Glutamate Transporter Research Articles

VGLUT2 and APP family: unraveling the neurobiochemical mechanisms of neurostimulation therapy to STZ-induced diabetes and neuropathy.

Diabetic Peripheral Neuropathy (DPN) poses an escalating threat to public health, profoundly impacting well-being and quality of life. Despite its rising prevalence, the pathogenesis of DPN remains enigmatic, and existing clinical interventions fall short of achieving meaningful reversals of the condition. Notably, neurostimulation techniques have shown promising efficacy in alleviating DPN symptoms, underscoring the imperative to elucidate the neurobiochemical mechanisms underlying DPN. This study employs an integrated multi-omics approach to explore DPN and its response to neurostimulation therapy. Our investigation unveiled a distinctive pattern of vesicular glutamate transporter 2 (VGLUT2) expression in DPN, rigorously confirmed through qPCR and Western blot analyses in DPN C57 mouse model induced by intraperitoneal Streptozotocin (STZ) injection. Additionally, combining microarray and qPCR methodologies, we revealed and substantiated variations in the expression of the Amyloid Precursor Protein (APP) family in STZ-induced DPN mice. Analyzing the transcriptomic dataset generated from neurostimulation therapy for DPN, we intricately explored the differential expression patterns of VGLUT2 and APPs. Through correlation analysis, protein-protein interaction predictions, and functional enrichment analyses, we predicted the key biological processes involving VGLUT2 and the APP family in the pathogenesis of DPN and during neurostimulation therapy. This comprehensive study not only advances our understanding of the pathogenesis of DPN but also provides a theoretical foundation for innovative strategies in neurostimulation therapy for DPN. The integration of multi-omics data facilitates a holistic view of the molecular intricacies of DPN, paving the way for more targeted and effective therapeutic interventions.

Effects of Tualang Honey Pre-Treatment on Cerebellar and Striatal Neuronal Changes and Excitatory Amino Acid Transporter-2 (EAAT2) Expression Following Kainic Acid Exposure in Rats

Effects of Tualang Honey Pre-Treatment on Cerebellar and Striatal Neuronal Changes and Excitatory Amino Acid Transporter-2 (EAAT2) Expression Following Kainic Acid Exposure in Rats

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

An ion-independent intermediate underlies the evolutionary switch between sodium and proton-coupled glutamate transporter homologues

An ion-independent intermediate underlies the evolutionary switch between sodium and proton-coupled glutamate transporter homologues

Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Modulation mechanism of a glutamate transporter homolog by fatty acids

Modulation mechanism of a glutamate transporter homolog by fatty acids

Ceftriaxone alters the gut microbiome composition and reduces alcohol intake in male and female Sprague–Dawley rats

Ceftriaxone is an antibiotic that increases central nervous system (CNS) protein expression of the glutamate transporters GLT-1 and xCT and ameliorates pathological behaviors in rodent models of neurological disease and substance use disorder. However, little ceftriaxone passes through the blood–brain barrier, the CNS binding partner of ceftriaxone is unknown, and ceftriaxone does not consistently upregulate GLT-1 and xCT in cell culture. Ceftriaxone alters the gut microbiome composition in rodents and humans, and the microbiome–gut–brain axis regulates drug-seeking. Thus, here we test the hypothesis that ceftriaxone reduces alcohol intake while ameliorating alcohol-induced disruption of the gut microbiome composition. Male and female Sprague–Dawley rats received intermittent access to alcohol (IAA) while controls received access to only water. Following 17 IAA sessions, ceftriaxone/vehicle treatment was given for 5 days. Analysis of the gut microbiome composition was assessed by 16S rRNA gene amplicon sequencing conducted on fecal pellets collected prior to and after alcohol consumption and following ceftriaxone treatment. Male rats displayed escalated alcohol intake and preference over the course of the 17 sessions; however, total alcohol intake did not differ between the sexes. Ceftriaxone reduced alcohol intake and preference in male and female rats. While alcohol affected a diverse set of amplicon sequencing variants (ASV), ceftriaxone markedly reduced the diversity of microbial communities reflected by a blooming of the Enterococcaceae family. The remaining effects of ceftriaxone, however, encompassed families both affected and unaffected by prior alcohol drinking and highlight the Ruminococcaceae and Muribaculaceae families as bidirectionally modulated by alcohol and ceftriaxone. Altogether, our study confirms that ceftriaxone reduces alcohol intake in rats and partially reverses alcohol-induced dysbiosis.

