eGFP Mice Research Articles

Heterogeneous Signaling at GABA and Glycine Co-releasing Terminals.

The corelease of several neurotransmitters from a single synaptic vesicle has been observed at many central synapses. Nevertheless, the signaling synergy offered by cotransmission and the mechanisms that maintain the optimal release and detection of neurotransmitters at mixed synapses remain poorly understood, thus limiting our ability to interpret changes in synaptic signaling and identify molecules important for plasticity. In the brainstem and spinal cord, GABA and glycine cotransmission is facilitated by a shared vesicular transporter VIAAT (also named VGAT), and occurs at many immature inhibitory synapses. As sensory and motor networks mature, GABA/glycine cotransmission is generally replaced by either pure glycinergic or GABAergic transmission, and the functional role for the continued corelease of GABA and glycine is unclear. Whether or not, and how, the GABA/glycine content is balanced in VIAAT-expressing vesicles from the same terminal, and how loading variability effects the strength of inhibitory transmission is not known. Here, we use a combination of loose-patch (LP) and whole-cell (WC) electrophysiology in cultured spinal neurons of GlyT2:eGFP mice to sample miniature inhibitory post synaptic currents (mIPSCs) that originate from individual GABA/glycine co-releasing synapses and develop a modeling approach to illustrate the gradual change in mIPSC phenotypes as glycine replaces GABA in vesicles. As a consistent GABA/glycine balance is predicted if VIAAT has access to both amino-acids, we test whether vesicle exocytosis from a single terminal evokes a homogeneous population of mixed mIPSCs. We recorded mIPSCs from 18 individual synapses and detected glycine-only mIPSCs in 4/18 synapses sampled. The rest (14/18) were co-releasing synapses that had a significant proportion of mixed GABA/glycine mIPSCs with a characteristic biphasic decay. The majority (9/14) of co-releasing synapses did not have a homogenous phenotype, but instead signaled with a combination of mixed and pure mIPSCs, suggesting that there is variability in the loading and/or storage of GABA and glycine at the level of individual vesicles. Our modeling predicts that when glycine replaces GABA in synaptic vesicles, the redistribution between the peak amplitude and charge transfer of mIPSCs acts to maintain the strength of inhibition while increasing the temporal precision of signaling.

Fetal microchimerism in mouse caerulein-induced pancreatitis model.

Objective(s):Fetal microchimerism is the persistence of allogeneic cell population that transfer from the fetus to the mother. The aim of this study was to evaluate the presence of fetal microchimerism in the pancreas of the mouse with acute pancreatitis (AP).Materials and Methods:In this experimental study, female wild-type mice were mated with male EGFP+. AP model was obtained by injection of caerulein two days after delivery. Sixty mice were divided into 3 groups: the virgin pancreatitis-induced animals, pregnant pancreatitis-induced animals mated with transgenic EGFP mice, and pregnant sham animals. To prove pancreatitis induction, the blood amylase and lipase were assessed; and pancreas was removed from a subpopulation of each group for histopathological examinations after 6 hr. The remaining mice were kept for 3 weeks and histopathological exanimation, immunohistochemistry, and PCR were performed.Results:EGFP+ cells were found in acini and around the blood vessels in the pancreas of pregnant pancreatitis-induced animals. They differentiated to acinar, adipocyte-like, and mesenchymal-like cells. PCR showed that 20% of the pregnant pancreatitis-induced animals were EGFP+. The histopathological study showed improvement in pancreatitis scores in the mice with history of pregnancy. Conclusion:It seems that pregnancy has a beneficial impact on caerulein-induced pancreatitis and improves the pancreatitis score in mouse.

Immediate effects of maternal separation on the development of interneurons derived from medial ganglionic eminence in the neonatal mouse hippocampus.

Aversive experiences, including maternal separation (MS), have been known as a risk for abnormal hippocampus development. Given that impairment of GABA inhibitory system is known as one of the common features of the abnormal hippocampal development induced by MS, we examined whether the MS on 4-day-old (P4) mice for 24hr abolishes the interneuron development. We observed that the MS reduced the volume of dorsal hippocampus on P14 as long-term effects. In addition, the MS decreased the number of parvalbumin (PV)-positive interneuron on P14 and P28 in the dorsal hippocampus. We further examined the immediate effects of MS by measuring the percentage of glutamic acid decarboxylase (GAD) 67-positive interneurons among the immature interneurons derived from medial ganglionic eminence (MGE) progenitors marked in nkx2.1cre;β-geo EGFP mice. During normal development from P4 to P5, the percentage of GAD67-positive interneurons among the MGE-derived interneurons in the dorsal hippocampus was significantly increased from 42.29% to 70.73% in the stratum pyramidale of the CA1 and increased from 46.4% to 56.99% in the stratum pyramidale of the CA2/3 region. However, the increase was not observed on P5 among the mice treated with the MS. These results suggest that the maturation of interneurons was suppressed by the MS. The suppressed maturation of interneurons may be one of the causes of the reduced volume of the hippocampus and PV+ interneurons observed in the hippocampus on P14 and P28 as a consequence of the MS during neonatal stage.

