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In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during in vitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte–cumulus–granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500 µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P < 0.05). Regardless of Ax treatment, 17β-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P < 0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P < 0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P < 0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P < 0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells. In vitro growth (IVG) culture is not established for bovine oocytes and development of the culture system is necessary. This study investigated the effect of astaxanthin (Ax), a strong antioxidant, on the developmental competence of bovine oocytes during IVG. Ax supplementation of IVG medium improved the quality of bovine oocytes due to suppression of the luteinisation of granulosa cells. [ABSTRACT FROM AUTHOR]

Phyto-oestrogens are biologically active components of many human and laboratory animal diets. In the present study, we investigated, in adult male mice with C57 BL/6 genetic background, the effects of a reduced phyto-oestrogens intake on anxiety-related behaviour and associated gene expression in the amygdala. After 6 weeks on a low-phyto-oestrogen diet (< 20 μg/g cumulative phyto-oestrogen content), animals showed reduced centre exploration in an open-field task compared to their littermates on a soybean-based standard diet (300 μg/g). Freezing behaviour in an auditory fear memory task, in contrast, was not affected. We hypothesised that this mildly increased anxiety may involve changes in the function of GABAergic local circuit neurones in the amygdala. Using GAD67+/GFP mice, we could demonstrate reduced transcription of the GAD67 gene in the lateral and basolateral amygdala under the low-phyto-oestrogen diet. Analysis of mRNA levels in microdissected samples confirmed this regulation and demonstrated concomitant changes in expression of the second glutamic acid decarboxylase (GAD) isoform, GAD65, as well as the anxiolytic neuropeptide Y. These molecular and behavioural alterations occurred without apparent changes in circulating oestrogens or testosterone levels. Our data suggest that expression regulation of interneurone-specific gene products in the amygdala may provide a mechanism for the control of anxiety-related behaviour through dietary phyto-oestrogens. [ABSTRACT FROM AUTHOR]

Irradiation test results demonstrate the validity of a scan FF technology for separately evaluating SET and SEU soft error rates (SERs) in logic VLSI systems. The SET and SEU soft errors mean the upset caused by latching an SET pulse that originates in combinational logic cell blocks and the upset caused by a direct ion hit to the FF, respectively. A test chip is fabricated using a 0.2-μm fully-depleted silicon-on-insulator standard cell library and irradiated under an LET of 40 MeV-cm2 /mg. The SET and SEU soft error rates are successfully measured by the scan FFs on the test chip. A theoretical SET SER estimation from measured SET-pulse widths is also experimentally validated. [ABSTRACT FROM AUTHOR]

Abstract: Despite increasingly widespread use of recombinant adeno-associated virus (AAV) and lentiviral (LV) vectors for transduction of neurons in a wide range of brain structures and species, the diversity of cell types within a given brain structure is rarely considered. For example, the ability of a vector to transduce neurons within a brain structure is often assumed to indicate that all neuron types within the structure are transduced. We have characterized the transduction of mouse somatosensory cortical neuron types by recombinant AAV pseudotyped with serotype 1 capsid (rAAV2/1) and by recombinant lentivirus pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein. Both vectors used human synapsin (hSyn) promoter driving DsRed-Express. We demonstrate that high titer rAAV2/1-hSyn efficiently transduces both cortical excitatory and inhibitory neuronal populations, but use of lower titers exposes a strong preference for transduction of cortical inhibitory neurons and layer 5 pyramidal neurons. In contrast, we find that VSV-G-LV-hSyn principally labels excitatory cortical neurons at the highest viral titer generated. These findings demonstrate that endogenous tropism of rAAV2/1 and VSV-G-LV can be used to obtain preferential gene expression in mouse somatosensory cortical inhibitory and excitatory neuron populations, respectively. [Copyright &y& Elsevier]

Abstract: Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient N-methyl-d-aspartic acid (NMDA) exposure resulted in irreversible loss of synaptic function and loss of microtubule associated protein 2 (MAP2) from apical dendrites. The present study examined the initiation and progression of dendritic injury in mouse hippocampal slices following this excitotoxic stimulus. NMDA exposure (30 μM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20–90 min) involving most proximal dendrites. Over the same time course, immunoreactivity for the microtubule-associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the time course of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Δneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slices. [Copyright &y& Elsevier]

Heavy-ion-induced SET-pulse widths in NOR-logic cells fabricated by a 0.2-μm FD-SOI technology are directly measured by using an on-chip self-triggering Flip-Flop circuit. The pulse widths are distributed from 0.3 to 1.0 ns under a constant LET of 40 MeV · cm²/mg. [ABSTRACT FROM AUTHOR]

Abstract: Cerebellar Purkinje cells have the most elaborate dendritic trees among the neurons in the CNS. To investigate the dynamic aspects of dendritic morphogenesis of Purkinje cells, we performed a long-term analysis of living cells in cerebellar cell cultures derived from glutamate decarboxylase 67–green fluorescent protein mice. Most Purkinje cells had several primary dendrites during the 25-day culture period. Repeated observation of green fluorescent protein-expressing Purkinje cells over a period of 10–25 days in vitro demonstrated that not only extension, but also retraction of primary dendrites occurred during this culture period. Interestingly, both extension and retraction of primary dendrites were active between 10 and 15 days in vitro, and retraction of a primary dendrite occurred concomitantly with elongation of other primary dendrites in the same cell. Analysis of the morphological characteristics of the retracted primary dendrites demonstrated that shorter and less branched primary dendrites tended to retract. Furthermore, treatment with an inhibitor of calcium/calmodulin-dependent protein kinase II reduced the number of primary dendrites specifically during 5–15 days in vitro, the culture period when the extension and retraction of primary dendrites occurred actively. Blockade of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-type glutamate receptors also reduced the number of primary dendrites during the same culture period, while inhibition of glutamate transporters increased the number. These findings suggest that the final morphology of Purkinje cells is achieved not only through extension, but also through retraction of their dendrites, and that calcium/calmodulin-dependent protein kinase II and neuronal activity are involved in this dendritic morphogenesis. [Copyright &y& Elsevier]

