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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]

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]

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]

Citrin is the liver-type aspartate-glutamate carrier that resides within the inner mitochondrial membrane. Citrin deficiency (due to homozygous or compound heterozygous mutations in the gene SLC25A13) causes both adult-onset type II citrullinaemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). Clinically, CTLN2 is characterized by hyperammonaemia and citrullinaemia, whereas NICCD has a much more varied and transient presentation that can include multiple aminoacidaemias, hypoproteinaemia, galactosaemia, hypoglycaemia, and jaundice. Personal histories from CTLN2 patients have repeatedly described an aversion to carbohydrate-rich foods, and clinical observations of dietary and therapeutic outcomes have suggested that their unusual food preferences may be directly related to their pathophysiology. In the present study, we monitored the food intake of 18 Japanese citrin-deficient subjects whose ages ranged from 1 to 33 years, comparing them against published values for the general Japanese population. Our survey confirmed a marked decrease in carbohydrate intake, which accounts for a smaller proportion of carbohydrates contributing to the total energy intake (PFC ratio) as well as a shift towards a lower centile distribution for carbohydrate intake relative to age- and sex-matched controls. These results strongly support an avoidance of carbohydrate-rich foods by citrin-deficient patients that may lead to worsening of symptoms. [ABSTRACT FROM AUTHOR]

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]

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]

Menkes disease (MNK) is a lethal, X-linked recessive disorder of copper metabolism dominated by neurodegenerative symptoms and connective tissue disturbances. The incidence of MNK in Asia is not known. Most patients die by the age of 3 years if adequate treatment is not carried out. Early parenteral administration of copper can prevent the neurological disturbances and lead to a better outcome. In the present study, a survey on MNK in Japan was performed. There were in total 53 live-born Japanese patients with MNK collected from 1990 to 2003, including two females. The incidence of live-born MNK patients between 1992 and 2002 was 2.8 per million live births (95% confidence interval (CI): 1.8 to 3.7), 4.9 per million male live births (95% CI: 3.2 to 6.6). One-third of the patients were born before 37 weeks or weighing less than 2500 g. Seventeen per cent were born both before 37 gestational weeks and weighing less than 2500 g. These proportions were higher than those in Japanese live-birth babies according to a nationwide estimate. The hair on these Japanese patients appeared not only as white or grey but also brown and blond. We also found that many signs had been noted before the patient was brought to a hospital with typical symptoms. These signs may be a clue to early diagnosis of MNK. [ABSTRACT FROM AUTHOR]

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]

Feedback regulation from the higher association areas is thought to control the primary sensory cortex, contribute to the cortical processing of sensory information, and work for higher cognitive functions such as multimodal integration and attentional control. However, little is known about the underlying neural mechanisms. Here, we show that the posterior parietal cortex (PPC) persistently inhibits the activity of the primary visual cortex (V1) in mice. Activation of the PPC causes the suppression of visual responses in V1 and induces the short-term depression, which is specific to visual stimuli. In contrast, pharmacological inactivation of the PPC or disconnection of cortical pathways from the PPC to V1 results in an effect of transient enhancement of visual responses in V1. Two-photon calcium imaging demonstrated that the cortical disconnection caused V1 excitatory neurons an enhancement of visual responses and a reduction of orientation selectivity index (OSI). These results show that the PPC regulates the response properties of V1 excitatory neurons. Our findings reveal one of the functions of the PPC, which may contribute to higher brain functions in mice., (© 2019 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

While it has been suggested that IL-33 plays pathogenic roles in various disorders, the factors that stimulate IL-33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL-33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High-dose LPS treatment induced IL-33 and TNF protein production in RAW264.7 macrophages. In contrast, low-dose LPS failed to induce IL-33 production while significantly inducing TNF production. In the presence of the membrane-permeable cAMP analog 8-Br-cAMP, low-dose LPS induced vigorous IL-33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP-increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL-33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8-Br-cAMP and adrenaline on IL-33 production, suggesting that PKA is involved in IL-33 induction. Taken together, cAMP-mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL-33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL-33; this process may be associated with the pathogenesis of various disorders involving IL-33., (© 2016 The Societies and John Wiley & Sons Australia, Ltd.)