Transplantation of Normal Adipose Tissue Improves Blood Flow and Reduces Inflammation in High Fat Fed Mice With Hindlimb Ischemia.

Background: Fat deposition is associated with peripheral arterial disease. Adipose tissue has recently been implicated in vascular remodeling and angiogenic activity. We hypothesized that the transplantation of adipose tissues from normal mice improves blood flow perfusion and neovascularization in high-fat diet fed mice.Methods: After 14 weeks of high-fat diet (HFD)-fed mice, unilateral hind limb ischemia was performed. Subcutaneous white adipose tissue (WAT) and brown adipose tissue (BAT) fat pads were harvested from normal EGFP mice, and subcutaneously transplanted over the region of the adductor muscles of HFD mice. Blood flow was measured using Laser Doppler Scanner. Vascular density, macrophages infiltration, and macrophage polarization were examined by RT-qPCR, and immunohistochemistry.Results: We found that the transplantation of WAT derived from normal mice improved functional blood flow in HFD-fed mice compared to mice transplanted with BAT and sham-treated mice. WAT transplantation increased the recruitment of pericytes associated with nascent blood vessels, but did not affect capillary formation. Furthermore, transplantation of WAT ameliorated HFD-induced insulin resistance, M2 macrophage predominance and the release of arteriogenic factors in ischemic muscles. Mice receiving WAT also displayed a marked reduction in several proinflammatory cytokines. In contrast, mice transplanted with BAT were glucose intolerant and demonstrated increased IL-6 levels in ischemic muscles.Conclusion: These results indicate that transplantation of adipose tissue elicits improvements in blood perfusion and beneficial effects on systemic glucose homeostasis and could be a promising therapeutic option for the treatment of diabetic peripheral arterial disease.

Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion.

Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1+ ) progenitor stem cell involvement during pathologic arterial remodeling. Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH. The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1+ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1+ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1+ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1+ adventitial progenitor stem cells invitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. EtOH reduces SHh-responsive Sca1+ progenitor cell myogenic differentiation/expansion invitro and during arterial remodeling in response to ligation injury invivo. Regulation of vascular Sca1+ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.

Mitochondrial uncoupling in the melanocortin system differentially regulates NPY and POMC neurons to promote weight-loss

ObjectiveThe mitochondrial uncoupling agent 2,4-dinitrophenol (DNP), historically used as a treatment for obesity, is known to cross the blood-brain-barrier, but its effects on central neural circuits controlling body weight are largely unknown. As hypothalamic melanocortin neuropeptide Y/agouti-related protein (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons represent key central regulators of food intake and energy expenditure we investigated the effects of DNP on these neurons, food intake and energy expenditure. MethodC57BL/6 and melanocortin-4 receptor (MC4R) knock-out mice were administered DNP intracerebroventricularly (ICV) and the metabolic changes were characterized. The specific role of NPY and POMC neurons and the ionic mechanisms mediating the effects of uncoupling were examined with in vitro electrophysiology performed on NPY hrGFP or POMC eGFP mice. ResultsHere we show DNP-induced differential effects on melanocortin neurons including inhibiting orexigenic NPY and activating anorexigenic POMC neurons through independent ionic mechanisms coupled to mitochondrial function, consistent with an anorexigenic central effect. Central administration of DNP induced weight-loss, increased BAT thermogenesis and browning of white adipose tissue, and decreased food intake, effects that were absent in MC4R knock-out mice and blocked by the MC4R antagonist, AgRP. ConclusionThese data show a novel central anti-obesity mechanism of action of DNP and highlight the potential for selective melanocortin mitochondrial uncoupling to target metabolic disorders.

