma-amino butyric acid (GABA) negatively regulates the excitatory activity of neurons and is a predominant neurotransmitter in the nervous system. The olfactory bulb, the main center in the olfactory system, is modulated by inhibitory interneurons that use GABA as their main neurotransmitter. The present study aimed to evaluate GABAergic transmission in the main olfactory bulb (MOB) of the Korean roe deer ( Capreolus pygargus ) by examining the immunohistochemical localization of GABAergic key molecules, including glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT), GABA transporters (GATs; GAT-1 and GAT-3), and potassium sodium chloride co-transporter 2 (KCC2). GAD, VGAT, and KCC2 were expressed in the glomerular layer (GL), external plexiform layer (ePL), mitral cell layer (ML), and granule cell layer (GrL). Intense GAT-1 expression was observed in the GL; GAT-1 expression was discernible in the ePL, ML, and GrL. However, intense GAT-3 expression was extensively observed in all layers of the MOB. These results suggest that substantial GABAergic synapses are present in the GL, ePL, ML, and GrL. Furthermore, the released GABA may be removed by GAT-1 and GAT-3 in the GL, and the majority of GABA, which is present in the ePL to GrL, may undergo reuptake by GAT-3. This is the first morphological and descriptive study of GABAergic transmission in the MOB of Korean roe deer. [ABSTRACT FROM AUTHOR]

Abstract: Gamma-amino butyric acid (GABA) mediates the hyperpolarization of membrane potential, negatively regulating glutamatergic activity in the adult brain, whereas, mediates depolarization in the immature brain. This developmental shift in GABA actions is induced by the expression of potassium chloride co-transporter 2 (KCC2). In this study, we focused on the developing mouse somatosensory cortex, where the barrel structure in layer 4 is altered by the whisker-lesion during the critical period, before postnatal day 4 (P4). First, to clarify the time-course of postnatal changes in GABA actions, we investigated the developmental localization of KCC2. Second, to reveal its spatial and temporal relationship with GABA synapse formation, we examined the developmental localization of GABA and vesicular GABA transporter. KCC2 was localized within the pyramidal cells in layer 5 after P3, granule cells in layer 4 after P5 and neurons in layers 2 and 3 after P7, indicating that KCC2 was expressed in the chronological order of neuronal settling at the destination. The onset of KCC2 localization was almost concomitant with the formation of GABA synapses, suggesting that GABA was inhibitory after GABA synapse formation. Furthermore, extrasynaptically released GABA might be involved in the maintenance of activity-dependent plasticity as an excitatory transmitter during the critical period. [Copyright &y& Elsevier]

Abstract: Developmental shift in GABA actions from depolarization to hyperpolarization occurs as a result of decreasing the intracellular Cl− concentration regulated by K+-Cl− co-transporter 2 (KCC2). To clarify the time-course of the developmental shift on the Purkinje cells, we examined KCC2-localization in the embryonic mouse cerebellum. The KCC2 was first detected within the Purkinje cells in the Purkinje cell layer of the hemisphere at embryonic day 15 (E15) and the vermis at E17, but the ventricular and intermediate zones were negative. These results suggest that GABA might become inhibitory on the Purkinje cells after their settling in the Purkinje cell layer. [Copyright &y& Elsevier]

Abstract: In the brain, γ-amino butyric acid (GABA), released extrasynaptically and synaptically from GABAergic neurons, plays important roles in morphogenesis, expression of higher functions and so on. In the GABAergic transmission system, plasma membrane GABA transporters (GATs) mediate GABA-uptake from the synaptic cleft in the mature brain and are thought to mediate diacrine of cytosolic GABA in the immature brain. In the present study, we focused on two GATs (GAT-1 and GAT-3) in the mouse cerebellar cortex, which are widely localized in neural and glial cells. Firstly, we examined the localization of GATs in the dendrites and cell bodies of developing GABAergic neurons, where GABA is extrasynaptically distributed, to clarify the GABA-diacrine before synaptogenesis. Secondly, we examined the developmental changes in the localization of GATs to reveal the development of the GABA-uptake system. Neither transporter was detected within the dendrites and cell bodies of GABAergic neurons, including Purkinje, stellate, basket and Golgi cells, in the immature cerebellar cortex. GAT-1 was observed within the Golgi cell axon terminals after postnatal day 5 (P5) and presynaptic axons of stellate and basket cells after P7. GAT-3 was localized within the astrocyte processes, sealing the GABAergic synapses in the Purkinje cell and granular layers after P10. These results indicated that GABA-diacrine did not work in the mouse cerebellar cortex. The onset of GAT-1-expression was prior to that of GAT-3. GAT-1 started to be localized within the GABAergic axon terminals during synapse formation. GAT-3 started to be localized within astrocyte processes when they sealed the synapses. [Copyright &y& Elsevier]