Chemically Functionalized Single-Walled Carbon Nanotubes Prevent the Reduction in Plasmalemmal Glutamate Transporter EAAT1 Expression in, and Increase the Release of Selected Cytokines from, Stretch-Injured Astrocytes in Vitro.

We tested the effects of water-soluble single-walled carbon nanotubes, chemically functionalized with polyethylene glycol (SWCNT-PEG), on primary mouse astrocytes exposed to a severe in vitro simulated traumatic brain injury (TBI). The application of SWCNT-PEG in the culture media of injured astrocytes did not affect cell damage levels, when compared to those obtained from injured, functionalization agent (PEG)-treated cells. Furthermore, SWCNT-PEG did not change the levels of oxidatively damaged proteins in astrocytes. However, this nanomaterial prevented the reduction in plasmalemmal glutamate transporter EAAT1 expression caused by the injury, rendering the level of EAAT1 on par with that of control, uninjured PEG-treated astrocytes; in parallel, there was no significant change in the levels of GFAP. Additionally, SWCNT-PEG increased the release of selected cytokines that are generally considered to be involved in recovery processes following injuries. As a loss of EAATs has been implicated as a culprit in the suffering of human patients from TBI, the application of SWCNT-PEG could have valuable effects at the injury site, by preventing the loss of astrocytic EAAT1 and consequently allowing for a much-needed uptake of glutamate from the extracellular space, the accumulation of which leads to unwanted excitotoxicity. Additional potential therapeutic benefits could be reaped from the fact that SWCNT-PEG stimulated the release of selected cytokines from injured astrocytes, which would promote recovery after injury and thus counteract the excess of proinflammatory cytokines present in TBI.

Repeated cold stress, an animal model for fibromyalgia, elicits proprioceptor-induced chronic pain with microglial activation in mice

BackgroundFibromyalgia is characterized by chronic pain, fatigue, and other somatic symptoms. We have recently revealed that proprioceptor hyperactivation induces chronic pain in a rat model of myalgic encephalomyelitis. The present study explores whether similar proprioceptor-induced pain is elicited in a mouse model of fibromyalgia.MethodsRepeated cold stress (RCS) was used as a fibromyalgia model. Pain behavior was examined using the von Frey test, and neuronal activation was examined immunohistochemically as activating transcription factor (ATF)3 expression. The Atf3:BAC transgenic mouse, in which mitochondria in hyperactivated neurons are specifically labeled by green fluorescent protein, was used to trace the activated neuronal circuit. PLX3397 (pexidartinib) was used for microglial suppression.ResultsRCS elicited long-lasting pain in mice. ATF3, a marker of cellular hyperactivity and injury, was expressed in the lumbar dorsal root ganglion (DRG) 2 days after RCS initiation; the majority of ATF3-expressing DRG neurons were tropomyosin receptor kinase C- and/or vesicular glutamate transporter 1-positive proprioceptors. Microglial activation and increased numbers of microglia were observed in the medial part of the nucleus proprius 5 days after RCS initiation, and in the dorsal region of the ventral horn 7 days after RCS. In the ventral horn, only a subset of motor neurons was positive for ATF3; these neurons were surrounded by activated microglia. A retrograde tracer study revealed that ATF3-positive motor neurons projected to the intrinsic muscles of the foot (IMF). Using Atf3:BAC transgenic mice, we traced hyperactivated neuronal circuits along the reflex arc. Green fluorescent protein labeling was observed in proprioceptive DRG neurons and their processes originating from the IMF, as well as in motor neurons projecting to the IMF. Microglial activation was observed along this reflex arc, and PLX3397-induced microglial ablation significantly suppressed pain behavior.ConclusionProprioceptor hyperactivation leads to local microglial activation along the reflex arc; this prolonged microglial activation may be responsible for chronic pain in the present model. Proprioceptor-induced microglial activation might be the common cause of chronic pain in both the fibromyalgia and myalgic encephalomyelitis models, although the experimental models are different.