Intestinal Regulation of C. albicans by PP Langerin+ DCs

Abstract As the largest mucosal compartment, the human gastrointestinal tract (GI) is highly colonized with a complex microbial ecosystem that includes fungal organisms. Although fewer in numbers and less extensively studied in the context of the intestinal mucosa, it is unclear whether commensal members such as Candida species play a role in intestinal mucosal immunity. Recent studies have suggested that Candida sp. in the intestinal mucosa are associated with a number of human diseases such as gastric ulcers, Crohn’s disease and Hirschsprung-associated enterocolitis. We have recently identified, a specific non-macrophage dendritic cell (DC) population within intestinal Peyer’s patches (PP) that expresses the C-type lectin receptor (CLR)-Langerin and are able to specifically uptake Candida albicans, Candida tropicalis and yeast purified β-1,3-glucan particles (GPs). We hypothesize that Langerin+ DCs are central in the sampling, presentation and regulation of C. albicans within PPs. To address this hypothesis, we are currently defining the role of Langerin+ DCs in the processing of Candida sp. after uptake using the Langerin-DTR- EGFP mouse model as well as the Langerin-DTR- EGFP-DSS model. We are also characterizing the local gene expression profiles of PP B and T- lymphocytes as well as Langerin+ DCs in response to C. albicans. Additionally, we are investigating whether Langerin+ DCs are a migratory cell subset that not only transports C. albicans near germinal centers but can also take them to tolerogenic sites such as the MLN. These studies will advance our knowledge of how commensal members that are also part of the “mycobiome” are sampled, what mucosal immune responses are elicited and how these fungal organisms are tolerated.

Generation of GABAergic interneuron-specific PGC-1α knockout mice

To generate mice which are specific for peroxisomproliferator-activated receptor-γ coactivator-1(PGC-1α) knockout in the GABAergic interneuron. Conditional mice specific for PGC-1αf/+ were introduced from the Jackson Laboratory, USA and initially inbred to obtain homozygote PGC-1α f/f mice. The PGC-1αf/f conditional mice were further crossed with Dlx5/6-Cre-IRES-EGFP transgenic mice to achieve specific knockout of PGC-1α in the GABAergic interneuron. The offspring with specific knockout PGC-1α gene were successful for the generation of GABAergic interneuron, with the resulting genotype being PGC-1α f/f;Dlx5/6-Cre-IRES-EGFP. The PGC-1α f/f;Dlx5/6-Cre-IRES-EGFP mice were obtained through a proper crossing strategy, which has provided a suitable platform for studying the function of PGC-1α in neuropsychiatric diseases.

Foxn1 and Mmp-9 expression in intact skin and during excisional wound repair in young, adult, and old C57Bl/6 mice.

The transcription factor Foxn1 is essential for skin development. Our previous studies performed on young C57BL/6J mice model showed that Foxn1 acts as regulator of the skin wound healing process. The present study extended our initial research regarding the expression and potential role of Foxn1 in the intact and wounded skin as a function of animal age and stage of the wound healing process. We analyzed Foxn1 and Mmp-9 expression in the intact and postinjured skin of young, adult, and old C57BL/6J and transgenic Foxn1::Egfp mice. The similar levels of epidermal Foxn1 mRNA expression were detected in young and adult C57BL/6J mice and higher levels in old animals. Postinjured skin tissues displayed a gradual decrease of Foxn1 mRNA expression at Days 1, 5, and 7 after injury. Foxn1-eGFP positive cells were abundant at wound margin and in re-epithelialized epidermis at postwounded Days 1, 5, and 7 and colocalized with E-cadherin and Mmp-9. Postwounded skin at Days 14-36 displayed Foxn1-eGFP cells in the epidermis and in the dermal part of the skin (papillary dermis). Asubset of Foxn1-eGFP positive cells in the papillary dermis expressed the myofibroblast marker αSMA. Flow cytometric analysis of cells isolated from postwounded (Day 7) skin tissues showed a significant increase in the percentage of Foxn1-eGFP positive cells with phenotype of double positivity for E-cadherin/N-cadherin (epithelial/mesenchymal markers). Collectively, these data identify the transcription factor Foxn1 as a potential key epidermal regulator modifying both epidermal and dermal healing processes after cutaneous wounding.

ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter.

The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Skin-Derived Precursors as a Source of Progenitors for Corneal Endothelial Regeneration.