Abstract: In the adult brain, γ-amino butyric acid (GABA) is synaptically released and mediates inhibitory transmission. Recent studies have revealed that GABA is a trophic factor for brain development. To reveal the distribution of GABA and its secretion mechanisms during brain development, we investigated the immunohistochemical localization of two molecules, GABA and vesicular GABA transporter (VGAT), which is a GABAergic vesicle protein, in the developing mouse cerebellum by means of newly developed antibodies. Furthermore, we tested the relationship between developmental changes in distribution of above two molecules in the presynapses and ontogeny of GABAergic synapses. GABAergic synapses were detected by immunohistochemistry for the GABAA receptor α1 subunit, which is an essential subunit for inhibitory synaptic transmission in the mature cerebellar cortex. Until postnatal day 7 (P7), GABA was localized throughout the GABAergic neurons, and VGAT accumulated at axon varicosities and growth cones, where the α1 subunit did not accumulate. After P10, both GABA and VGAT became confined to the terminal sites where the α1 subunit was localized. These results suggested that GABA was extrasynaptically released from axon varicosities and growth cones by vesicular secretion ‘exocytosis’ and from all parts of GABAergic neurons during the cerebellar development by non-vesicular secretion ‘diacrine’. [Copyright &y& Elsevier]

In the adult central nervous system (CNS), γ-amino butyric acid (GABA) is a predominant inhibitory neurotransmitter, which regulates glutamatergic activity. Recent studies have revealed that GABA serves as an excitatory transmitter in the immature CNS, and is involved in brain morphogenesis. To elucidate how GABA exerts its effect on immature neurons and how GABAergic synapses are formed, we examined both development of pre- and post-synaptic elements of the GABAergic synapses formed between granule and Golgi cells in the mouse cerebellar granular layer. Immunohistochemistry for glutamic acid decarboxylase (GAD) demonstrated that GABA was localized throughout the Golgi cells before postnatal day 7 (P7), and became confined to the axon terminals during the second postnatal week. Electron microscopic analysis demonstrated that GABAergic synapses were clearly detected at P10. In situ hybridization and immunohistochemistry for the GABAA receptor α1 and α6 subunits, which are mainly involved in inhibitory synaptic transmission, demonstrated that both subunits appeared at P7. Distribution of both subunits expanded in the granular layer with special reference to the development of GABAergic synapses. Furthermore, the majority of the subunits accumulated adjacent to the GABAergic terminals. These results suggested that in the granular layer, GABA might be non-synaptically secreted from Golgi cell axons and dendrites during the first postnatal week. From the second postnatal week, GABA is synaptically released and begins to mediate inhibitory transmission. Furthermore, it was suggested that GABAergic innervation could initiate expression and trafficking of the GABAA receptors containing the α1 and α6 subunits. [Copyright &y& Elsevier]

In the adult mammalian brain, synaptic transmission mediated by γ-amino butyric acid (GABA) plays a role in inhibition of excitatory synaptic transmission. During brain development, GABA is involved in brain morphogenesis. To clarify how GABA exerts its effect on immature neurons, we examined the expression of the GABAA receptor α2 and α3 subunits, which are abundantly expressed before α1 and α6 subunits appear, in the developing mouse cerebellum using in situ hybridization. Proliferating neuronal precursors in the ventricular zone and external granular layer expressed neither α2 nor α3 subunits. Hybridization signals for the α2 and α3 subunit mRNAs first appeared in the differentiating zone at embryonic day 13 (E13). The α2 subunit was detected in the migrating and differentiating granule cells and cerebellar nucleus neurons until postnatal day 14 (P14). Hybridization signals for the α3 subunit mRNA, on the other hand, were localized in the developing Purkinje cells and cerebellar nucleus neurons, and disappeared from Purkinje cells by the end of first postnatal week. Taken together, this indicated that the α2 and α3 subunits were abundantly expressed in distinct types of cerebellar neurons after completing cell proliferation while forming the neural network. These results suggest that GABA might extrasynaptically activate the GABAA receptors containing α2 and/or α3 subunits on the differentiating neurons before finishing the formation of synapses and networks, and could be involved in neuronal differentiation and maturation in the cerebellum. [Copyright &y& Elsevier]