Glaucoma Animal Models beyond Chronic IOP Increase.

Glaucoma is a complex and multifactorial disease defined as the loss of retinal ganglion cells (RGCs) and their axons. Besides an elevated intraocular pressure (IOP), other mechanisms play a pivotal role in glaucoma onset and progression. For example, it is known that excitotoxicity, immunological alterations, ischemia, and oxidative stress contribute to the neurodegeneration in glaucoma disease. To study these effects and to discover novel therapeutic approaches, appropriate animal models are needed. In this review, we focus on various glaucoma animal models beyond an elevated IOP. We introduce genetically modified mice, e.g., the optineurin E50K knock-in or the glutamate aspartate transporter (GLAST)-deficient mouse. Excitotoxicity can be mimicked by injecting the glutamate analogue N-methyl-D-aspartate intravitreally, which leads to rapid RGC degeneration. To explore the contribution of the immune system, the experimental autoimmune glaucoma model can serve as a useful tool. Here, immunization with antigens led to glaucoma-like damage. The ischemic mechanism can be mimicked by inducing a high IOP for a certain amount of time in rodents, followed by reperfusion. Thereby, damage to the retina and the optic nerve occurs rapidly after ischemia/reperfusion. Lastly, we discuss the importance of optic nerve crush models as model systems for normal-tension glaucoma. In summary, various glaucoma models beyond IOP increase can be utilized.

FBXL20 promotes synaptic impairment in depression disorder via degrading vesicle-associated proteins

BackgroundSynaptic plasticity changes in presynaptic terminals or postsynaptic membranes play a critical role in cognitive impairments and emotional disorders, but the underlying molecular mechanisms in depression remain largely unknown. MethodsThe regulation effects of F-box and leucine-rich repeat protein 20 (FBXL20), vesicular glutamate transporter 1 (VGLUT1) and vesicle-associated membrane protein 1 (VAMP1) on synaptic plasticity and depressive-like behaviors examined by proteomics analysis, viral stereotaxic injection, transmission electron microscope and biochemical methods. The glutamate release detected by fluorescent probe in cultured primary pyramidal neurons. ResultsWe found that chronic unpredictable mild stress (CUMS) induced significant synaptic deficits within hippocampus of depressed rats, accompanied with the decreased expression of VGLUT1 and VAMP1. Moreover, knockdown of VGLUT1 or VAMP1 in hippocampal pyramidal neurons resulted in abnormal glutamatergic neurotransmitter release. In addition, we found that the E3 ubiquitin ligase FBXL20 was increased within hippocampus, which may promote ubiquitination and degradation of VGLUT1 and VAMP1, and thus resulted in the reduction of glutamatergic neurotransmitter release, the disruptions of synaptic transmission and the induction of depression-like behaviors in rats. In contrast, shRNA knockdown of FBXL20 within the hippocampus of depressed rats significantly ameliorated synaptic damage and depression-like behaviors. LimitationOnly one type of depression model was used in the present study, while other animal models should be used in the future to confirm the underlying mechanisms reported here. ConclusionsThis study provides new insights that inhibiting FBXL20 pathway in depressed rats may be an effective strategy to rescue synaptic transmission and depression-like behaviors.

Morphological, molecular, and functional characterization of mouse glutamatergic myenteric neurons.