Glutamate decarboxylase (GAD), which has two isoforms, GAD65, and GAD67, is responsible for synthesis of the major inhibitory neurotransmitter, γ-aminobutyric acid. GAD is expressed predominantly in the central nervous system; recent reports suggest that GAD is also expressed in non-neuronal organs including the pancreas. In the pancreatic islets, GAD serves as one of the autoantigens in type I diabetes mellitus. Recent flow cytometric analyses have shown that a variety of self-antigens, including GAD, are ectopically transcribed and expressed in particular cell populations of the thymus, although consensus concerning the cellular phenotype has not been obtained. The aim of this study was to clarify the localization and cellular phenotype of GAD67-expressing cells in the thymus at a cellular level with a novel approach using GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP is expressed specifically in GAD67-positive cells. GFP-positive cells were detected in the thymic medulla and were identified as epithelial cells by immunohistochemistry. Almost all GFP-positive cells were positive for major histocompatibility complex (MHC) class II antigen staining and were positive for both cytokeratin and Ulex Europaeus Agglutinin I, markers of medullary thymic epithelial cells, but were negative for CD11c, Gr-1, and CD45, markers of dendritic cells, macrophages, and B-lymphocytes, respectively. [ABSTRACT FROM AUTHOR]

Background: Upper respiratory tract infections (URTIs) account for at least half of all acute illnesses. Specific antiviral therapy has not been developed against most respiratory viruses thought to cause URTIs. The pharmacologic action of glycyrrhizin has been shown to produce anti-inflammatory activity, modulation of the immune system, inhibition of virus growth, and inactivation of viruses.Objective: The aim of this study was to assess the tolerability, efficacy, and cost of glycyrrhizin in improving the severity and duration of signs and symptoms of URTIs. The primary end point was tolerability, and the secondary and points included improvement in signs and symptoms of URTI and cost.Methods: Members of the Japanese Maritime Self-Defense Force (SDF) treated for URTIs from January 2002 to May 2002 in the SDF Etajima Hospital (Hiroshima, Japan) were eligible for this prospective, randomized, double-blind, controlled, parallel-group, alternate-day treatment assignment study. All patients in this study fulfilled the following enrollment criteria: admitted to the hospital on the first arrival day as an outpatient; fever (body temperature <38.0°C) with signs and symptoms of URTI (headache, sore throat, rhinorrhea, pharyngitis); and had not received antibiotics or oseltamivir phosphate for 4 weeks before the study. Patients who were admitted on an even day received an IV drip infusion of 40 mL of glycyrrhizin (0.2%) and 500 mL of lactated Ringer's solution daily during hospitalization (glycyrrhizin group). Patients who were admitted on an odd day received an IV drip infusion of 500 mL/d of lactated Ringer's solution only (control group). Adverse effects were assessed by the physicians during hospitalization, using patient interview and laboratory analysis.Results: Forty-one consecutive patients entered the study; 15 patients (15 men, 0 women; mean [SD] age, 25.2 [1.5] years) were assigned to the glycyrrhizin group and 269 patients (24 men, 2 women; mean [SD] age, 22.6 [0.9] years) were assigned to the control group. The 2 groups were similar in terms of baseline characteristics. The mean duration of hospitalization was shorter (P = 0.01), the mean maximum body temperature 24 to 48 hours after admission was less (P = 0.05), and the cost of therapy (P = 0.03) was less in the glycyrrhizin group than the control group. No AEs were reported.Conclusions: In this study of hospitalized patients with URTIs, glycyrrhizin therapy was associated with a shorter hospitalization, lower-grade fever, and lower cost of therapy compared with controls, showing that it may be beneficial to patients with URTIs without acute bacterial infections. [ABSTRACT FROM AUTHOR]

The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors is implicated in synaptic plasticity. Although we have found that brain-derived neurotrophic factor (BDNF) triggers surface translocation of AMPA receptor proteins, the physiological significance of the BDNF effect remained to be determined. The present immunohistochemical studies revealed that cortical GABAergic neurons exhibited the most striking response to BDNF. Accordingly, we monitored AMPA-triggered currents through GABAergic neurons: Chronic BDNF treatment increased the AMPA-triggered currents but not NMDA-triggered currents in culture. In parallel, the amplitude, but not frequency, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) was elevated in GABAergic neurons. In agreement, BDNF enhanced GABA release triggered by AMPA compared to the amount triggered by high potassium. Conversely, there was a significant decrease in the mEPSC amplitude of GABAergic neurons in heterozygous BDNF-knockout mice. These findings indicate that the neurotrophin enhances the input sensitivity of GABAergic neurons to facilitate their inhibitory function in the neocortex. [Copyright &y& Elsevier]

Time-resolved fluorescence SNOM is used to probe the mesoscopic structure and dynamics of long-chain merocyanine (C[sub 18]MC) J-aggregates on glass plates prepared by spin coating, casting, and casting of water-soluble polymer films. A globular structure with an average diameter of ∼ 1 µm and a height of ∼ 50 nm was attributed to the J-aggregate of C[sub 18]MC in the spin-coating film. In polymer films, the bandwidth of the absorption of J-aggregate is much narrower in polyvinyl alcohol (PVA, ∼ 20 nm) than that in polyvinyl sulphate (PVS, ∼60 nm). We have demonstrated that the large bandwidth of the spectrum is due to the inhomogeneous distribution of the J-aggregate. The fluorescence image of the J-aggregate in PVA film was rather uniform, whereas non-uniform distribution of the fluorescence was observed in PVS film. The fluorescence of C[sub 18]MC J-aggregate in a small domain of PVA film was a single exponential decay with a lifetime as short as 19 ps, which was shorter than that in PVS film with a two-exponential decay (average lifetime of ∼25 ps). The fluorescence lifetime of the J-aggregate and its single exponential behaviour are considered to be indicators of the uniform distribution of the J-aggregate. The non-uniform distribution of the J-aggregate in PVS film was interpreted in terms of electrostatic interaction between PVS and merocyanine. [ABSTRACT FROM AUTHOR]