Synaptic transmission mediated by γ-amino butyric acid (GABA) plays an important role in inhibition of glutamatergic excitatory transmission and expression of higher brain functions, such as memory, learning and anxiety. To elucidate mechanisms underlying formation of the postsynaptic elements for GABAergic transmission, we employed the reeler mutant mice in this study. In the reeler cerebellum, abnormal cytoarchitecture and an aberrant environment affect the formation of neural networks and maturation of neurons. We examined the expression and localization of GABAA receptor α subunits in the reeler cerebellum and determined whether various abnormalities in the reeler mice affected formation of the postsynaptic elements. In situ hybridization analysis revealed that the specific expression of α subunit mRNAs in each neuronal type was preserved. Abnormal expression of α subunits was not detected, although GABAergic networks were altered and neuronal maturation was severely disturbed. Immunohistochemistry for the α1 and α6 subunits, which were expressed abundantly in the reeler cerebellum, revealed that both subunit proteins accumulated at positions adjacent to GABAergic terminals. These results, taken together, suggested that expression of the GABAA receptor subunits in postsynaptic neurons might be genetically determined, but trafficking and accumulation of the subunit proteins at the GABAergic synapse may be induced by GABAergic innervation. [Copyright &y& Elsevier]

• We injured tibial nerve in heterozygous KCC2-knockout (HT) and wild-type (WT) mice. • Motor dysfunction and decreased ChAT (Ach synthesis) were less severe in HT mice. • EM and immunostaining revealed faster degeneration, regeneration, and myelination. • Nerve injury may induce earlier and longer reduction of the KCC2 in HT mice. • Reduced KCC2 expression may accelerate axonal regeneration via GABA/Gly excitation. Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-] i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index—indicating lower limb motor function—was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-] i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation. [ABSTRACT FROM AUTHOR]

• Thalamic reticular nucleus (TRN) neurons are GABAergic and regulate thalamic neurons. • Avp-Vgat−/− mice show selective suppression of GABAergic transmission from the TRN. • Simple operant learning of lever pressing is normal in Avp-Vgat−/− mice. • Learning to press three levers in a correct order is impaired in Avp-Vgat−/− mice. • GABAergic input from the TRN to thalamus is critical for action sequence learning. The thalamic reticular nucleus (TRN) is a thin sheet of GABAergic neurons surrounding the thalamus, and it regulates the activity of thalamic relay neurons. The TRN has been reported to be involved in sensory gating, attentional regulation, and some other functions. However, little is known about the contribution of the TRN to sequence learning. In the present study, we examined whether the TRN is involved in reward-based learning of action sequence with no eliciting stimuli (operant conditioning), by analyzing the performance of male and female Avp-Vgat−/− mice (Vgatflox/flox mice crossed to an Avp-Cre driver line) on tasks conducted in an operant box having three levers. Our histological and electrophysiological data demonstrated that in adult Avp-Vgat−/− mice, vesicular GABA transporter (VGAT) was absent in most TRN neurons and the GABAergic transmission from the TRN to the thalamus was largely suppressed. The performance on a task in which mice needed to press an active lever for food reward showed that simple operant learning of lever pressing and learning of win-stay and lose-shift strategies are not affected in Avp-Vgat−/− mice. In contrast, the performance on a task in which mice needed to press three levers in a correct order for food reward showed that learning of the order of lever pressing (action sequence learning) was impaired in Avp-Vgat−/− mice. These results suggest that the TRN plays an important role in action sequence learning. [ABSTRACT FROM AUTHOR]