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission.NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Interhemispheric reactivity of the subthalamic nucleus sustains progressive dopamine neuron loss in asymmetrical parkinsonism

Parkinson's disease (PD) is characterized by the progressive and asymmetrical degeneration of the nigrostriatal dopamine neurons and the unilateral presentation of the motor symptoms at onset, contralateral to the most impaired hemisphere. We previously developed a rat PD model that mimics these typical features, based on unilateral injection of a substrate inhibitor of excitatory amino acid transporters, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), in the substantia nigra (SN). Here, we used this progressive model in a multilevel study (behavioral testing, in vivo 1H-magnetic resonance spectroscopy, slice electrophysiology, immunocytochemistry and in situ hybridization) to characterize the functional changes occurring in the cortico-basal ganglia-cortical network in an evolving asymmetrical neurodegeneration context and their possible contribution to the cell death progression. We focused on the corticostriatal input and the subthalamic nucleus (STN), two glutamate components with major implications in PD pathophysiology. In the striatum, glutamate and glutamine levels increased from presymptomatic stages in the PDC-injected hemisphere only, which also showed enhanced glutamatergic transmission and loss of plasticity at corticostriatal synapses assessed at symptomatic stage. Surprisingly, the contralateral STN showed earlier and stronger reactivity than the ipsilateral side (increased intraneuronal cytochrome oxidase subunit I mRNA levels; enhanced glutamate and glutamine concentrations). Moreover, its lesion at early presymptomatic stage halted the ongoing neurodegeneration in the PDC-injected SN and prevented the expression of motor asymmetry. These findings reveal the existence of endogenous interhemispheric processes linking the primary injured SN and the contralateral STN that could sustain progressive dopamine neuron loss, opening new perspectives for disease-modifying treatment of PD.

Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy

The growing recognition of a dichotomous role of astrocytes in neurodegenerative processes has heightened the need for unraveling distinct astrocytic subtypes in neurological disorders. In multiple system atrophy (MSA), a rare, rapidly progressing atypical Parkinsonian disease characterized by increased astrocyte reactivity. However the specific contribution of astrocyte subtypes to neuropathology remains elusive. Hence, we first set out to profile glial fibrillary acidic protein levels in astrocytes across the human post mortem motor cortex, putamen, and substantia nigra of MSA patients and observed an overall profound astrocytic response. Matching the post mortem human findings, a similar astrocytic phenotype was present in a transgenic MSA mouse model. Notably, MSA mice exhibited a decreased expression of the glutamate transporter 1 and glutamate aspartate transporter in the basal ganglia, but not the motor cortex. We developed an optimized astrocyte isolation protocol based on magnetic-activated cell sorting via ATPase Na+/K+ transporting subunit beta 2 and profiled the transcriptomic landscape of striatal and cortical astrocytes in transgenic MSA mice. The gene expression profile of astrocytes in the motor cortex displayed an anti-inflammatory signature with increased oligodendroglial and pro-myelinogenic expression pattern. In contrast, striatal astrocytes were defined by elevated pro-inflammatory transcripts accompanied by dysregulated genes involved in homeostatic functions for lipid and calcium metabolism. These findings provide new insights into a region-dependent, dichotomous astrocytic response—potentially beneficial in the cortex and harmful in the striatum—in MSA suggesting a differential role of astrocytes in MSA-related neurodegenerative processes.

Scaling of smaller pyramidal neuron size and lower energy production in schizophrenia