Thin films of water-soluble free-base porphyrin, 5,10,15,20-tetraphenyl-21H, 23H-porphinetetrasulphonic acid (TPPS) mixed with poly(diallyldimethyl ammonium chloride) (PDDA) have been prepared by a spin-coating method, in which the monomeric species were observed in the spin-coat film, whereas dimer was formed in the cast film prepared from TPPS/PDDA solution. Mesoscopic structures and dynamics of excitation energy migration and trapping of TPPS/PDDA spin-coat film have been analysed by time-resolved scanning near-field optical microspectroscopy (SNOM) and atomic force microscope. The observed film structure can be classified roughly into two parts: one is a large, flocculated polymer part, and the other is a smooth part widely spread around the flocculated polymers. In the smooth part, the observed spindle-like structure and circular hills and dips are essentially due to PDDA. The ellipsoidal small structures with ∼2µm length and <1 µm width in the flocculated polymer part show non-exponential fluorescence decays. The non-exponential dynamics originates from the excitation energy migration among TPPS monomers and energy trapping to dimers. From the analysis of fluorescence decay curves based on the equation developed by Klafter and Blumen, the spectral dimension has been estimated to be ∼1.46 for ellipsoidal structures. These results indicate that the distribution of the chromophore is inhomogeneous and a fractal-like structure exists even in the small domains determined by the resolution of the SNOM tip. [ABSTRACT FROM AUTHOR]

Objective:To clarify the incidence and characteristics of spinal cord concussion in an urban city in Japan.Methods:The current retrospective study was conducted by reviewing the medical records of patients with spinal cord injuries admitted between January 2007 and December 2010. The subjects were divided into two groups: a spinal cord concussion group and a spinal cord injury group.Results:There were 36 patients with spinal cord injuries admitted during this period. Among them, there were 8 patients with spinal cord concussions. No significant differences were apparent between the two groups with regard to sex, mechanism of injury, Glasgow Coma Scale, incidence of shock, inhospital Frankel classification, level of spinal cord injury, frequency of vertebral dislocation/fracture, surgery and the survival rate. However, the mean age in the spinal cord concussion group was lower than that in the spinal cord injury group. The systolic blood pressure, the heart rate and the diameter of minimum spinal canal in the concussion group were all higher than those in the spinal cord injury group. Half of the subjects in the spinal cord concussion group demonstrated immediate neurological improvement between prehospital and inhospital findings, however none of the subjects in the spinal cord injury group demonstrated such improvement. This difference was statistically significant.Conclusion:Spinal cord concussion is not rare in urban Japan. Checking neurological findings at the scene leads to the revelation of the true high incidence of spinal cord concussion among spinal cord injuries. [ABSTRACT FROM AUTHOR]

Superior colliculus (SC) is a midbrain structure that integrates sensory inputs and generates motor commands to initiate innate motor behaviors. Its retinorecipient superficial layers (sSC) receive dense cholinergic projections from the parabigeminal nucleus (PBN). Our previous in vitro study revealed that acetylcholine induces fast inward current followed by prominent GABAergic inhibition within the sSC circuits (Endo T, Yanagawa Y, Obata K, Isa T. J Neurophysiol 94: 3893–3902, 2005). Acetylcholine-mediated facilitation of GABAergic inhibition may play an important role in visual signal processing in the sSC; however, both the anatomical and physiological properties of cholinergic inputs from PBN have not been studied in detail in vivo. In this study, we specifically visualized and optogenetically manipulated the cholinergic neurons in the PBN after focal injections of Cre-dependent viral vectors in mice that express Cre in cholinergic neurons. We revealed that the cholinergic projections terminated densely in the medial part of the mouse sSC. This suggests that the cholinergic inputs mediate visual processing in the upper visual field, which would be critical for predator detection. We further analyzed the physiological roles of the cholinergic inputs by recording looming-evoked visual responses from sSC neurons during optogenetic activation or inactivation of PBN cholinergic neurons in anesthetized mice. We found that optogenetic manipulations in either direction induced response suppression in most neurons, whereas response facilitation was observed in a few neurons after the optogenetic activation. These results support a circuit model that suggests that the PBN cholinergic inputs enhance functions of the sSC in detecting visual targets by facilitating the center excitation–surround inhibition. NEW & NOTEWORTHY The modulatory role of the cholinergic inputs from the parabigeminal nucleus in the visual responses in the superficial superior colliculus (sSC) remains unknown. Here we report that the cholinergic projections terminate densely in the medial sSC and optogenetic manipulations of the cholinergic inputs affect the looming-evoked response and enhance surround inhibition in the sSC. Our data suggest that cholinergic inputs to the sSC contribute to the visual detection of predators. [ABSTRACT FROM AUTHOR]

Abstract: It has been reported in the cat and rat that inhibitory premotor neurons, which send their axons to motoneurons of the trigeminal motor nucleus (Vm) are distributed in the reticular regions around the Vm, especially in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint). In the present study, we examined neuronal connections of GABAergic neurons in the Vsup and Vint in the mouse by utilizing the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter [Yanagawa, Y., Kaneko, K., Kanbara, N., Totsuka, M., Yagi, T., Obata, K., 2001. Development of mouse expressing GFP in GABAergic neurons. Neurosci. Res. Suppl. 25, S77; Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., Kaneko, T., 2003. Green fluorescent protein expression and colocalization with calretinin, parvalbumin and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60–79]. The connections were examined light- and electron-microscopically by combining the anterograde or the retrograde tract-tracing method with the immunohistochemical method for GFP. The data indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side and sent their axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons. [Copyright &y& Elsevier]

Inhibitory neurotransmitters, γ-aminobutyric acid (GABA) and glycine, are transported into synaptic vesicles by the vesicular GABA transporter (VGAT). Glutamate decarboxylase (GAD) is a GABA-synthesizing enzyme and two isoforms of GAD, GAD65 and GAD67 are encoded by two independent genes. There was virtually no GABA content in GAD65/GAD67 double knockout (GADs DKO) mouse brains. Neither GABAergic nor glycinergic inhibitory postsynaptic currents were almost detected in VGAT knockout (KO) mouse cultured neurons and spinal cords. GAD67 KO and VGAT KO mice displayed developmental abnormalities, cleft palate and omphalocele, suggesting that GABAergic transmission is involved in palate and abdominal wall formations. However, the incidence and severity of both failures in GAD67 KO mice were lower and less than those in VGAT KO mice. These results raise the possibility that GABAergic transmission mediated by GAD65-produced GABA and/or glycinergic transmission contributed to both palate and abdominal wall formations. However, it still remains unclear whether GABAergic transmission mediated by GAD65 and glycinergic transmission contribute to those formations. Here, to answer these questions, we generated GADs DKO mice and compared the phenotypes of GADs DKO mice with those of GAD67 KO and VGAT KO mice. Our anatomical analyses demonstrated that the incidence of cleft palate and omphalocele in GAD67 KO mice was 65.8% and 58.9%, respectively, but the incidence of both phenotypes in GADs DKO and VGAT KO mice was 100%. The severity of cleft palate and omphalocele was evaluated by elevation of palate shelves and size and liver inclusion of omphalocele, respectively. We observed that the phenotypes of cleft palate and omphalocele in GADs DKO mice were more and less severe than those in GAD67 KO and VGAT KO mice, respectively. These results indicate the significant contribution of not only GAD65-mediated GABAergic but also glycinergic transmissions to both palate and abdominal wall formations. [ABSTRACT FROM AUTHOR]

Am J Forensic Med Pathol 1991;12:252-4. 4 Tanoue S, Yanagawa Y. Ischemic stroke with left hemiparesis or shock should be evaluated by computed tomography for aortic dissection. Sir, Ischemic stroke is occasionally complicated with acute aortic dissection. However, hemorrhagic stroke with acute aortic dissection was extremely rare.[[1]],[[2]],[[3]] We herein report a fatal case of the concurrence of intracranial hemorrhaging and Stanford type A acute aortic dissection with cardiac tamponade, confirmed by computed tomography (CT). [Extracted from the article]

GABAergic neurons of the amygdala are thought to play a critical role in establishing networks for feedback and feedforward inhibition and in mediating rhythmic network activity patterns relevant for emotional behavior, determination of stimulus salience, and memory strength under stressful experiences. These functions are typically fulfilled in interplay of amygdala and hippocampus. Therefore, we explored the putative connectivity of GABAergic neurons with the hippocampo-amygdalar projection with the anterograde tracers Phaseolus vulgaris leucoagglutinin (Phal) and Miniruby injected to GAD67-GFP knock-in mice in which GABAergic neurons are labeled by the expression of the gene for green fluorescent protein (GFP) inserted to the GAD1 gene locus (Tamamaki et al. J Comp Neurol 467:60-79, ). We found that, while hippocampal axons target all nuclei of the amygdala, the densest fiber plexus was found in the posterior basomedial nucleus. Electron microscopy revealed that the vast majority of contacts in this nucleus were formed by thin fibers making small asymmetrical contacts, predominantly on GFP-negative profiles. However, several asymmetrical contacts could also be seen on GFP-positive profiles. A surprising result was the occasional occurrence of anterogradely labeled symmetrical synapses indicating a GABAergic contribution to the projection from the hippocampus to the amygdala. While hippocampal input to the amygdala appears to be largely excitatory and targets non-GABAergic neurons, our data provide evidence for a direct involvement of GABAergic neurons in the interplay of these regions, either as target in the amygdala or as projection neurons from the hippocampus. These particular 'interface neurons' may be of relevance for the information processing in the amygdalo-hippocampal system involved in emotional behavior and memory formation. [ABSTRACT FROM AUTHOR]

Abstract: The identification and characterization of excitatory and inhibitory neurons are significant steps in understanding neural network functions. In this study, we investigated the intrinsic electrophysiological properties of neurons in the prepositus hypoglossi nucleus (PHN), a brainstem structure that is involved in gaze holding, using whole-cell recordings in brainstem slices from vesicular GABA transporter (VGAT)-Venus transgenic rats, in which inhibitory neurons express the fluorescent protein Venus. To characterize the intrinsic properties of these neurons, we recorded afterhyperpolarization (AHP) profiles and firing patterns from Venus-expressing [Venus(+)] and Venus-non-expressing [Venus(−)] PHN neurons. Although both types of neurons showed a wide variety of AHP profiles and firing patterns, oscillatory firing was specific to Venus(+) neurons, while a firing pattern showing only a few spikes was specific to Venus(−) neurons. In addition, AHPs without a slow component and delayed spike generation were preferentially displayed by Venus(+) neurons, whereas a firing pattern with constant interspike intervals was preferentially displayed by Venus(−) neurons. We evaluated the mRNAs expression of glutamate decarboxylase (GAD65, GAD67) and glycine transporter 2 (GlyT2) to determine whether the recorded Venus(+) neurons were GABAergic or glycinergic. Of the 67 Venus(+) neurons tested, GlyT2 expression alone was detected in only one neuron. Approximately 40% (28/67) expressed GAD65 and/or GAD67 (GABAergic neuron), and the remainder (38/67) expressed both GAD(s) and GlyT2 (GABA&GLY neuron). These results suggest that most inhibitory PHN neurons use either GABA or both GABA and glycine as neurotransmitters. Although the overall distribution of firing patterns in GABAergic neurons was similar to that of GABA&GLY neurons, only GABA&GLY neurons exhibited a firing pattern with a long first interspike interval. These differential electrophysiological properties will be useful for the identification of specific types of PHN neurons. [Copyright &y& Elsevier]

The differentiation of a vegetative cell and a generative cell is a critical event during pollen development. The Lilium GlsA is known to localize in pollen and is considered to be involved in development of the generative cell. Here, we cloned a glsA ortholog from Alstroemeria, a commercially important cut flower. The expression of AaglsA ( Alstroemeria aurea glsA) transcripts increased gradually after pollen mitosis I (PMI) and reached a significant level when the generative cell started to elongate. Analysis of the promoter of AaglsA suggests that AaglsA expression is controlled by several cis-regulatory elements during pollen development. This is the first investigation of reproductive factors regulating male gametogenesis in Alstroemeria. [ABSTRACT FROM AUTHOR]

Two kinds of feldspars, such as microcline and albite, were irradiated with g-rays of 250 Gy in 77 K and followed by ESR-measurements at 77 K. First ESR-spectra showed an Al-Oh+ center peak at a centeral part and hydrogen radical peaks having hyperfine (HF) splitting width of 50.2 mT. Second ESR-spectra measured after warming up to room temperature once showed eleven absorption peaks owing to an Al-Oh+-Al center, together with complete disappearance of H0-radicals. The Al-Oh+-Al signals in ESR-spectra showed similar dose response curves in both feldspars, giving almost linear relationships up to several hundred Gy and saturation behavior beyond 1 kGy. Thermal treatment of the feldspars induced an interesting appearance of two new hydrogen radicals in the ESR spectrum, which possess broader HF splitting in the ESR-spectra and relatively stable property at room temperature. The correlation of Al-Oh+-Al centers and the BTL intensities around 180 °C peak showed linear dependency. As a result, it is concluded that the Al-Oh+-Al centers should be main BTL-luminescence centers around the 180 °C peak in the feldspars. [ABSTRACT FROM AUTHOR]