Summary: The prevalences of obesity and type 2 diabetes mellitus are dramatically increasing, and there is a strong need for more effective and safer therapies. However, some of drugs show limited efficacy and considerable adverse effects. Furthermore, artificial energy-dense foods and non-caloric foods may promote overeating and weight gain. In this context, a natural food-based approach may represent a valuable means of tackling the obesity–diabetes syndrome. Although recent studies have shown that brown rice improves glucose intolerance and prevents obesity and type 2 diabetes in humans, the underlying molecular mechanisms remain unclear. We found that one of the major components of brown rice, γ-oryzanol (Orz), plays an important role in the metabolically beneficial effects of brown rice. Orz acts as a chemical chaperone and decreases high fat diet (HFD)-induced endoplasmic reticulum (ER) stress in the hypothalamus, thereby leading to a significant shift in preference from fatty to healthy foods. Orz also decreases HFD-induced ER stress in pancreatic β-cells and improves β-cell function. Notably, Orz directly acts on pancreatic islets and enhances glucose-stimulated insulin secretion (GSIS). This evidence highlights food preference as a promising therapeutic target in obesity–diabetes syndrome and suggests that brown rice and Orz may have potential for the treatment of obesity and type 2 diabetes in humans. [Copyright &y& Elsevier]

[Display omitted] • Galanin and microglia increased but ChAT and KCC2 decreased during regeneration. • ChAT level gradually recovered during regeneration and myelination of axons. • Excitatory shift of GABA/glycine by KCC2 reduction may induce nerve regeneration. • Loose ligation resulted in slowly progressing nerve degeneration and regeneration. • Degeneration after nerve compression may cause motor dysfunction in human diseases. Peripheral nerve injury affects motor functions. To reveal the mechanisms underlying motor dysfunction and recovery after nerve compression, which have not been precisely examined, we investigated the temporal relationship among changes in motor function, nerve histopathology, and marker molecule expression in the spinal cord after loose ligation of the mouse sciatic nerve. After ligation, sciatic motor function suddenly declined, and axons gradually degenerated. During degeneration, galanin was localized in motor neuron cell bodies. Then, in the ventral horn, microglia were activated, and expression of choline acetyltransferase (ChAT), a synthetic enzyme of acetylcholine, and potassium chloride co-transporter 2 (KCC2), which shifts the action of γ-amino butyric acid (GABA) and glycine to inhibitory, decreased. Motor function recovery was insufficient although axonal regeneration was complete. ChAT levels gradually recovered during axonal regeneration. When regeneration was nearly complete, microglial activation declined, and KCC2 expression started to increase. The KCC2 level sufficiently recovered when axonal regeneration was complete, suggesting that the excitatory action of GABA/glycine may participate in axonal regeneration. Furthermore, these changes proceeded slower than those after severance, suggesting that loose ligation, compression, may mediate slower progression of degeneration and regeneration than severance, and these changes may cause the motor dysfunction and its recovery. [ABSTRACT FROM AUTHOR]

• We examined the PC-specific VGAT knockout (L7-VGAT) mice, in which GABA exocytosis was age-dependently diminished. • GABAergic synapse formation was completed by P2M, but glycinergic synapse formation continued until P13M in control CN. • The number of glycinergic terminals was significantly higher in the L7-VGAT FN than the control FN at P13M. • Increased glycinergic terminals in the L7-VGAT FN may derive from interneurons in the FN and may also release GABA. • The deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals in the L7-VGAT FN. GABA and glycine are inhibitory neurotransmitters. However, the mechanisms underlying the formation of GABAergic and glycinergic synapses remain unclear. The influence of GABAergic input deprivation on inhibitory terminal formation was investigated using Purkinje cell (PC)-specific vesicular GABA transporter (VGAT) knockout (L7-VGAT) mice, in which GABA release from PCs diminishes in an age-dependent manner. We compared the late development of GABAergic and glycinergic terminals in the cerebellar nucleus (CN) between control and L7-VGAT mice. In the control CN, the density of glutamate decarboxylase (GAD)-positive dots remained unchanged between postnatal 2 months (P2M) and 13 months (P13M), whereas glycine transporter 2 (GlyT2)-positive dots increased in density during this time frame. No difference in the density of GlyT2-positive dots was observed between control and L7-VGAT mice at P2M, but the density was significantly higher in the L7-VGAT fastigial nuclei (FN) than the control FN at P13M. When VGAT was absent from PC terminals, GlyT2-positive dots included GAD and VGAT and formed synapses. These results indicated that GABAergic terminals were formed by P2M, glycinergic terminals were actively formed after P2M, and more glycinergic terminals were formed in the L7-VGAT FN than in the control FN, suggesting that the increased glycinergic terminals may derive from interneurons within the FN and may also release GABA. These results suggest that the deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals derived from interneurons and that the inhibitory terminal numbers and types may be regulated by the quantity of functional GABAergic inputs. [ABSTRACT FROM AUTHOR]

The inhibitory neurotransmitter gamma-amino butyric acid (GABA) plays important roles in energy balance and feeding behavior in the hypothalamus. To reveal the time course of GABAergic network formation, we examined the immunohistochemical localization of glutamic acid decarboxylase (GAD), a GABAergic neuron marker, vesicular GABA transporter (VGAT), a marker of inhibitory terminals, and K + -Cl − -cotransporter2 (KCC2), which shifts GABA action from excitation to inhibition, in the developing mouse hypothalamus. GABAergic terminals, seen as GAD- and VGAT-positive dots, increased in density during embryonic development. Moreover, the onset of KCC2 localization was almost concomitant with GABAergic terminal formation, and KCC2-positive profiles increased in density during development. This suggested that after the formation of GABAergic terminals, GABAergic action may change to inhibition in the hypothalamus. This maturation appears to proceed as follows: the lateral hypothalamus (LH) matures first, followed by the paraventricular nucleus (PVN) by the time of birth, while the ventromedial hypothalamus (VMH) and the arcuate nucleus (Arc) are not fully mature at the time of birth. Our findings suggest that GABAergic networks in the “feeding center” (LH) and the “exit” (PVN) may mature before birth, while those in the “satiety center” (VMH) and “higher control center” (Arc) may mature after birth. [ABSTRACT FROM AUTHOR]

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension. [ABSTRACT FROM AUTHOR]

In the spinal cord, glycine and γ-amino butyric acid (GABA) are inhibitory neurotransmitters. However, the ontogeny of the glycinergic network remains unclear. To address this point, we examined the developmental formation of glycinergic terminals by immunohistochemistry for glycine transporter 2 (GlyT2), a marker of glycinergic terminals, in developing mouse cervical spinal cord. Furthermore, the developmental localization of GlyT2 was compared with that of glutamic acid decarboxylase (GAD), a marker of GABAergic terminals, and vesicular GABA transporter (VGAT), a marker of inhibitory terminals, by single and double immunolabeling. GlyT2-positive dots (glycinergic terminals) were first detected in the marginal zone on embryonic day 14 (E14). In the ventral horn, they were detected at E16 and increased in observed density during postnatal development. Until postnatal day 7 (P7), GAD-positive dots (GABAergic terminals) were dominant and GlyT2 immunolabeling was localized at GAD-positive dots. During the second postnatal week, GABAergic terminals markedly decreased and glycinergic terminals became dominant. In the dorsal horn, glycinergic terminals were detected at P0 in lamina IV and P7 in lamina III and developmentally increased. GlyT2 was also localized at GAD-positive dots, and colocalizing dots were dominant at P21. VGAT-positive dots (inhibitory terminals) continued to increase until P21. These results suggest that GABAergic terminals first appear during embryonic development and may often change to colocalizing terminals throughout the gray matter during development. The colocalizing terminals may remain in the dorsal horn, whereas in the ventral horn, colocalizing terminals may give rise to glycinergic terminals. [ABSTRACT FROM AUTHOR]

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid neuropeptide, synthesized by alternative splicing of calcitonin gene mRNA. CGRP is characteristically distributed in the nervous system, and its function varies depending on where it is expressed. To reveal developmental formation of the CGRP network and its function in neuronal maturation, we examined the immunohistochemical localization of CGRP in the developing mouse cervical spinal cord and dorsal root ganglion. CGRP immunolabeling (IL) was first detected in motor neurons on E13, and in ascending axons of the posterior funiculus and DRG neurons on E14. CGRP-positive sensory axon fibers entered Laminae I and II on E16, and Laminae I through IV on E18. The intensity of the CGRP-IL gradually increased in both ventral and dorsal horns during embryonic development, but markedly decreased in the ventral horn after birth. These results suggest that CGRP is expressed several days after neuronal settling and entry of sensory fibers, and that the CGRP network is formed in chronological and sequential order. Furthermore, because CGRP is markedly expressed in motor neurons when axons are vastly extending and innervating targets, CGRP may also be involved in axonal elongation and synapse formation during normal development. [ABSTRACT FROM AUTHOR]