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine responsible for both immune regulation and tissue repair. Although TGF-β consists of TGF-β1, -β2, and -β3 in mammals, isoform-selective transcriptional regulation is less well documented in myeloid linage cells such as macrophages. In the present study, the effect of the stress-related catecholamine adrenaline on the expression of TGF-β isoforms in RAW264.7 macrophages and murine bone marrow-derived macrophages was examined. Treatment with adrenaline markedly increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Agonist and antagonist studies indicated that adrenaline-induced TGF-β3 mRNA expression is mediated via β2 -adrenoceptor. Protein kinase A (PKA) inhibitor H89 was found to block an increase in adrenoceptor-mediated TGF-β3 mRNA expression. The membrane-permeable cAMP analog 8-Br-cAMP increased the mRNA expression of TGF-β3 but not of TGF-β1 and -β2. Thus, the β2 -adrenoceptor-mediated cAMP-PKA pathway appears to enhance TGF-β3 mRNA expression in macrophages. Adrenoceptor-mediated TGF-β3 expression by macrophages may influence immune regulation and tissue repair in conditions of stress, during which the sympathetic-nervous system releases catecholamines., (© 2015 The Societies and John Wiley & Sons Australia, Ltd.)

Although it has been proposed that neurons that contain both acetylcholine (ACh) and γ-aminobutyric acid (GABA) are present in the prepositus hypoglossi nucleus (PHN), these neurons have not been characterized because of the difficulty in identifying them. In the present study, PHN neurons that express both choline acetyltransferase and the vesicular GABA transporter (VGAT) were identified using double-transgenic rats, in which the cholinergic and inhibitory neurons express the fluorescent proteins tdTomato and Venus, respectively. To characterize the neurons that express both tdTomato and Venus (D+ neurons), the afterhyperpolarization (AHP) profiles and firing patterns of these neurons were investigated via whole-cell recordings of brainstem slice preparations. Regarding the three AHP profiles and four firing patterns that the D+ neurons exhibited, an AHP with an afterdepolarization and a firing pattern that exhibited a delay in the generation of the first spike were the preferential properties of these neurons. In the three morphological types classified, the multipolar type that exhibited radiating dendrites was predominant among the D+ neurons. Immunocytochemical analysis revealed that the VGAT-immunopositive axonal boutons that expressed tdTomato were primarily located in the dorsal cap of inferior olive (IO) and the PHN. Although the PHN receives cholinergic inputs from the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, D+ neurons were absent from these brain areas. Together, these results suggest that PHN neurons that co-express ACh and GABA exhibit specific electrophysiological and morphological properties, and innervate the dorsal cap of the IO and the PHN., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The superior colliculus (SC) is critical in localizing salient visual stimuli and making decisions on the location of the next saccade. Lateral interactions across the spatial map of the SC are hypothesized to help mediate these processes. Here, we investigate lateral interactions within the SC by applying whole-cell recordings in horizontal slices of mouse SC, which maintained the local structure of the superficial (SCs) visual layer, which is hypothesized to participate in localizing salient stimuli, and the intermediate (SCi) layer, which is supposed to participate in saccade decision-making. When effects of either electrical or chemical (uncaging of free glutamate) stimuli were applied to multiple sites with various distances from the recorded cell, a pattern of center excitation-surround inhibition was found to be prominent in SCs. When the interactions of synaptic effects induced by simultaneous stimulation of two sites were tested, non-linear facilitatory or inhibitory interactions were observed. In contrast, in the SCi, stimulation induced mainly excitation, which masked underlying inhibition. The excitatory synaptic effects of stimulation applied at remote sites were summed in a near linear manner. The result suggested that SCs lateral interactions appear suitable for localizing salient stimuli, while the lateral interactions within SCi are more suitable for faithfully accumulating subthreshold signals for saccadic decision-making. Implementation of this laminar-specific organization makes the SC a unique structure for serially processing signals for saliency localization and saccadic decision-making., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Activation of mu-opioid receptor (MOR) disinhibits dopaminergic neurons in the ventral tegmental area (VTA) through inhibition of γ-aminobutyric acid (GABA)ergic neurons. This mechanism is thought to play a pivotal role in mediating reward behaviors. Here, we characterised VTA-projecting enkephalinergic neurons in the anterior division of the bed nucleus of the stria terminalis (BST) and investigated their targets by examining MOR expression in the VTA. In the BST, neurons expressing preproenkephalin mRNA were exclusively GABAergic, and constituted 37.2% of the total GABAergic neurons. Using retrograde tracer injected into the VTA, 21.6% of VTA-projecting BST neurons were shown to express preproenkephalin mRNA. Enkephalinergic projections from the BST exclusively formed symmetrical synapses onto the dendrites of VTA neurons. In the VTA, 74.1% of MOR mRNA-expressing neurons were GABAergic, with the rest being glutamatergic neurons expressing type-2 vesicular glutamate transporter mRNA. However, MOR mRNA was below the detection threshold in dopaminergic neurons. By immunohistochemistry, MOR was highly expressed on the extrasynaptic membranes of dendrites in GABAergic VTA neurons, including dendrites innervated by BST-VTA projection terminals. MOR was also expressed weakly on GABAergic and glutamatergic terminals in the VTA. Given that GABAA α1 is expressed at GABAergic BST-VTA synapses on dendrites of GABAergic neurons [T. Kudo et al. (2012) J. Neurosci., 32, 18035-18046], our results collectively indicate that the BST sends dual inhibitory outputs targeting GABAergic VTA neurons; GABAergic inhibition via 'wired' transmission, and enkephalinergic inhibition via 'volume' transmission. This dual inhibitory system provides the neural substrate underlying the potent disinhibitory control over dopaminergic VTA neurons exerted