Corneal blindness is the fourth leading cause of blindness in the world. Current treatment is allogenic corneal transplantation, which is limited by shortage of donors and immunological rejection. Skin‐derived precursors (SKPs) are postnatal stem cells, which are self‐renewing, multipotent precursors that can be isolated and expanded from the dermis. Facial skin may therefore be an accessible autologous source of neural crest derived cells. SKPs were isolated from facial skin of Wnt1‐Cre/Floxed EGFP mouse. After inducing differentiation with medium containing retinoic acid and GSK 3‐β inhibitor, SKPs formed polygonal corneal endothelial‐like cells (sTECE). Expression of major corneal endothelial markers were confirmed by Reverse transcription polymerase chain reaction (RT‐PCR) and quantitative Real time polymerase chain reaction (qRT‐PCR). Western blots confirmed the expression of Na, K‐ATPase protein, the major functional marker of corneal endothelial cells. Immunohistochemistry revealed the expression of zonular occludens‐1 and Na, K‐ATPase in cell‐cell junctions. In vitro functional analysis of Na, K‐ATPase pump activity revealed that sTECE had significantly high pump function compared to SKPs or control 3T3 cells. Moreover, sTECE transplanted into a rabbit model of bullous keratopathy successfully maintained corneal thickness and transparency. Furthermore, we successfully induced corneal endothelial‐like cells from human SKPs, and showed that transplanted corneas also maintained corneal transparency and thickness. Our findings suggest that SKPs may be used as a source of autologous cells for the treatment of corneal endothelial disease. Stem Cells Translational Medicine 2017;6:788–798

AB315. SPR-42 Cyclophosphamide-induced overactive bladder via downregulation of relaxation factors in detrusor PDGFRα+ cells

ObjectiveMorphology and functional role of PDGFRα+ cells have been recently characterized in the detrusor muscle layer. Detrusor relaxation is caused by activation of small conductance Ca2+ activated-K+ (SK) channels and purinergic inhibitory responses in detrusor PDGFRα+ cells. Loss of PDGFRα+ cells or alteration of P2Y receptors and SK channels will affect detrusor excitability. Cyclophosphamide (CYP)-treated animals exhibited overactive bladder (OAB). We hypothesized that the downregulation of P2Y receptors and/or SK channels in PDGFRα+ cells will display the phenotype of CYP-induced OAB.MethodsCYP was injected intraperitoneally in PDGFRα+/eGFP and SMC/eGFP mice. We harvested the detrusor muscle without urothelium and disperse the cells for the fluorescence activated cell sorting (FACS). Sorted PDGFRα+ cells and smooth muscle cells (SMCs) were used for molecular study to compare the changes in transcripts between CYP-injected and control group. Transcripts were examined included; Pdgfrα, P2ry1, P2ry2, P2ry4, Kcnn1, Kcnn2, Kcnn3 and inflammation marker (Il-6). Immunohistochemistry, mechanical contractility and ex vivo cystometry were also performed.ResultsQuantitative analysis of PCR revealed that CYP-injected detrusor muscle increased transcriptional expression of Il-6, but decreased the expression of Pdgfrα. Transcriptional changes in CYP-injected sorted PDGFRα+ cells from PDGFRα+/eGFP mice showed Pdgfα, Kccn3 (SK3), P2ry1, P2ry2 and P2ry4 genes were decreased compared with saline-injected control. Sorted SMCs from SMC/eGFP mice did not show significant expression of those genes and no detectable changes. Immunohistochemistry showed SK3 in PDGFRα immunoreactivity was downregulated in CYP-injected detrusor muscle. Apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP-injected mice compared to saline-injected mice. In ex vivo cystometry, increased spontaneous non-voiding contractions and less apamin sensitivity were observed in CYP-injected mice.ConclusionsThese findings are the first report to investigate the role of PDGFRα+ cells in relation to OAB mechanisms. In conclusion, we found that CYP-induced OAB is resulted from down regulation of PDGFRα, P2Y receptors and SK channels in CYP-injected bladder. These results provide novel mechanisms of functional role of PDGFRα+ cells on OAB.Funding Source(s)NIDDK, RO1 DK098388 and Urology Care Foundation Research Scholar Award (Interstitial Cystitis Association)

Descending projections from the inferior colliculus to the dorsal cochlear nucleus are excitatory.

Ascending projections of the dorsal cochlear nucleus (DCN) target primarily the contralateral inferior colliculus (IC). In turn, the IC sends bilateral descending projections back to the DCN. We sought to determine the nature of these descending axons in order to infer circuit mechanisms of signal processing at one of the earliest stages of the central auditory pathway. An anterograde tracer was injected in the IC of CBA/Ca mice to reveal terminal characteristics of the descending axons. Retrograde tracer deposits were made in the DCN of CBA/Ca and transgenic GAD67-EGFP mice to investigate the cells giving rise to these projections. A multiunit best frequency was determined for each injection site. Brains were processed by using standard histologic methods for visualization and examined by fluorescent, brightfield, and electron microscopy. Descending projections from the IC were inferred to be excitatory because the cell bodies of retrogradely labeled neurons did not colabel with EGFP expression in neurons of GAD67-EGFP mice. Furthermore, additional experiments yielded no glycinergic or cholinergic positive cells in the IC, and descending projections to the DCN were colabeled with antibodies against VGluT2, a glutamate transporter. Anterogradely labeled endings in the DCN formed asymmetric postsynaptic densities, a feature of excitatory neurotransmission. These descending projections to the DCN from the IC were topographic and suggest a feedback pathway that could underlie a frequency-specific enhancement of some acoustic signals and suppression of others. The involvement of this IC-DCN circuit is especially noteworthy when considering the gating of ascending signal streams for auditory processing. J. Comp. Neurol. 525:773-793, 2017. © 2016 Wiley Periodicals, Inc.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia–axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