GABA acts as inhibitory neurotransmitter in the adult central nervous system but as excitatory neurotransmitter during early postnatal development. This shift in GABA’s action from excitation to inhibition is caused by a decrease in intracellular chloride concentration ([Cl − ] i ), which in turn is caused by changes in the relative expression levels of the K + –Cl − co-transporter (KCC2) and the Na + , K + –2Cl − co-transporter (NKCC1) proteins. Previous studies have used slices containing the medullary pre-Bötzinger complex (pre-BötC) to record respiration-related rhythmic activity (RRA) from the hypoglossal nucleus (12 N). The role of GABAergic transmission in the regulation of medullary RRA neonatally, however, is yet to be determined. Here, we examined how GABA and chloride co-transporters contribute to RRA during development in the 12 N where inspiratory neurons reside. We recorded extracellular RRA in medullary slices obtained from postnatal day (P) 0–7 mice. RRA was induced by soaking slices in artificial cerebrospinal fluid (aCSF) containing 8 mM-K + . Application of GABA significantly increased the frequency of RRA after P3, whereas application of a KCC2 blocker ( R (+)-[(2- n -butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H -indenyl-5-yl)oxy]acetic acid (DIOA)) significantly decreased the frequency of RRA after P1. In addition, dense KCC2 immunolabeling was seen in the superior longitudinalis (SL) of the 12 N, which is responsible for retraction of the tongue, from P0 and P7. These results indicate that GABA administration can increase RRA frequency during the first week following birth. This in turn suggests that decreasing [Cl − ] i levels caused by increasing KCC2 levels in the 12 N could play important roles in regulating the frequency of RRA during development. [ABSTRACT FROM AUTHOR]

Highlights: [•] Developmental changes in GABAergic transmission were investigated in the mouse SpVi. [•] The SpVi may first receive GABAergic projection fibers from extra-nuclear area. [•] GABAergic neurons were localized after E15 and may form synapses after E17. [•] GABA action may shift from excitatory to inhibitory between E13 and E17. [ABSTRACT FROM AUTHOR]

Abstract: Trimethyltin (TMT) is an organotin neurotoxicant with effects that are selectively localized to the limbic system (especially the hippocampus), which produces memory deficits and temporal lobe seizures. Galectin-3 (Gal-3) is a beta-galactoside-binding lectin that is important in cell proliferation and regulation of apoptosis. The present study evaluated the temporal expression of Gal-3 in the hippocampus of adult BALB/c mice after TMT treatment (i.p., 2.5mg/kg). Western blotting analyses showed that Gal-3 immunoreactivity began to increase 2days after treatment; the immunoreactivity peaked significantly within 4days after treatment but significantly declined between days 4 and 8. Immunohistochemical analysis indicated that Gal-3 expression was very rare in the hippocampi of vehicle-treated controls. However, Gal-3 immunoreactivity appeared between 2 and 8days after TMT treatment and was primarily localized to the hippocampal dentate gyrus (DG), in which neuronal degeneration occurred. The immunoreactivity was detected predominantly in most of the Iba1-positive microglia and in some GFAP-positive astrocytes of the hippocampal DG. Furthermore, Gal-3 expression co-localized with the pro-inflammatory enzymes cyclooxygenase-2 and inducible nitric oxide synthase in the hippocampal DG. Therefore, we suggest that Gal-3 is involved in the inflammatory process of neurodegenerative disorder induced by organotin intoxication. [Copyright &y& Elsevier]