by the BST., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Cerebellar function is regulated by cholinergic mossy fiber inputs that are primarily derived from the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN). In contrast to the growing evidence surrounding cholinergic transmission and its functional significance in the cerebellum, the intrinsic and synaptic properties of cholinergic projection neurons (ChPNs) have not been clarified. In this study, we generated choline acetyltransferase (ChAT)-tdTomato transgenic rats, which specifically express the fluorescent protein tdTomato in cholinergic neurons, and used them to investigate the response properties of ChPNs identified via retrograde labeling using whole-cell recordings in brainstem slices. In response to current pulses, ChPNs exhibited two afterhyperpolarisation (AHP) profiles and three firing patterns; the predominant AHP and firing properties differed between the MVN and PHN. Morphologically, the ChPNs were separated into two types based on their soma size and dendritic extensions. Analyses of the firing responses to time-varying sinusoidal current stimuli revealed that ChPNs exhibited different firing modes depending on the input frequencies. The maximum frequencies in which each firing mode was observed were different between the neurons that exhibited distinct firing patterns. Analyses of the current responses to the application of neurotransmitter receptor agonists revealed that the ChPNs expressed (i) AMPA- and NMDA-type glutamate receptors, (ii) GABAA and glycine receptors, and (iii) muscarinic and nicotinic acetylcholine receptors. The current responses mediated by these receptors of MVN ChPNs were not different from those of PHN ChPNs. These findings suggest that ChPNs receive various synaptic inputs and encode those inputs appropriately across different frequencies., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice-patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti-motilin receptor antibody. Bath application of motilin (0.1 μm) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose-dependent manner (K(d) , 0.03 μm). The motilin action on spontaneous action potentials was blocked by apamin (100 nm), a blocker of small-conductance Ca(2+) -activated K(+) channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate-γ-S (1 mm) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild-type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ-aminobutyric acid transporter-Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small-conductance Ca(2+) -activated K(+) channels and postsynaptic γ-aminobutyric acid receptors through heterotrimeric guanosine triphosphate-binding protein-coupled receptor in a group of glutamatergic MVNs., (© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Numerous studies have investigated the effects of lesions of the primary visual cortex (V1) on visual responses in neurons of the superficial layer of the superior colliculus (sSC), which receives visual information from both the retina and V1. However, little is known about the changes in the local circuit dynamics of the sSC after receiving V1 lesions. Here, we show that surround inhibition of sSC neurons is transiently enhanced following V1 lesions in mice and that this enhancement may be attributed to alterations in the balance between excitatory and inhibitory inputs to sSC neurons. Extracellular recordings in vivo revealed that sSC neuronal responses to large visual stimuli were transiently reduced at about 1 week after visual cortical lesions compared with normal mice and that this reduction was partially recovered at about 1 month after the lesions. By using whole-cell patch-clamp recordings from sSC neurons in slice preparations obtained from mice that had received visual cortical lesions at 1 week prior to the recordings, we found cell type-dependent changes in the balance between excitation and inhibition. In non-GABAergic cells, inhibition predominated over excitation, whereas the excitation-inhibition balance did not change in GABAergic neurons. These results suggest that enhanced inhibition may be partially responsible for the reduced responses to large visual stimuli in some sSC neurons. Thus, we propose that the enhanced surround inhibition shortly after visual cortical lesions may prevent hyperexcitability in the sSC local circuit, contributing to reconstructing the finely tuned receptive field organization of sSC neurons after the visual cortical lesions., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Neurons in the intermediate gray layer (SGI) of the mammalian superior colliculus (SC) receive dense cholinergic innervations from the brainstem parabrachial region. Such cholinergic inputs may influence execution of orienting behaviors. To obtain deeper insights into how the cholinergic inputs modulate the SC local circuits, we analysed the cholinergic responses in identified γ-aminobutyric acid (GABA)ergic and non-GABAergic neurons using SC slices obtained from GAD67-GFP knock-in mice. The responses of SGI neurons to cholinergic agonists were various combinations of fast inward currents mediated mainly via α4β2 and partly by α7 nicotinic receptors (nIN), slow inward currents caused by activation of M1 plus M3 muscarinic receptors (mIN), and slow outward currents caused by activation of M2 muscarinic receptors (mOUT). The most common cholinergic responses in non-GABAergic neurons was nIN + mIN + mOUT (38/68), followed by nIN + mIN (16/68), nIN + mOUT (11/68), nIN only (2/68), and no response (1/68). On the other hand, the major response pattern in GABAergic neurons was either nIN only (26/54) or nIN + mIN (21/54), followed by nIN + mOUT (4/54), mOUT only (2/54), and no response (1/54). Thus, major effects of cholinergic inputs to both SGI GABAergic and non-GABAergic neurons are excitatory, but the response patterns in these two types of SGI neurons are different. Thus, actions of the cholinergic inputs to non-GABAergic and GABAergic SGI neurons are not simple push-pull mechanisms, like excitation vs inhibition, but might cooperate to balance the level of excitation and inhibition for setting the state of the response property of the local circuit., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The present study elucidated whether early life stress alters the extracellular signal-regulated kinase (ERK) pathway that underlies fear retrieval and fear extinction based on a contextual fear conditioning paradigm, using a juvenile stress model. Levels of phospho-ERK (pERK), the