Dear Editor, Cerebral ischemia is a common cause of transient hemiparesis. CT angiography was performed to evaluate the aorta and internal carotid arteries. J Huazhong Univ Sci Technolog Med Sci 2014;34:582-5. 5 Tanoue S, Yanagawa Y. Ischemic stroke with left hemiparesis or shock should be evaluated by computed tomography for aortic dissection. [Extracted from the article]

Sensory information arising from the upper neck is important in the reflex control of posture and eye position. It has also been linked to the autonomic control of the cardiovascular and respiratory systems. Whiplash associated disorders (WAD) and cervical dystonia, which involve disturbance to the neck region, can often present with abnormalities to the oromotor, respiratory and cardiovascular systems. We investigated the potential neural pathways underlying such symptoms. Simulating neck afferent activity by electrical stimulation of the second cervical nerve in a working heart brainstem preparation (WHBP) altered the pattern of central respiratory drive and increased perfusion pressure. Tracing central targets of these sensory afferents revealed projections to the intermedius nucleus of the medulla (InM). These anterogradely labelled afferents co-localised with parvalbumin and vesicular glutamate transporter 1 indicating that they are proprioceptive. Anterograde tracing from the InM identified projections to brain regions involved in respiratory, cardiovascular, postural and oro-facial behaviours-the neighbouring hypoglossal nucleus, facial and motor trigeminal nuclei, parabrachial nuclei, rostral and caudal ventrolateral medulla and nucleus ambiguus. In brain slices, electrical stimulation of afferent fibre tracts lateral to the cuneate nucleus monosynaptically excited InM neurones. Direct stimulation of the InM in the WHBP mimicked the response of second cervical nerve stimulation. These results provide evidence of pathways linking upper cervical sensory afferents with CNS areas involved in autonomic and oromotor control, via the InM. Disruption of these neuronal pathways could, therefore, explain the dysphagic and cardiorespiratory abnormalities which may accompany cervical dystonia and WAD. [ABSTRACT FROM AUTHOR]

The bed nucleus of the stria terminalis (BNST) is an important region for 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) predator odor-induced stress responses in mice. It is sexually dimorphic and a region for corticotropin-releasing factor (CRF)-enhanced stress responses. Dense GABAergic and CRF input from the amygdala to the BNST gives point to relevant interactions between CRF and GABA activity in these brain regions. Hence, to investigate sexual dimorphism of stress-induced neuronal changes, we studied effects of acute TMT exposure on CRF mRNA expression in stress-related brain regions in male and female GAD67 mice and their wild-type littermates. In GAD67 mice, heterozygous knock-in of GFP in GABAergic neurons caused a 50% decrease of GAD67 protein level in the brain [91,99]. Results show higher CRF mRNA levels in the BNST of male but not female GAD67 mice after TMT and control odor exposure. While CRF neurons in the BNST are predominantly GABAergic and CRF enhances GABAergic transmission in the BNST [20,51], the deficit in GABAergic transmission in GAD67 mice could induce a compensatory CRF increase. Sexual dimorphism of the BNST with greater density of GABA-ir neurons in females could explain the differences in CRF mRNA levels between male and female GAD67 mice. Effects of odor exposure were studied in a radial arm maze (RAM) task. Results show impaired retrieval of spatial memory after acute TMT exposure in both sexes and genotypes. However, only GAD67 mice show increased working memory errors after control odor exposure. Our work elicits GAD67 mice as a model to further study interactions of GABA and CRF in the BNST for a better understanding of how sex-specific characteristics of the brain may contribute to differences in anxiety- and stress-related psychological disorders. [ABSTRACT FROM AUTHOR]

Opioids are the most widely used analgesics in the treatment of severe acute and chronic pain. However, opioids have many adverse side effects, including the development of antinociceptive tolerance after long-term use. The antinociceptive tolerance of opioids has limited their clinical use. A recent study has reported that autophagy is responsible for morphine-induced neuronal injury. However, little is known about the role of autophagy in morphine antinociceptive tolerance. In the present study, chronic morphine administration was found to induce the expression of autophagy-related proteins, including Beclin1 and microtubule-associated protein light chain 3 (LC3)-II, in GABAergic interneurons in the superficial layer (lamina I-II) of the spinal cord. A single intrathecal administration of autophagy inhibitors, 3-methyladenine (3MA) or wortmannin, inhibited the development of antinociceptive tolerance in a dose-dependent manner. Autophagy in the lamina I-II neurons was associated with increased level of cathepsin B (CatB), a lysosomal cysteine protease. The pharmacological blockade or gene deletion of CatB markedly prevented the development of morphine antinociceptive tolerance. Furthermore, the intrathecal administration of 3MA suppressed the upregulation of CatB 5 days after morphine administration. Finally, CatB deficiency inhibited the increased release probability of glutamate in the lamina I neurons after chronic morphine treatment. These observations suggest that the dysfunction of the spinal GABAergic system induced by CatB-dependent excessive autophagy is partly responsible for morphine antinociceptive tolerance following chronic treatment. [ABSTRACT FROM AUTHOR]

Reduced glutamic acid decarboxylase ( GAD)67 expression may be causally involved in the development of social withdrawal in neuropsychiatric states such as autism, schizophrenia and bipolar disorder. In this study, we report disturbance of social behavior in male GAD67 haplodeficient mice. GAD67+/− mice, compared to GAD67+/+ littermates, show reduced sociability and decreased intermale aggression, but normal nest building and urine marking behavior, as well as unchanged locomotor activity and anxiety-like behavior. Moreover, the mutants display a reduced sensitivity to both social and non-social odors, indicating a disturbance in the detection and/or processing of socially relevant olfactory stimuli. Indeed, we observed reduced activation of the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, medial and cortical amygdala upon exposure of GAD67+/− mice to social interaction paradigm, as indicated by c-Fos immunohistochemistry. These data suggest a disturbance of stimulus processing in the brain circuitry controlling social behavior in GAD67+/− mice, which may provide a useful model for studying the impact of a reduced GAD67 expression on alterations of social behavior related to neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