Abstract: In the adult brain, γ-amino butyric acid (GABA) is an inhibitory neurotransmitter, whereas it acts as an excitatory transmitter in the immature brain, and may be involved in morphogenesis. In the present study, we immunohistochemically examined the developmental changes in GABA signaling in the embryonic mouse cervical spinal cord. Glutamic acid decarboxylase and GABA were markers for GABA neurons. Vesicular GABA transporter was a marker for GABAergic and glycinergic terminals. Potassium chloride cotransporter 2 was a marker for GABAergic inhibition. We found five points: (1) In the ventral part, GABA neurons were divided into three groups. The first differentiated group sent commissural axons after embryonic day 11 (E11), but disappeared or changed their transmitter by E15. The second and third differentiated groups were localized in the ventral horn after E12, and sent axons to the ipsilateral marginal zone. There was a distal-to-proximal gradient in varicosity formation in GABAergic axons and a superficial-to-deep gradient in GABAergic synapse formation in the ventral horn; (2) In the dorsal horn, GABA neurons were localized after E13, and synapses were diffusely formed after E15; (3) GABA may be excitatory for several days before synapses formation; (4) There was a ventral-to-dorsal gradient in the development of GABA signaling. The GABAergic inhibitory network may develop in the ventral horn between E15 and E17, and GABA may transiently play crucial roles in inhibitory regulation of the motor system in the mouse fetus; (5) GABA signaling continued to develop after birth, and GABAergic system diminished in the ventral horn. [Copyright &y& Elsevier]

Abstract: In the adult central nervous system (CNS), GABA and glycine (Gly) are predominant inhibitory neurotransmitters, negatively regulating glutamatergic transmission. In the immature CNS, on the other hand, they act as trophic factors, mediating morphogenesis. In the present study, to investigate their involvement in axonal regeneration, we morphologically examined changes in their signaling in mouse hypoglossal nuclei during degeneration and regeneration of hypoglossal nerves. We found that (1) expression and localization of presynaptic elements were not changed, (2) localization of gephyrin, which anchors GABA and Gly receptors, was spread on the surface of motor neuron cell bodies and dendrites, (3) KCC2-expression markedly decreased, (4) choline acetyltransferase, which mediates acetylcholine-synthesis, immediately disappeared from the motor neurons, and (5) the synaptic cleft of both excitatory and inhibitory synapses became irregularly wider, in the hypoglossal nuclei of the sutured side after the operation. These changes gradually normalized during regeneration. These results suggested that synthesis of acetylcholine may be stopped in the motor neuron after axotomy. GABA/Gly may be normally released from presynaptic terminals, be spilled over the original synaptic cleft, be diffused into the neighboring space, bind to extrasynaptically localized receptors, and mediate depolarization of the membrane potential of motor neurons during degeneration and regeneration. Furthermore, it was suggested that GABA/Gly signaling in postsynaptic motor neurons went back to being immature after axotomy, and may play an important role in axonal regeneration. [Copyright &y& Elsevier]

• Tibial nerve injury induced allodynia, KCC2 reduction, and microglial activation. • Above changes remained after ligation but were recovered 90 days after severance. • Microglia density was high in tibial zone until D21 but in sural zone after D28. • Allodynia may be commonly caused by a reduction of GABAergic synaptic inhibition. • Development and persistence of allodynia may be induced by different mechanisms. Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter in the mature brain, but is excitatory during development and after motor nerve injury. This difference in GABAergic action depends on the intracellular chloride ion concentration ([Cl−] i), primarily regulated by potassium chloride co-transporter 2 (KCC2). To reveal precise processes of the neuropathic pain through changes in GABAergic action, we prepared tibial nerve ligation and severance models using male mice, and examined temporal relationships amongst changes in (1) the mechanical withdrawal threshold in the sural nerve area, (2) localization of the molecules involved in GABAergic transmission and its upstream signaling in the dorsal horn, and (3) histology of the tibial nerve. In the ligation model, tibial nerve degeneration disappeared by day 56, but mechanical allodynia, reduced KCC2 localization, and increased microglia density remained until day 90. Microglia density was higher in the tibial zone than the sural zone before day 21, but this result was inverted after day 28. In contrast, in the severance model, all above changes were detected until day 28, but were simultaneously and significantly recovered by day 90. These results suggested that in male mice, allodynia may be caused by reduced GABAergic synaptic inhibition, resulting from elevated [Cl−] i after the reduction of KCC2 by activated microglia. Furthermore, our results suggested that factors from degenerating nerve terminals may diffuse into the sural zone, whereby they induced the development of allodynia in the sural nerve area, while other factors in the sural zone may mediate persistent allodynia through the same pathway. [ABSTRACT FROM AUTHOR]