BackgroundDorsolateral prefrontal cortex (DLPFC) dysfunction in schizophrenia appears to reflect alterations in layer 3 pyramidal neurons (L3PNs), including smaller cell bodies and lower expression of mitochondrial energy production genes. However, prior somal size studies used biased strategies for identifying L3PNs, and somal size and levels of energy production markers have not been assessed in individual L3PNs. Study designWe combined fluorescent in situ hybridization (FISH) of vesicular glutamate transporter 1 (VGLUT1) mRNA and immunohistochemical-labeling of NeuN to determine if the cytoplasmic distribution of VGLUT1 mRNA permits the unbiased identification and somal size quantification of L3PNs. Dual-label FISH for VGLUT1 mRNA and cytochrome C oxidase subunit 4I1 (COX4I1) mRNA, a marker of energy production, was used to assess somal size and COX4I1 transcript levels in individual DLPFC L3PNs from schizophrenia (12 males; 2 females) and unaffected comparison (13 males; 1 female) subjects. Study resultsMeasures of L3PN somal size with NeuN immunohistochemistry or VGLUT1 mRNA provided nearly identical results (ICC = 0.96, p < 0.0001). Mean somal size of VGLUT1-identified L3PNs was 8.7% smaller (p = 0.004) and mean COX4I1 mRNA levels per L3PN were 16.7% lower (p = 0.01) in schizophrenia. These measures were correlated across individual L3PNs in both subject groups (rrm = 0.81–0.86). ConclusionsThis preliminary study presents a novel method for combining unbiased neuronal identification with quantitative assessments of somal size and mRNA levels. We replicated findings of smaller somal size and lower COX4I1 mRNA levels in DLPFC L3PNs in schizophrenia. The normal scaling of COX4I1 mRNA levels with somal size in schizophrenia suggests that lower markers of energy production are secondary to L3PN morphological alterations in the illness.

Bilateral and symmetric glycinergic and glutamatergic projections from the LSO to the IC in the CBA/CaH mouse.

Auditory space has been conceptualized as a matrix of systematically arranged combinations of binaural disparity cues that arise in the superior olivary complex (SOC). The computational code for interaural time and intensity differences utilizes excitatory and inhibitory projections that converge in the inferior colliculus (IC). The challenge is to determine the neural circuits underlying this convergence and to model how the binaural cues encode location. It has been shown that midbrain neurons are largely excited by sound from the contralateral ear and inhibited by sound leading at the ipsilateral ear. In this context, ascending projections from the lateral superior olive (LSO) to the IC have been reported to be ipsilaterally glycinergic and contralaterally glutamatergic. This study used CBA/CaH mice (3-6 months old) and applied unilateral retrograde tracing techniques into the IC in conjunction with immunocytochemical methods with glycine and glutamate transporters (GlyT2 and vGLUT2, respectively) to analyze the projection patterns from the LSO to the IC. Glycinergic and glutamatergic neurons were spatially intermixed within the LSO, and both types projected to the IC. For GlyT2 and vGLUT2 neurons, the average percentage of ipsilaterally and contralaterally projecting cells was similar (ANOVA, p = 0.48). A roughly equal number of GlyT2 and vGLUT2 neurons did not project to the IC. The somatic size and shape of these neurons match the descriptions of LSO principal cells. A minor but distinct population of small (< 40 μm2) neurons that labeled for GlyT2 did not project to the IC; these cells emerge as candidates for inhibitory local circuit neurons. Our findings indicate a symmetric and bilateral projection of glycine and glutamate neurons from the LSO to the IC. The differences between our results and those from previous studies suggest that species and habitat differences have a significant role in mechanisms of binaural processing and highlight the importance of research methods and comparative neuroscience. These data will be important for modeling how excitatory and inhibitory systems converge to create auditory space in the CBA/CaH mouse.

Suppressive effect of Yokukansan on glutamate released from canine keratinocytes.

Canine atopic dermatitis (CAD) is caused by skin barrier dysfunction due to allergen exposure. Excessive glutamate release in the skin is associated with delayed skin barrier function recovery and epidermal thickening and lichenification. Treatment with Yokukansan (YKS), a traditional Japanese medicine, reduces dermatitis severity and scratching behavior in NC/Nga mice by decreasing epidermal glutamate levels. However, the association between canine keratinocytes and glutamate and the mechanism by which YKS inhibits glutamate release from keratinocytes remains unknown. We aimed to investigate glutamate release from canine progenitor epidermal keratinocytes (CPEKs) and the inhibitory effect of YKS on this release. We also explored the underlying mechanism of YKS to enable its application in CAD treatment. Glutamate produced from CPEKs in the medium at 24 hours was measured. The measurement conditions varied in terms of cell density and YKS concentration. CPEKs were treated with a glutamate receptor antagonist (MK-801), a glutamate transporter antagonist (THA), and a glutamate dehydrogenase inhibitor (epigallocatechin gallate; EGCG), and the inhibitory effect of YKS, YKS + THA, MK-801, and EGCG on this release was determined. MK-801 and glutamate dehydrogenase inhibitor were tested alone, and THA was tested in combination with YKS. Finally, glutamine incorporated into CPEKs at 24 hours was measured using radioisotope labeling. CPEKs released glutamate in a cell density-dependent manner, inhibited by YKS in a concentration-dependent manner. Moreover, YKS reduced the intracellular uptake of radioisotope-labeled glutamine in a concentration-dependent manner. No involvement of glutamate receptor antagonism or activation of glutamate transporters was found, as suggested by previous studies. In addition, EGCG could inhibit glutamate release from CPEKs. Our findings indicated that glutamate release from CPEKs could be effectively inhibited by YKS, suggesting the utility of YKS in maintaining skin barrier function during CAD. In addition, CPEKs are appropriate for analyzing the mechanism of YKS. However, we found that the mechanism of action of YKS differs from that reported in previous studies, suggesting that it may have had a similar effect to EGCG in this study. Further research is warranted to understand the exact mechanism and clinical efficacy in treating CAD.

Functional evaluation of Bacillus licheniformis PF9 for its potential in controlling enterotoxigenic Escherichia coli in weaned piglets.

During the bacterial selection, isolate PF9 demonstrated tolerance to low pH and high bile salt and an ability to extend the lifespan of Caenorhabditis elegans infected with enterotoxigenic Escherichia coli (ETEC; P < 0.05). Thirty-two weaned piglets susceptible to ETEC F4 were randomly allocated to four treatments as follows: 1) non-challenged negative control group (NNC; basal diet and piglets gavaged with phosphate-buffered saline), 2) negative control group (NC; basal diet and piglets challenged with ETEC F4, 3 × 107 CFU per pig), 3) positive control (PC; basal diet + 80 mg·kg-1 of avilamycin and piglets challenged with ETEC F4), and 4) probiotic candidate (PF9; control basal diet + 2.5 × 109 CFU·kg-1 diet of B. licheniformis PF9 and piglets challenged with ETEC F4). The infection of ETEC F4 decreased average daily gain and gain:feed in the NC group when compared to the NNC group (P < 0.05). The inoculation of ETEC F4 induced severe diarrhea at 3h postinoculum (hpi), 36, 40 hpi in the NC group when compared to the NNC group (P < 0.05). The supplementation of B. licheniformis PF9 significantly relieved diarrhea severity at 3 hpi when compared to the NC group (P < 0.05). The inoculation of ETEC F4 reduced duodenal, jejunal, and ileal villus height (VH) in the NC group when compared to the NNC group. A significant (P < 0.05) decrease was detected in the duodenal VH in the PC and NNC groups. Moreover, the NNC group had a reduced relative mRNA level of Na+-glucose cotransporter 1 (SGLT1) when compared to the NC group (P < 0.05). Compared to the NC and NNC groups, the supplementation of B. licheniformis PF9 increased the relative mRNA levels of aminopeptidase N, occludin, zonula occludens-1, and SGLT1 (P < 0.05). The supplementation of B. licheniformis PF9 also significantly increased the relative mRNA level of excitatory amino acid transporter 1 when compared to the NC group (P < 0.05). Piglets supplemented with B. licheniformis PF9 showed lower relative abundance of Bacteroidetes in the colon than piglets from the NNC group (P < 0.05). The NNC group had a higher relative abundance of Firmicutes in the ileum than all the challenged piglets (P < 0.05); however, a lower relative abundance of Proteobacteria in the ileum and colon was observed in the NC group (P < 0.05). This study provides evidence that B. licheniformis PF9 has the potential to improve the gut health of piglets under challenging conditions.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites β-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.