active form of ERK, increased after fear retrieval in the hippocampal CA1 region but not in the medial prefrontal cortex (mPFC). ERK activation in the CA1 following fear retrieval was not observed in adult rats who received aversive footshock (FS) stimuli during the second postnatal period (2wFS), which exhibited low levels of freezing. In fear extinction, pERK levels in the CA1 were increased by repeated extinction trials, but they were not altered after extinction retrieval. In contrast, pERK levels in the mPFC did not change during extinction training, but were enhanced after extinction retrieval. These findings were compatible in part with electrophysiological data showing that synaptic transmission in the CA1 field and mPFC was enhanced during extinction training and extinction retrieval, respectively. ERK activation in the CA1 and mPFC associated with extinction processes did not occur in rats that received FS stimuli during the third postnatal period (3wFS), which exhibited sustained freezing behavior. The repressed ERK signaling and extinction deficit observed in the 3wFS group were ameliorated by treatment with the partial N-methyl-D-aspartate receptor agonist D-cycloserine. These findings suggest that early postnatal stress induced the downregulation of ERK signaling in distinct brain regions through region-specific regulation, which may lead to increased behavioral abnormalities or emotional vulnerabilities in adulthood., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The direction and amplitude of saccadic eye movements are determined by the location of the center of gravity of burst activity over a neuronal population on the spatial map of the intermediate gray layer (SGI) of the superior colliculus (SC). GABAergic interneurons might play critical roles in shaping the activation field on the topographical map but, to understand the mechanism, basic information on the organization of inhibitory circuits is essential. In the present study, we investigated the electrophysiological and morphological properties of GABAergic neurons in SGI by whole-cell patch-clamp recordings and intracellular staining using biocytin in GAD67-GFP knock-in mice (PND17-22), in which GABAergic neurons specifically express GFP fluorescence. The most common firing properties among these GABAergic neurons (n=231) were fast spiking (58%), followed by burst spiking (29%), late spiking (8%) and, the least common, regular spiking (2%) and rapid spike inactivation (3%). Morphological analysis of axonal trajectories of intracellularly-labeled GABAergic neurons revealed three major subclasses: (i) intralaminar interneurons, which were further divided into two subclasses, local and horizontal interneurons; (ii) interlaminar interneurons; and (iii) commissural and tectofugal neurons. These results reveal distinct subsets of GABAergic neurons including neurons that mediate local and long-range inhibition in the SC, neurons that potentially modulate visual and other sensory inputs to the SC, and neurons that project to nuclei outside the SC., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive cells attaching to neuronal perikarya immunostained for microtubule-associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2',3'-Cyclic nucleotide 3'-phosphodiesterase-positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain-type lipid binding protein and glial fibrillary acidic protein), microglia (Iba-1) and NG2(+) glia. However, almost all perineuronal oligodendrocytes expressed glia-specific or glia-enriched metabolic enzymes, i.e. the creatine synthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase and L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase. As to molecules participating in the glutamate-glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT-1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV-VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, and found a novel outward sPSC mediated by the GABA(B) receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule-specific difference is at least partly attributable to differences in the density of GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABA(B) receptor currents (larger in lobules 9 and 10 than in lobule 3). The G-protein-coupled inward rectifying K(+) channel (GIRK) is known to be activated by GABA(B) receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs(+) block, and was observed when Cs(+) was used as a charge carrier. These results suggest that a K(+) channel other than GIRK could be activated by GABA(B) receptors. KCNK13 is a Cs(+)-permeable K(+) channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co-expression of G(βγ) subunits and was observed when Cs(+) was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs(+)-permeable K(+) channel activated by GABA(B) receptors, presumably via G(βγ) subunits in Purkinje cells., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The Ca(2+)-binding proteins (CBPs) calbindin D28k, calretinin and parvalbumin are phenotypic markers of functionally diverse subclasses of neurons in the adult brain. The developmental dynamics of CBP expression are precisely timed: calbindin and calretinin are present in prospective cortical interneurons from mid-gestation, while parvalbumin only becomes expressed during the early postnatal period in rodents. Secretagogin (scgn) is a CBP cloned from pancreatic beta and neuroendocrine cells. We hypothesized that scgn may be expressed by particular neuronal contingents during prenatal development of the mammalian telencephalon. We find that scgn is expressed in neurons transiting in the subpallial differentiation zone by embryonic day (E)11 in mouse. From E12, scgn(+) cells commute towards the extended amygdala and colonize the bed nucleus of stria terminalis, the interstitial nucleus of the posterior limb of the anterior commissure, the dorsal substantia innominata (SI) and the central and medial amygdaloid nuclei. Scgn(+) neurons can acquire a cholinergic phenotype in the SI or differentiate into GABA cells in the central amygdala. We also uncover phylogenetic differences in scgn expression as this CBP defines not only neurons destined to the extended amygdala but also cholinergic projection cells and cortical pyramidal cells in the fetal nonhuman primate and human brains, respectively. Overall, our findings emphasize the developmentally shared origins of neurons populating the extended amygdala, and suggest that secretagogin can be relevant to the generation of functional modalities in specific neuronal circuitries.

Inhibitory synaptic transmission mediated by gamma-aminobutyric acid (GABA)(A) receptors is involved in regulation of experience-dependent cortical plasticity. However, little information is available on their presynaptic and postsynaptic developmental profiles. The present study aims to investigate the developmental changes of miniature and unitary inhibitory postsynaptic currents (mIPSCs and uIPSCs) in mouse barrel cortex. mIPSCs recorded from supragranular pyramidal neurons showed a gradual increase in frequency during postnatal days 6-15 (PD6-15) followed by a marked increase at PD16-20, and mIPSCs frequency reached a plateau at about PD21-30. The amplitude of mIPSCs showed a transient decrease at PD10-12 followed by an increase during PD13-30, and reached a plateau at about PD30. Their decay time constant progressively decreased during the first 30 days postnatally, and reached a steady level at about PD30. Paired recordings from interneurons and synaptically coupled target pyramidal cells revealed that uIPSC amplitude increased with age up to PD30. In contrast, failure rate and coefficient of variation decreased during PD7-15 and showed little change at a later stage. Short-term depression induced by presynaptic stimulation at 33 Hz progressively decreased during PD6-20, and was stabilized at about PD21-30. Quantal analysis revealed that the number of release sites increased with age up to PD30, while the release probability increased during PD6-12 and then reached a plateau level. These results suggest that the number of release sites and release probability of GABA and GABA(A)-mediated IPSC kinetics show distinct developmental profiles, which could play roles in regulating the onset and offset of critical periods for experience-dependent cortical plasticity.

Purpose: Although the presence of gamma-aminobutyrate acid (GABA) in amacrine cells and its co-localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA-ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse., Methods: Whole mounts of the retina were prepared and the GFP-positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3., Results: Displaced GABA-ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 +/- 170 cells/mm(2). In the inner nuclear layer (INL), the density of amacrine cells was 8821 +/- 448 cells/mm(2) in the central region and 6825 +/- 408 cells/mm(2) in the peripheral region. GFP-positive amacrine cells co-localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co-localization was seen with antibodies against PV, TH, and VGluT 1-3., Conclusions: This study presents the first quantitative data concerning the co-localization of GABA-ergic neurons in the mouse retina with various neuronal markers.

Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro. Small (10-15 microm) or medium-sized (15-25 microm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3-12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.

The characteristics of GABAergic neurons involved in respiratory control have not been fully understood because identification of GABAergic neurons has so far been difficult in living tissues. In the present in vitro study, we succeeded in analysing the electrophysiological as well as morphological characteristics of GABAergic neurons in the pre-Bötzinger complex. We used 67-kDa isoform of glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) (Delta neo) knock-in (GAD67(GFP/+)) mice, which enabled us to identify GABAergic neurons in living tissues. We prepared medullary transverse slices that contained the pre-Bötzinger complex from these neonatal mice. The fluorescence intensity of the pre-Bötzinger complex region was relatively high among areas of the ventral medulla. Activities of GFP-positive neurons in the pre-Bötzinger complex were recorded in a perforated whole-cell patch-clamp mode. Six of 32 GFP-positive neurons were respiratory and the remaining 26 neurons were non-respiratory; the respiratory neurons were exclusively inspiratory, receiving excitatory post-synaptic potentials during the inspiratory phase. In addition, six inspiratory and one expiratory neuron of 30 GFP-negative neurons were recorded in the pre-Bötzinger complex. GFP-positive inspiratory neurons showed high membrane resistance and mild adaptation of spike frequency in response to depolarizing current pulses. GFP-positive inspiratory neurons had bipolar, triangular or crescent-shaped somata and GFP-negative inspiratory neurons had multipolar-shaped somata. The somata of GFP-positive inspiratory neurons were smaller than those of GFP-negative inspiratory neurons. We suggest that GABAergic inhibition not by expiratory neurons but by inspiratory neurons that have particular electrophysiological and morphological properties is involved in the respiratory neuronal network of the pre-Bötzinger complex.

Gamma-aminobutyric acid (GABA)ergic neurons in the neocortex have been mainly regarded as interneurons and thought to provide local interactions. Recently, however, glutamate decarboxylase (GAD) immunocytochemistry combined with retrograde labeling experiments revealed the existence of GABAergic projection neurons in the neocortex. We further studied the network of GABAergic projection neurons in the neocortex by using GAD67-green fluorescent protein (GFP) knock-in mice for retrograde labeling and a novel neocortical GABAergic neuron labeling method for axon tracing. Many GFP-positive neurons were retrogradely labeled after Fast Blue injection into the primary somatosensory, motor and visual cortices. These neurons were labeled not only around the injection site, but also at a long distance from the injection site. Of the retrogradely labeled GABAergic neurons remote from the injection sites, the vast majority (91%) exhibited somatostatin immunoreactivity, and were preferentially distributed in layer II, layer VI and in the white matter. In addition, most of GABAergic projection neurons were positive for neuropeptide Y (82%) and neuronal nitric oxide synthase (71%). We confirmed the long-range projections by tracing GFP-labeled GABAergic neurons with axon branches traveled rostro-caudally and medio-laterally. Axon branches could be traced up to 2 mm. Some (n = 2 of 4) were shown to cross the areal boundaries. The GABAergic projection neurons preferentially received neocortical inputs. From these results, we conclude that GABAergic projection neurons are distributed throughout the neocortex and are part of a corticocortical network.

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor-mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann-BSA), Escherichia coli(E. coli), or ovalbumin-containing immune complex (OVA-IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA-IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA-IC was significantly inhibited by anti-Fcgamma receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor-mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19-treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome-associated membrane protein-1 (Lamp-1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp-1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.

The dorsal lateral geniculate nucleus (dLGN), the major thalamic station of the visual pathway, contains a fairly large number of dopaminergic terminals, and dopamine was found to reduce spontaneous and visually evoked activity in the dLGN in vivo. The cellular basis of this influence remained unknown. Here we have used whole cell patch-clamp techniques to analyse the effects of dopamine (DA) on GABAergic transmission in dLGN slices of juvenile postnatal day (P) 12-P24 Long-Evans rats or juvenile (P12-P22) GAD67-GFP (Deltaneo) mice. Spontaneous inhibitory postsynaptic currents (sIPSCs) were increased in frequency by the D(2)-like agonist quinpirole (QUIN) in rat (n = 6), as well as in mouse (n = 5) thalamic slices. This effect was blocked in the presence of the D(2)-like antagonist sulpiride (SULP, n = 5) and was absent in the ventrobasal complex (VB) of rat (n = 7) and mouse (n = 4) thalamus, which is devoid of GABAergic interneurons. Direct recordings from labelled GABAergic neurons in the dLGN of GAD67-GFP mice revealed a QUIN-mediated membrane depolarization (n = 12), which was attenuated by SULP (n = 6). These data demonstrate that DA through activation of D(2)-like receptors in GABAergic interneurons induces an increase in inhibitory interactions most likely at F2 dendrodendritic terminals, thereby providing a cellular correlate of the observation made in vivo that DA predominantly acts through inhibition of relay cell activity in the dLGN.

Activity plays important roles in the formation and maturation of synaptic connections. We examined these roles using solitary neocortical excitatory neurons, receiving only self-generated synaptic inputs, cultured in a microisland with and without spontaneous spike activity. The amplitude of excitatory postsynaptic currents (EPSCs), evoked by applying brief depolarizing voltage pulses to the cell soma, continued to increase from 7 to 14 days in culture. Short-term depression of EPSCs in response to paired-pulse or 10-train-pulse stimulation decreased with time in culture. These developmental changes were prevented when neurons were cultured in a solution containing tetrodotoxin (TTX). The number of functional synapses estimated by recycled synaptic vesicles with FM4-64 was significantly smaller in TTX-treated than control neurons. However, the miniature EPSC amplitude remained unchanged during development, irrespective of activity. Transmitter release probability, assessed by use-dependent blockade of N-methyl-D-aspartate receptor-mediated EPSCs with MK-801, was higher in TTX-treated than control neurons. Therefore, the activity-dependent increase in EPSC amplitude was mainly ascribed to the increase in synapse number, while activity-dependent alleviation of short-term depression was mostly ascribed to the decrease in release probability. The effect of activity blockade on short-term depression, but not EPSC amplitude, was reversed after 4 days of TTX removal, indicating that synapse number and release probability are controlled by activity in very different ways. These results demonstrate that activity regulates the conversion of immature synapses transmitting low-frequency input signals preferentially to mature synapses transmitting both low- and high-frequency signals effectively, which may be necessary for information processing in mature cortex.

Brain-derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67-GFP knock-in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layer IV stimulation in most GFP-positive neurons in layer II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti-TrkB IgG, indicating that the acute inhibitory action of BDNF is activity-dependent and mediated through TrkB. Paired-pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10-100 ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca(2+) chelator, suggesting that a rise in Ca(2+) is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)-positive neurons, but in only 19% of PV-negative neurons, indicating that the depressive action is biased to PV-positive GABAergic neurons.

To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C.perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C.perfringens isolates from those samples were grouped by slide agglutination and by pulsed-field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C.perfringens, and not by a food-borne outbreak.

The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.

Biopsy specimens of human gastric mucosa of patients with gastric complaints and subjected to endoscopic examination were cultured microaerobically, and Campylobacter pyloridis was detected in 46 out of 80 cases (57.5%). The organism was found in 13 out of 22 patients with gastritis, 11 out of 16 with gastric ulcer scar, 7 out of 16 with gastric ulcer, 3 out of 9 with gastric polyp, 4 out of 5 with gastric carcinoma, 2 out of 2 with esophagus carcinoma, and 6 out of 9 with other gastric diseases. The isolates were identified as C. pyloridis, demonstrating its characteristic features such as positive for oxidase and catalase, negative for reduction of nitrite and nitrate, positive for urease, no growth at 25 C, growth at 37 C, not tolerant to 1% glycine, and resistant to nalidixic acid. Positive alkaline phosphatase activity was considered as an additional feature characteristic for the strains of C. pyloridis. The major cellular fatty acids were tetradecanoic acid and 19-carbon-cyclopropane acid. This pattern is unique among Campylobacter species. The survival of the organism for a longer period than 60 min at pH 2.5 indicates its significant resistance to acidic environment.