Highlights: [•] GluN2A, B and D receptors regulate distinct orofacial movement topographies. [•] GAD65-GABA exerts subtle effects on orofacial movement topographies. [•] These effects do not involve direct interactions with D1-like dopamine receptors. [•] Orofacial movements and locomotion are regulated via distinct neuronal systems. [•] GluN2 receptors and GAD65-GABA differentially regulate orofacial pattern generators. [ABSTRACT FROM AUTHOR]

Abstract: Schizophrenia (Sz), along with other neuropsychiatric disorders, is associated clinically with abnormalities in neocortical gamma frequency (30–80 Hz) oscillations. In Sz patients, these abnormalities include both increased and decreased gamma activity, and show a strong association with Sz symptoms. For several decades, administration of sub-anesthetic levels of ketamine has provided the most comprehensive experimental model of Sz-symptoms. While acute application of ketamine precipitates a psychotic-like state in a number of animal models, as well as humans, the underlying mechanisms behind this effect, including alteration of neuronal network properties, are incompletely understood, making an in vitro level analysis particularly important. Previous in vitro studies have had difficulty inducing gamma oscillations in neocortical slices maintained in submerged-type recording chambers necessary for visually guided whole-cell recordings from identified neurons. Consequently, here, we validated a modified method to evoke gamma oscillations using brief, focal application of the glutamate receptor agonist kainate (KA), in slices prepared from mice expressing green fluorescent protein in GABAergic interneurons (GAD67-GFP knock-in mice). Using this method, gamma oscillations dependent on activation of AMPA and GABAA receptors were reliably elicited in slices containing mouse prelimbic cortex, the rodent analogue of the human dorsolateral prefrontal cortex. Examining the effects of ketamine on this model, we found that bath application of ketamine significantly potentiated KA-elicited gamma power, an effect mimicked by selective NMDAR antagonists including a selective antagonist of NMDARs containing the NR2B subunit. Importantly, ketamine, unlike more specific NMDAR antagonists, also reduced the peak frequency of KA-elicited oscillatory activity. Our findings indicate that this effect is mediated not through NMDAR, but through slowing the decay kinetics of GABAA receptor-mediated inhibitory postsynaptic currents in identified GABAergic interneurons. These in vitro findings may help explain the complexities of gamma findings in clinical studies of Sz and prove useful in developing new therapeutic strategies. [Copyright &y& Elsevier]

Abstract: GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profile expression of Shaw-related Kv3.1b and Kv3.3 K+-channel subunits in dorsal horn interneurones. Neuronal expression of these subunits confers specific biophysical characteristic referred to as ‘fast-spiking’. Immuno-labelling for Kv3.1b or Kv3.3 revealed the presence of both of these subunits across the dorsal horn, most abundantly in laminae I–III. Co-localization studies in transgenic mice indicated that Kv3.1b but not Kv3.3 was associated with GAD65-GFP and GAD67-GFP immunopositive neurones. For comparison the distributions of Kv4.2 and Kv4.3 K+-channel subunits which are linked to an excitatory neuronal phenotype were characterized. No co-localization was found between GAD-GFP +ve neurones and Kv4.2 or Kv4.3. In functional studies to evaluate whether either GABAergic population is activated by noxious stimulation, hindpaw intradermal injection of capsaicin followed by c-fos quantification in dorsal horn revealed co-expression c-fos and GAD65-GFP (quantified as 20–30% of GFP +ve population). Co-expression was also detected for GAD67-GFP +ve neurones and capsaicin-induced c-fos but at a much reduced level of 4–5%. These data suggest that whilst both GAD65-GFP and GAD67-GFP +ve neurones express Kv3.1b and therefore may share certain biophysical traits, their responses to peripheral noxious stimulation are distinct. [Copyright &y& Elsevier]

Abstract: Stress affects learning and there are both facilitating and impairing actions of stressors on memory processes. Here we investigated the influence of acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), an ethological relevant stressor for rodents, on spatial memory formation and performance in a radial arm maze (RAM) task and studied TMT effects on corticosterone levels in GAD67-GFP knock-in mice and their wildtype littermates. Our results suggest that predator odor-exposure differently affects consolidation and retrieval of memory in a hippocampus-dependent spatial learning task in adult male mice, independently from their genotypes. Acute TMT-stress before retrieval facilitates performance, whereas repeated TMT-stress during consolidation exerts no influence. Additionally, we found genotype specific effects of TMT on corticosterone release. While TMT-stress tend to result in increased corticosterone release in wildtypes there was a significant decrease in transgenic mice. Taken together, these findings indicate that biologically significant predator odor-induced stress can have different actions on the strength of spatial memory formation depending on the timing with regard to memory phases. Furthermore, we suppose an impact of GABAergic mechanisms on HPA-stress axis activation to TMT resulting in absent peripheral corticosterone release of GAD67-GFP mice. [Copyright &y& Elsevier]

Abstract: Sensory experience influences brain organization and function. A particularly striking example is in the olfactory bulb where reduction of odorant sensory signals profoundly down-regulates dopamine in glomerular neurons. There are two large populations of glomerular inhibitory interneurons: (1) GABAergic periglomerular (PG) cells, whose processes are limited to a single glomerulus, regulate intraglomerular processing and (2) DAergic-GABAergic short axon (SA) cells, whose processes contact multiple glomeruli, regulate interglomerular processing. The inhibitory neurotransmitter GABA is synthesized from L-glutamic acid by the enzyme glutamic acid decarboxylase (GAD) of which there are two major isoforms: GAD65 and GAD67. GAD65 is expressed in uniglomerular PG cells. GAD67 is expressed by SA cells, which also co-express the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH). Deafferentation or sensory deprivation decreases TH expression but it is not known if sensory input alters GAD isoforms. Here we report that either deafferentation or reduction of sensory input by nares occlusion significantly reduced GAD67 protein and the number of SA cells expressing GAD67. However, neither manipulation altered GAD65 protein or the number of GAD65 PG cells. These findings show that sensory experience strongly impacts transmitter regulation in the circuit that controls neural processing across glomeruli but not in the circuit that regulates intraglomerular processing. [Copyright &y& Elsevier]

Abstract: The non-competitive N-methyl-d-aspartate NMDA receptor antagonist ketamine, a dissociative anesthetic capable of inducing analgesia, is known to have psychotomimetic actions, but the detailed mechanisms remain unclear because of its complex properties. The present study elucidated neural mechanisms of the effect of ketamine, at doses that exert psychotomimetic effects without anesthetic and analgesic effects, by evaluating cortical synaptic responses in vivo. Systemic administration (i.p.) of low (1 and 5 mg/kg), subanesthetic (25 mg/kg) and anesthetic (100 mg/kg) doses of ketamine dose-dependently decreased hippocampal stimulation-evoked potential in the medial prefrontal cortex (mPFC) in freely moving rats. The behavioral analysis assessed by prepulse inhibition (PPI) of acoustic startle response showed that ketamine (5 and 25 mg/kg, i.p.) produced PPI deficit. Thus, the psychotomimetic effects observed in ketamine-treated groups (5 and 25 mg/kg, i.p.) are associated with the induction of synaptic depression in the hippocampus-mPFC neural pathway. Based on these results, we further examined the underlying mechanisms of the ketamine-induced synaptic depression under anesthesia. Ketamine (5 and 25 mg/kg, i.p.) caused increases in dialysate dopamine in the mPFC in anesthetized rats. Moreover, the ketamine-induced decreases in the evoked potential, at the dose 5 mg/kg which has no anesthetic and analgesic effects, were indeed absent in dopamine-lesioned rats pretreated with 6-hydroxydopamine (6-OHDA; 150 μg/rat, i.c.v.). Ketamine (5 mg/kg, i.p.)-induced synaptic depression was blocked by pretreatment with dopamine D1 receptor antagonist SCH 23390 (10 μg/rat, i.c.v.) but not dopamine D2 receptor antagonist haloperidol (1.5 mg/kg, i.p.), suggesting that dopaminergic modulation mediated via D1 receptors are involved in the synaptic effects of ketamine. Furthermore, ketamine (5 mg/kg, i.p.)-induced synaptic depression was prevented also by GABAA receptor antagonist bicuculline (0.2 or 2 μg/rat, i.c.v.). These findings suggest that ketamine at the dose that exerts psychotomimetic symptoms depresses hippocampus-mPFC synaptic transmission through mechanisms involving dopaminergic modulation mediated via D1 receptors, which may lead to a net augmentation of synaptic inhibition mediated via GABAA receptors. [Copyright &y& Elsevier]

Abstract: Traumatic events during early life may affect the neural systems associated with memory function, including extinction, and lead to altered sensitivity to stress later in life. We recently reported that changes in prefrontal synaptic efficacy in response to extinction trials did not occur in adult rats exposed to early postnatal stress (i.e. footshock [FS] stress during postnatal day 21–25 [3W-FS group]). However, identifying neurocircuitry and neural mechanisms responsible for extinction retrieval after extinction training have not been precisely determined. The present study explored whether synaptic transmission in the hippocampal-medial prefrontal cortex (mPFC) neural pathway is altered by extinction retrieval on the day after extinction trials using electrophysiological approaches combined with behavioral analysis. We also elucidated the effects of early postnatal stress on the synaptic response in this neural circuit underlying extinction retrieval. Evoked potential in the mPFC was enhanced following extinction retrieval, accompanied by reduced freezing behavior. This synaptic facilitation (i.e. a long-term potentiation [LTP]-like response) did not occur; rather synaptic inhibition was observed in the 3W-FS group, accompanied by sustained freezing. The behavioral deficit and synaptic inhibition observed in the 3W-FS group were time-dependently ameliorated by the partial N-methyl-d-aspartate (NMDA) receptor agonist d-cycloserine (15 mg/kg, i.p.). These findings suggest that the LTP-like response in the hippocampal-mPFC pathway is associated with extinction retrieval of context-dependent fear memory. Early postnatal stress appears to induce neurodevelopmental dysfunction of this neural circuit and lead to impaired fear extinction later in life. The present data indicate that psychotherapy accompanied by pharmacological interventions that accelerate and strengthen extinction, such as d-cycloserine treatment, may have therapeutic potential for the treatment of anxiety disorders, including posttraumatic stress disorder. [Copyright &y& Elsevier]

Abstract: Glutamatergic synapses form onto both glutamatergic and GABAergic neurons. These two types of glutamatergic synapses differ in their electrical responses to high-frequency stimulation and postsynaptic density protein composition. However, it is not known whether they differ in the actin cytoskeleton composition. In the present study, we used hippocampal neuronal cultures prepared from glutamate decarboxylase 67 (GAD67)-GFP knock-in mice and analyzed the differences in the actin cytoskeleton at glutamatergic synapses contacting GABAergic and glutamatergic neurons. Drebrin-binding actin filaments enriched in dendritic spines are known to play a pivotal role in spine formation. Immunocytochemical analyses demonstrated that drebrin accumulated at glutamatergic synapses on GABAergic neurons as well as at those on glutamatergic neurons. However, the density of drebrin clusters along dendrites in GABAergic neurons was significantly lower than those of glutamatergic neurons. Furthermore, the level of drebrin accumulating at glutamatergic synapses was lower on GABAergic neurons than on glutamatergic neurons. In neurons overexpressing drebrin, drebrin cluster density and accumulation levels in GABAergic and glutamatergic neurons were similar, suggesting that the low drebrin levels in the glutamatergic postsynaptic sites on GABAergic neurons may be because GABAergic neurons express low levels of drebrin. On the other hand, pharmacological analysis demonstrated that the postsynaptic localization of drebrin depended on actin cytoskeleton organization in both GABAergic and glutamatergic neurons. Together these results indicated that, although GABAergic and glutamatergic neurons share common regulatory systems affecting drebrin localization, the density of drebrin-positive glutamatergic synapses formed on GABAergic neurons is lower than those on glutamatergic neurons. This is probably due to the low expression of drebrin in GABAergic neurons. [ABSTRACT FROM AUTHOR]

Abstract: The medial septum diagonal band complex (MS/DB) projects via cholinergic and GABAergic pathways to the hippocampus and plays a key role in the hippocampal theta rhythm. In the MS/DB we have previously described a population of fast spiking GABAergic neurons that contain parvalbumin and mediate theta frequency activity in vitro. The Kv3.1 potassium channel is a delayed rectifier channel that plays a major role in fast spiking neurons in the CNS, and has previously been localized in the MS/DB. To determine which cell types in the MS/DB express the Kv3.1b ion channel subunit, transgenic mice in which the expression of GABAergic and glutamate markers are associated with the expression of green fluorescent protein (GFP; GAD67-GFP and VGluT2-GFP mice, respectively) were used for immunofluorescence and axonal tract tracing. Electrophysiological studies were also carried out on rat MS/DB slices to examine the role of the Kv3.1 channel in theta frequency oscillations. The results for the MS/DB were as follows: (1) cholinergic cells did not express GFP in either GAD67-GFP or VGluT2-GFP mice, and there was GAD67 immunoreactivity in GFP-positive neurons in GAD67-GFP mice and in a small proportion (6%) of GFP-positive neurons in VGluT2-GFP mice. (2) Kv3.1b immunofluorescence was associated with the somata of GABAergic neurons, especially those that contained parvalbumin, and with a minority of glutamatergic neurons, but not with cholinergic neurons, and with GABAergic axonal terminal-like processes around certain GABAergic neurons. (3) Both Kv3.1b-positive and -negative GABAergic neurons were septo-hippocampal, and there was a minor projection to hippocampus from VGluT2-GFP neurons. (4) Kainate-induced theta oscillations in the MS/DB slice were potentiated rather than inhibited by the Kv3.1 blocker 4-aminopyridine, and this agent on its own produced theta frequency oscillations in MS/DB slices that were reduced by ionotropic glutamate and GABA receptor antagonists and abolished by low extracellular calcium. These studies confirm the presence of heterogeneous populations of septo-hippocampal neurons in the MS/DB, and suggest that presence of Kv3.1 in the GABAergic neurons does not contribute to theta activity through fast spiking properties, but possibly by the regulation of transmitter release from axonal terminals. [Copyright &y& Elsevier]

Abstract: Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67–GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT–Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67–GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT–Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS. [Copyright &y& Elsevier]

Abstract: Predator odors, which are non-intrusive and naturalistic stressors of high ethological relevance, were used to study the neurobiology of innate fear in rodents. The present study investigates behavioral effects and the induction of c-fos mRNA in adult male predator naive mice caused by acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of the fox feces odor. On the behavioral level, TMT potently increased unconditioned freezing and decreased non-defensive grooming behavior. With quantitative real time PCR we established a strong TMT-induced activation in the bed nucleus of the stria terminalis (BNST) (eight-fold increase, p <0.016) and in the ventral olfactory bulb (two-fold increase, p <0.036). In contrast, no significant TMT-induced c-fos induction could be observed in the dorsal olfactory bulb or in the amygdala. Our results display robust fear responses of GAD67-GFP knock-in mice exposed to TMT and suggest that the ventral olfactory bulb and the BNST are strongly activated during the elicitation of fear through predator odor in these transgenic mice. [Copyright &y& Elsevier]

Abstract: GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67–green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67–GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via α1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period. [Copyright &y& Elsevier]

Diet is one of the important factors that modulate immune responses. In the present study, we have examined the capacity of dietary lipids to modify immune responses in mice and we have investigated the contribution of glycolipid-reactive natural killer T (NKT) cells in this process. Mice fed, high fat diet (HFD; 21.2% fat, 0.20% cholesterol) for 3 weeks, as compared with mice fed standard fat diet (SFD; 4.3% fat, 0.03% cholesterol), showed significantly reduced interferon-γ production in sera at 6 or 12 h after intraperitoneal injection of an NKT cell ligand, α-galactosylceramide. In contrast, production of interleukin-13 was significantly higher at 2 and 6 h in HFD fed mice compared with mice on SFD. No difference was detected in the serum interleukin-4 levels between these two groups of animals. The proportion of NKT cells in spleen and liver was reduced in mice fed HFD compared with those on SFD. In addition, activation of NKT cells assessed by up-regulation of CD69 was suppressed specifically in liver from mice fed HFD. Recall responses of conventional T cells and delayed-type hypersensitivity (Th1 type) against ovalbumin were significantly suppressed in mice fed HFD in comparison with those fed SFD. This suppression was not observed in CD1d−/− mice, suggesting that NKT cells in mice fed HFD played a role in suppressing Th1 responses. Taken together, our findings suggest a critical link between NKT cells, dietary lipid and adaptive immune responses. [ABSTRACT FROM AUTHOR]

Abstract: The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking glutamate decarboxylase 67. Eur J Neurosci 21:173–178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem–spinal cord blocks prepared from GAD65−/−:67−/− and vesicular GABA transporter (VGAT) −/− mice on embryonic day 14 (E14)–E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65−/−:67−/− preparations but not in VGAT−/− preparations. C4 discharges were observed in the knockout mice on E14–E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a “blind” patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65−/−:67−/− mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT−/− mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age. [Copyright &y& Elsevier]

Background: Activation of the cAMP signaling pathway by means of beta2-adrenoceptor agonists has been shown to up-regulate interleukin-6 (IL-6) gene expression and to stimulate IL-6 production in macrophage cells. However, whether beta2-adrenoceptor activation can also modify the rate of IL-6 production in macrophage cells activated by the bacterial endotoxins has not yet been determined. Using renal resident macrophage cells treated with endotoxin, lipopolysaccharide (LPS), and beta2-adrenoceptor agonist, terbutaline, we investigated the role of cAMP pathway, tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinase (MAPK) pathway (p42/p44) in regulating IL-6 production.Methods: IL-6 protein, mRNA, and promoter activity were measured in these cells exposed to LPS (1 microg/ml) and/or terbutaline (10(-9) to 10(-6) M). Furthermore, the time course effects of terbutaline on cAMP, MAPK (p42/p44), and TNF-alpha release were evaluated in the cells.Results: Terbutaline at high concentrations (10(-6) M) significantly up-regulated IL-6 by approximately 25% (P<0.05), whereas at a lower concentration (10(-8) M), it down-regulated IL-6 production by 42% (P<0.05). Terbutaline (10(-8) and 10(-6) M) caused a concentration- and time-dependent stimulation of cAMP (P<0.05) and TNF production (P<0.05) and a time-dependent decrease in MAPK activity (P<0.05). Following the addition of a cAMP inhibitor, IL-6 promoter activity was correlated with TNF-alpha levels and MAPK activity.Conclusions: A biphasic effect of beta2-adrenoceptor agonist on IL-6 production in renal resident macrophage cells became apparent when LPS was exposed to the cells. The terbutaline-induced down-regulation of IL-6 gene production was mediated by an inhibitory effect of terbutaline on TNF-alpha, which was exerted through the MAPK and cAMP pathways, whereas the up-regulation appeared to be due to a direct action of intracellular cAMP. [ABSTRACT FROM AUTHOR]