0109 : Connexin40 controls endothelial activation by dampening NF B activation

Endothelial cells (ECs) of healthy arteries express high levels of connexin40 (Cx40), but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is known to be atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. Here, we investigate the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around the right common carotid artery of mice expressing eGFP controlled by the Cx40 promoter. We found that Cx40 expression is down-regulated in regions of oscillatory shear stress (0.45±0.13, p=0.04), but is conserved in regions of high and low laminar shear stress (1.45±0.48 and 0.85±0.11, N=8) by en face immunofluorescence on Cx40 +/eGFP mice. Using a high-throughput phage display, we retrieved a consensus binding motif for the intracellular regulatory C-terminus of Cx40 (Cx40CT), i.e. HS[I,L,V][K,R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5 to 16 of IκBα, a member of the protein family that inhibit the nuclear translocation of NFκB. In vitro binding of this peptide was confirmed by crosslinking and by en face proximity ligation assay targeting IκBα and Cx40 on rat carotid arteries. Nuclear translocation of NFκB in mouse ECs in response to activation by TNFα was enhanced after reduction of Cx40 with siRNA. Transfection of Cx40 or Cx40CT in HeLa cells demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced of NFκB nuclear translocation. Finally, we observed an exaggarated induction of atherosclerotic plaques by LLSS or OSS in Apoe −/− mice with endothelial-specific deletion of Cx40. In conclusion, our data show a novel functional interaction between IκBα and Cx40 that may be relevant for the control of NFκB activation by shear stress in development of atherosclerotic disease. The author hereby declares no conflict of interest

Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina.

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst(2A) and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH-RFP mice and M1 ipRGCs in OPN4-EGFP mice. SRIF increases K(+) currents, decreases Ca(2+) currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst(2A) agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4'-piperidine]-1'-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N(2)-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-L-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain.

Oct4-GFP expression during transformation of gonocytes into spermatogonial stem cells in the perinatal mouse testis

Background/aimIn cryptorchidism perinatal failure to switch off Oct4, a germ cell (GC) marker, may lead to carcinoma in situ. We aimed to analyze Oct4 expression during mouse gonocyte transformation into spermatogonial stem cells (SSC). Materials and methodsTestes from OG2 (Oct4-promoter driven eGFP) mice at embryonic day (E) 17 and postnatal day P0–10 underwent immunohistochemistry and immunoblotting. Antibodies against MVH, AMH, Ki67, and c-Kit were visualized by confocal microscopy. Numbers of Oct4-GFP+ GC and Oct4-GFP− GC/tubule were counted using ImageJ. Data were analyzed using nonparametric one-way ANOVA. ResultsGC from E17-P4 were Oct4-GFP+. Numbers of Oct4-GFP− GC/tubule increased from P6–10, whereas Oct4-GFP+ GC/tubule numbers remained similar between P6 and P10. Sertoli cells proliferated from E17–P10, whereas GC only proliferated from P2. Gonocytes (Oct4-GFP+/c-Kit−) central in tubules migrated to the basement membrane to become prospermatogonia (Oct4-GFP+/c-Kit−) and then SSC (Oct4-GFP+/c-Kit+) from day 4 and further developed into Oct4-GFP−/c-Kit+ at P6. ConclusionIn Oct4-GFP mice both centrally located gonocytes and prospermatogonia located at the tubular basement membrane were Oct4-GFP+/c-Kit− before further developing into SSC (Oct4-GFP+/c-Kit+). This indicates that Oct4 is important in gonocyte transformation into SSC. Understanding this process will aid GC tumor diagnostics and fertility potential in boys with UDT undergoing orchidopexy.

Early Inflammatory Responses Following Cell Grafting in the CNS Trigger Activation of the Subventricular Zone: A Proposed Model of Sequential Cellular Events.

While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.

UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation.