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The administration of the α2-adrenergic receptor agonist clonidine via intracerebroventricular route produces hypotension in pentobarbital-anesthetized rats and pressor responses in conscious normotensive rats. We explored the impact of different anesthetics on the central nervous system-dependent cardiovascular effects of clonidine. Normotensive male Wistar rats with guide cannulas previously implanted in the cerebroventricular system were anesthetized with pentobarbital or ketamine/xylazine and prepared for blood pressure measurement. The animals received intracerebroventricular injections of 10 μg clonidine or 0.6 μg dexmedetomidine, and the effects on the systolic, diastolic, mean arterial pressure, and heart rate were evaluated. The influence of 5 μg yohimbine, a selective α2-adrenergic receptor antagonist, was also assessed. The i.c.v. microinjection of clonidine decreased all three components of systemic arterial pressure and the heart rate of pentobarbital-anesthetized rats. On the other hand, clonidine increased the blood pressure and generated a less intense reduction in the heart rate of ketamine/xylazine-anesthetized rats. The pressor and bradycardic effects of clonidine in ketamine/xylazine-anesthetized animals were reproduced by dexmedetomidine, a more selective α2-adrenergic receptor agonist. Notably, the previous intracerebroventricular injection of yohimbine significantly inhibited the hypertensive effect of clonidine and dexmedetomidine. This study discloses that while normotensive rats anesthetized with pentobarbital show hypotensive responses, the stimulation of α2-adrenergic receptors increases the blood pressure in rats under ketamine/xylazine-induced anesthesia, reproducing the effects seen in conscious normotensive animals. Recognizing the mechanisms involved in these differences may allow us to understand better the final effects of clonidine and other α2-adrenergic receptor agonists in the central nervous system, contributing to the repurposing of these drugs. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation. [ABSTRACT FROM AUTHOR]

Genetically modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Here we present a fully automated system for microinjection into the pronucleus to facilitate genetic modification. We first developed software that automatically controls the microinjection system hardware. The software permits automatic rotation of the zygote to move the pronucleus to the injection pipette insertion position. We also developed software that recognizes the pronucleus in 3-dimensional coordinates so that the injection pipette can be automatically inserted into the pronucleus, and achieved a 94% insertion rate by linking the 2 pieces of software. Next, we determined the optimal solution injection conditions (30 hPa, 0.8–2.0 s) by examining the survival rate of injected zygotes. Finally, we produced transgenic (traditional DNA injection and piggyBac Transposon system) and knock-in (genomic editing) mice using our newly developed Integrated Automated Embryo Manipulation System (IAEMS). We propose that the IAEMS will simplify highly reproducible pronuclear stage zygote microinjection procedures. [ABSTRACT FROM AUTHOR]

In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and animal proteins. The aim of the present study was to develop the earthworms Eisenia fetida Waki and Eisenia andrei Sagami as next-generation animal protein production hosts. These earthworms have been approved as model animals for acute toxicity tests by the Organization for Economic Co-operation and Development, and they have post-translational modification systems. However, so far, none of the studies have used earthworm transfection techniques. Thus, we developed a transfection method for E. fetida and E. andrei using microinjection and electroporation systems. The maximum survival rates and transfection efficiencies were 79.2% and 29.2% for E. fetida, and 95.8% and 50.0% for E. andrei, respectively. Furthermore, human erythropoietin was detected in the transformed earthworm tail fragments using an enzyme-linked immunosorbent assay. These results contribute to the development of a potential earthworm-based novel animal protein production system. [ABSTRACT FROM AUTHOR]

Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Gram-positive Enterococcus faecalis protoplasts and gram-negative Lelliottia amnigena spheroplasts had large and small vacuoles inside the cytoplasm, respectively. Although no vacuoles were found at the early stage of cell enlargement, all enlarged cells used in the microinjection procedures had vacuoles. The plasma membrane of L. amnigena was more flexible than that of E. faecalis. In addition, E. faecalis protoplasts had unique discoidal structures as well as spherical structures in the cytoplasm. Our findings showed that the number of vacuoles increased as the L. amnigena plasma membrane expanded and that the size of vacuoles increased as the E. faecalis plasma membrane expanded, suggesting that bacterial cell enlargement involved vacuole generation. Thus, biosynthesis of the plasma and vacuolar membranes was synchronous with the bacterial cell enlargement. Differences in the plasma membrane flexibility might influence the different types of vacuole generation. [ABSTRACT FROM AUTHOR]

Aberrant epigenetic modification, including N6-methylation of adenosine (m6A), has been frequently reported in embryos derived from parthenogenetic activation (PA). However, the role of Igf2bp1 expression pattern in m6A modification and the mechanism through which Igf2bp1 function is regulated in PA embryos remains elusive. Therefore, in this study, using si-Igf2bp1 and betaine (N,N,N-trimethylglycine, a major methyl donor), we investigated the effect of Igf2bp1 expression in m6A modification on the development of PA embryos. The results indicated that the down-regulation of Igf2bp1 reduced the cleavage and blastula rates of PA embryos. Moreover, m6A expression level was markedly down-regulated following microinjection with si-Igf2bp1. However, the treatment with betaine could significantly restore the m6A level. Further bioinformatics analysis revealed Igf2bp1 as the putative target of microRNA 670 (miR-670). Thus, to confirm this finding, mimics and inhibitor of miR-670 were microinjected into PA embryos. The results demonstrated that miR-670 inhibitor augmented the expression of Igf2bp1 and rescued cleavage and blastula rates. In addition, the miR-670 inhibitor promoted the m6A expression level. TUNEL assay revealed a loss of expression of Igf2bp1 induced cell apoptosis in PA embryos. Taken together, these results demonstrated that miR-670-3p functions as the regulator of Igf2bp1 expression and plays a crucial role in PA development through m6A modification. [ABSTRACT FROM AUTHOR]

Background: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development. Results: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes. Conclusions: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies. [ABSTRACT FROM AUTHOR]

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy)-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy-related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders. [ABSTRACT FROM AUTHOR]

In mitochondria, compatibility of proteins encoded in mitochondrial DNA and nuclear DNA is essential for the normal functioning of the organelle. Incompatibility between mitochondrial and nuclear DNA can lead to dysfunctional respiration, mitochondrial diseases, and lethal problems, which suggests that the presence of heterologous mitochondria is unfavorable. In a previous study, we established a transplant method for DNA‐lacking mitochondria (mitosomes) in the anaerobic protozoan Entamoeba histolytica. In this study, interspecies transplant of mitosomes from E. histolytica into Entamoeba invadens, which is a parasitic protozoon of reptiles, was performed using the microinjection method at various temperatures and injection volumes. When E. invadens was used as recipient, it showed higher tolerance to a lower temperature and larger injection volume, in comparison with E. histolytica. After microinjection, donor mitosomes expressing HA‐tag conjugated protein were observed in recipient cells by immunofluorescent staining. The heterologous mitosomes‐injected cells proliferated and growth rate of the microinjected‐cells was similar to that of intact cells. Therefore, we conclude that interspecies transplant of DNA‐lacking mitochondria does not result in incompatibility. [ABSTRACT FROM AUTHOR]

The human insula has been consistently reported to be overactivated in all anxiety disorders, activation which has been suggested to be proportional to the level of anxiety and shown to decrease with effective anxiolytic treatment. Nonetheless, studies evaluating the direct role of the insula in anxiety are lacking. Here, we set out to investigate the role of the rodent insula in anxiety by either inactivating different insular regions via microinjections of glutamatergic AMPA receptor antagonist CNQX or activating them by microinjection of GABA receptor antagonist bicuculline in rats, before measuring anxiety-like behavior using the elevated plus maze. Inactivation of caudal and medial insular regions induced anxiogenic effects, while their activation induced anxiolytic effects. In contrast, inactivation of more rostral areas induced anxiolytic effects and their activation, anxiogenic effects. These results suggest that the insula in the rat has a role in the modulation of anxiety-like behavior in rats, showing regional differences; rostral regions have an anxiogenic role, while medial and caudal regions have an anxiolytic role, with a transition area around bregma +0.5. The present study suggests that the insula has a direct role in anxiety. [ABSTRACT FROM AUTHOR]

Abstract Optic neuritis (ON) is an inflammatory, demyelinating, neurodegenerative, and presently untreatable condition of the optic nerve which might induce blindness. We analyzed the effect of environmental enrichment (EE) on visual pathway damage provoked by experimental ON induced by a microinjection of bacterial lipopolysaccharide (LPS) into the optic nerve. For this purpose, LPS was microinjected into the optic nerve from male Wistar rats. After injection, one group of animals was submitted to EE, and another group remained in standard environment (SE) for 21 days. EE prevented the decrease in pupil light reflex (PLR), visual evoked potentials, retinal anterograde transport, phosphorylated neurofilament immunoreactivity, myelination (luxol fast blue staining), and axon (toluidine blue staining) and retinal ganglion cell (Brn3a-immunoreactivity) number. EE also prevented microglial/macrophage reactivity (Iba-1- and ED1-immunoreactivity), and astrocytosis (glial fibrillary acidic protein-immunostaining) induced by experimental ON. LPS-injected optic nerves displayed oxidative damage and increased inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-1β and TNFα mRNA levels which were prevented by EE. EE increased optic nerve brain-derived neurotrophic factor levels. When EE started at 4 (but not 7) days post-injection of LPS, a preservation of the PLR was observed at 21 days post-LPS, which was blocked by the daily administration of ANA-12 from day 4 to day 7 post-LPS. Moreover, EE from day 4 to day 7 post-LPS significantly preserved the PLR at 21 days post-injection. Taken together, our data suggest that EE preserved visual functions and reduced neuroinflammation of the optic nerve. This article is part of the Special Issue entitled "Neurobiology of Environmental Enrichment". Highlights • Environmental enrichment (EE) protects visual functions against optic neuritis. • EE protects retinal anterograde transport and prevents microglial reactivity. • EE prevents astrocytosis, demyelination, and axon and retinal ganglion cell loss. • EE housing behaves as an anti-inflammatory, anti-oxidative, and pro-BDNF therapy. • A TrkB receptor antagonist blocks the protective effect of EE on visual function. [ABSTRACT FROM AUTHOR]

Genome editing technology is becoming increasingly accepted as a way to improve traits in marine fish aquaculture. In fish, microinjection is a major method for introducing RNA or protein into eggs for genome editing; however, this method has not yet been established in aquaculture fish. We successfully established microinjection methods achieving high survival rates for tiger pufferfish and red sea bream by optimizing the following three parameters: (1) the soaking solution of fertilized eggs during microinjection, (2) the elapsed time from in vitro fertilization to microinjection, (3) the elapsed time from stripping to microinjection. In tiger pufferfish, Iwamatsu solution or diluted sea water is effective as the soaking solution. In vitro fertilization can be performed at intervals of 15 min from fertilization until 2.5 h after stripping. Similarly, in red sea bream, Leibovitz's L-15 medium or Iwamatsu solution is effective as the soaking solution and in vitro fertilization can be performed at intervals of 10 min from fertilization until 2.5 h after stripping. We anticipate our findings will contribute to effectively establish genome edited aquaculture breeds. [ABSTRACT FROM AUTHOR]

Abstract In the present work, various blend ratios of poly (lactic acid) (PLA), triallylisocyanurate (TAIC) and octavinyl-POSS (OvPOSS) were prepared by melt compounding in an Xplore microcompounder and molded by an Xplore microinjection molding device. POSS is a new generation organic/inorganic hybrid nanoparticle having possibility to improve the performance of polymers without sacrificing mechanical and thermal properties. TAIC and OvPOSS were used as crosslinking agents. After the compounding process, PLA, PLA/TAIC and PLA/OvPOSS composites were exposed to a gamma irradiation at various adsorbed doses ranging from 0 to 100 kGy. The properties of neat PLA, PLA/TAIC and PLA/OvPOSS composites were investigated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and tensile testing. Results showed that both chain scission reactions and formation of network structure occurred simultaneously after gamma irradiation. The highest gel formation was obtained from PLA composites including 3 wt% OvPOSS at the dose of 100 kGy. DSC results indicated that a sharp decrease in the degree of crystallinity (X c %) of PLA in the presence of TAIC and OvPOSS was obtained after gamma irradiation process. All irradiated composites exhibited lower elongation at break and higher E-Modulus values at any irradiation doses. TGA results showed that irradiated samples were more thermally stable than the unirradiated ones. Moreover, the heat deflection temperature (HDT) of PLA in the presence of TAIC and OvPOSS was found higher than that of neat PLA. Brittle surface morphology of composites after gamma irradiation process was obtained from SEM analysis. Graphical abstract fx1 Highlights • Octavinyl-POSS (OvPOSS) was found as an effective crosslinker for PLA. • PLA/OvPOSS composites exhibited higher tensile strength at any irradiation doses. • Irradiated PLA composites were found more thermally stable. [ABSTRACT FROM AUTHOR]

Abstract Microinjection has proven useful for introduction of low-permeability cryoprotective agents (CPAs) into fish eggs or embryos for cryopreservation. In this work, we examined the suitable conditions for single or combined microinjection into the perivitelline space (PS) and the yolk mass (YM) of embryos of the Japanese whiting, an alternative marine fish model for embryo cryopreservation studies. The parameters examined were injection volume, CPA type and concentration, vehicle (diluent), and suitable developmental stage. Somites and tail elongation embryos tolerated single or combined injection with 2.1 and 15.6 nl in the PS and YM, respectively, whereas earlier embryonic stages tolerated only up to 8.2 nl in the YM. The injected solutions diffused rapidly throughout the PS and YM and remained contained within each compartment unless in the case of structural damage caused by injection of larger volumes. Yamamoto solution was marginally better as a vehicle for microinjection of CPAs than fish Ringer and phosphate buffer saline whereas ¼ artificial sea water was clearly unsuitable. Ethylene glycol was well tolerated by embryos in all developmental stages whereas 1, 2-propylene glycol was suitable only for early embryonic stages. Overall, microinjection was efficient in delivering high loads of CPAs inside whiting embryos more swiftly than previously obtained for this species by immersion-based impregnation protocols. Embryos microinjected with CPAs showed a decrease in embryo nucleation temperature and an increase in chilling tolerance. CPA-microinjected embryos will provide valuable materials to optimize the remaining parameters that are critical for successful cryopreservation such as cooling and warming strategies. [ABSTRACT FROM AUTHOR]

Abstract Micro features on surfaces are critical for the functionality of a product, such as micro channels for a microfluidic chip and micro lens arrays for an artificial compound eye lens. In the present work, we design a series of microfluidic and microstructured surface patterns to evaluate the influence of variotherm and vacuum venting on replication integrity of micro features. By using a process monitoring system, including machine process and cavity pressure/temperature measurements, the influence of warm and cold circuit settings, and vacuum venting are characterised in terms of injection pressure, cavity pressure and mould–polymer interface temperature. These are then correlated with the replication integrity of micro features by assessing feature replication height, cross-section area and surface morphology. The performance of anti-stick coatings on a micro mould tool insert was also characterised in terms of moulding cycles. After a series experiments and optimisation, we have significantly enhanced feature replication with 100% replication of proposed features without either significant distortion or tearing, which cannot easily be achieved using conventional injection moulding. Additionally, we have successfully moulded features having good integrity and a maximum aspect ratio of up to 10 at a width of less than 8 μm over areas as large as 5.25 mm2. [ABSTRACT FROM AUTHOR]

Abstract Hemorrhages occurring within the thalamus lead to a pain syndrome. Clinical treatment of thalamic pain is ineffective, at least in part, due to the elusive mechanisms that underlie the induction and maintenance of thalamic pain. The present study investigated the possible contribution of a protein-protein interaction between postsynaptic density protein 95 (PSD-95) and neuronal nitric oxide synthase (nNOS) to thalamic pain in mice. Thalamic hemorrhage was induced by microinjection of type IV collagenase into unilateral ventral posterior medial/lateral nuclei of the thalamus. Pain hypersensitivities, including mechanical allodynia, heat hyperalgesia, and cold allodynia, appeared at day 1 post-microinjection, reached a peak 5–7 days post-microinjection, and persisted for at least 28 days post-microinjection on the contralateral side. Systemic pre-treatment (but not post-treatment) of ZL006, a small molecule that disrupts PSD-95-nNOS interaction, alleviated these pain hypersensitivities. This effect is dose-dependent. Mechanistically, ZL006 blocked the hemorrhage-induced increase of binding of PSD-95 with nNOS and membrane translocation of nNOS in thalamic neurons. Our findings suggest that the protein-protein interaction between PSD-95 and nNOS in the thalamus plays a significant role in the induction of thalamic pain. This interaction may be a promising therapeutic target in the clinical management of hemorrhage-induced thalamic pain. Highlights • The thalamus hemorrhagic mice exhibit hyperalgesia phenomena. • Pre-treatment of ZL006 attenuates hemorrhage-induced thalamic pain. • ZL006 takes effect by disrupting the PSD-95-nNOS protein-protein interaction in thalamic neurons. [ABSTRACT FROM AUTHOR]

The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression. [ABSTRACT FROM AUTHOR]

Highlights • Intra-VTA blockade of CB1 receptors, inhibited the acquisition and expression of nicotine-induced place preference. • Intra-VTA blockade of OX2 receptors, decreased the acquisition and expression of nicotine-induced place preference. • There is a probable interaction between orexinergic and endocannabinoid systems in the VTA in nicotine addiction. Abstract In this study, the role of orexin-2 (OX2) and cannabinoid-1 (CB1) receptors and their potential interaction within the ventral tegmental area (VTA) on nicotine-induced place preference, was examined in male rats. A 5-day conditioned place preference (CPP) paradigm was used. Nicotine (0.5 mg/kg; s.c.) induced a significant CPP, without any effect on the locomotor activity during the testing phase. TCS-OX2-29 (0.4, 0.8 and 4 μg/rat), as a selective OX2 receptor antagonist and AM251 (0.2, 1 and 2 μg/rat), as a selective cannabinoid CB1 receptor antagonist, individually or simultaneously were microinjected bilaterally into the VTA. The results showed that administration of AM251 (1 and 2 μg/rat) or TCS-OX2-29 (0.4, 0.8 and 4 μg/rat) into the VTA, during the 3-day conditioning phase or testing day, could dose-dependently inhibit the development of nicotine-induced CPP, in the acquisition or expression, respectively. Concurrent administration of ineffective doses of TCS-OX2-29 and AM251 into the VTA could not affect conditioning scores. The findings of this study support the possible role of OX2 and CB1 receptors in the VTA, in the acquisition and the expression of nicotine-induced place preference. Furthermore, our data suggest that there is a possible interaction between the VTA orexinergic and cannabinoid systems in nicotine-induced place preference. [ABSTRACT FROM AUTHOR]

There is an increasing demand for genetically modified mice produced without crossing, for rapid phenotypic screening studies at the organismal level. For this purpose, generation of completely embryonic-stem-cell (ESC)-derived chimeric mice without crossing is now possible using a microinjection or aggregation method with 3i culture medium. However, the microinjection of ESCs into blastocyst, morula, or 8-cell-stage embryos requires a highly skilled operator. The aggregation method is an easier alternative, but the conventional aggregation protocol still requires special skills. To make the aggregation method easier and more precise, here we developed a micro-aggregation device. Unlike conventional 3-dimensional culture, which uses hanging-drop devices for aggregation, we fabricated a polystyrene funnel-like structure to smoothly drop ESCs into a small area (300-μm in diameter) at the bottom of the device. The bottom area was designed so that the surface tension of the liquid-air interface prevents the cells from falling. After aggregation, the cells can be recovered by simply exerting pressure on the liquid from the top. The microdevice can be set upon a regular 96-well plate, so it is compatible with multichannel pipette use or machine operation. Using the microdevice, we successfully obtained chimeric blastocysts, which when transplanted resulted in completely ESC-derived chimeric mice with high efficiency. By changing the number of ESCs in the aggregate, we found that the optimum number of co-cultured ESCs was around 90~120 per embryo. Under this condition, the efficiency of generating completely ESC-derived mice was the same or better than that of the injection method. These results indicated that our microdevice can be used to produce completely ESC-derived chimeric mice easily and with a high success rate, and thus represents a promising alternative to the conventional microinjection or aggregation method, especially for high-throughput, parallel experimental applications. [ABSTRACT FROM AUTHOR]

Background: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. Methodology/Principal findings: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. Conclusion and significance: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions. [ABSTRACT FROM AUTHOR]

Background/Aims: Mitochondria (MT) and mitochondrial DNA (mtDNA) show maternal inheritance in most eukaryotic organisms; the sperm mtDNA is usually delivered to the egg during fertilization and then rapidly eliminated to avoid heteroplasmy, which can affect embryogenesis. In our previous study, fertilization-delivered sperm mtDNA exhibited late elimination and transcriptional quiescence in cyprinid fish embryos. However, the mechanisms underlying elimination and transcriptional quiescence of paternal mtDNA are unclear. Methods: Goldfish and zebrafish were used to investigate the fate of mtDNAs with different parental origins delivered by fertilization or microinjection in embryos. Goldfish MT from heart, liver and spermatozoa were microinjected into zebrafish zygotes, respectively. Specific PCR primers were designed so that the amplicons have different sizes to characterize goldfish and zebrafish cytb genes or their cDNAs. Results: The MT injection-delivered paternal mtDNA from sperm, as well as those from the heart and liver, was capable of persistence and transcription until birth, in contrast to the disappearance and transcriptional quiescence at the heartbeat stage of fertilization-delivered sperm mtDNA. In addition, the exogenous MT-injected zebrafish embryos have normal morphology during embryonic development. Conclusions: The fate of paternal mtDNA in fishes is dependent on the delivery strategy rather than the MT source, suggesting that the presence of sperm factor(s) is responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA. These findings provide insights into the mechanisms underlying paternal mtDNA fate and heteroplasmy in cyprinid fishes. [ABSTRACT FROM AUTHOR]

Abstract The manipulation of single cells and whole tissues has been possible since the early 70's, when semi-automatic injectors were developed. Since then, microinjection has been used to introduce an ever-expanding range of colloids of up to 1000 nm in size into living cells. Besides injecting nucleic acids to study transfection mechanisms, numerous cellular pathways have been unraveled through the introduction of recombinant proteins and blocking antibodies. The injection of nanoparticles has also become popular in recent years to investigate toxicity mechanisms and intracellular transport, and to conceive semi-synthetic cells containing artificial organelles. This article reviews colloidal systems such as proteins, nucleic acids and nanoparticles that have been injected into cells for different research aims, and discusses the scientific advances achieved through them. The colloids' intracellular processing and ultimate fate are also examined from a drug delivery perspective with an emphasis on the differences observed for endocytosed versus microinjected material. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Background and Objective: The most important alkaloid of opium family, morphine, may show unfavorable effect on the reproductive organs. This research investigated the effect of microinjection of morphine into the rat’s ventromedial hypothalamus (VMH) on cystic genesis in the ovary and the health of neurons of VMH. Materials and Methods: Female rats (Wistar, weighing 200-250 g) kept under standard conditions were cannulated under anesthesia by Stoelting stereotaxic instrument at the coordinates anterior-posterior: −1.92, ventral: 9, lateral: 0.5. After being recovered, they were microinjected single morphine (0.1-0.4 µg/rat, once intra-VMH) and/or naloxone hydrochloride (0.1-0.4 µg/rat, once intra-VMH) using a 5-µL Hamilton syringe with the polyethylene tubing. The control group solely given physiological saline (1 µL/rat, intra-VMH). Three days after the experiment, both ovary and brain samples were collected from the control and the experimental groups, and they were studied histopathologically. The brain samples were checked out with the aid of the cresyl violet, and the ovaries were stained by the hematoxylin and eosin. The samples were also biometrically examined to compare the ovaries’ cystic formations. Also, the number of healthy or injured neurons in the nuclei was compared. Results: The ovaries of morphine-treated rats showed polycystic characteristics in comparison with the control samples. In addition, the brain slices of the morphine-treated rats illustrated a significant decrease in intact neurons. Both mal effects were resolved in the presence of naloxone. Conclusion: These results may show that the morphine induces anovulatory infertility probably by hypothalamus–pituitary–ovary axis dysfunction. [ABSTRACT FROM AUTHOR]

Embryo micromanipulation was developed after introduction of microinjection to overcome infertility. Embryo micromanipulation may be performed at any embryo stage from pronuclear to blastocyst. The technique started out as basic and turned out to be increasingly more complex. Embryo micromanipulation at the cleavage-stage includes a wide range of techniques, from opening the zona pellucida in order to improve the chance of implantation, to removing detrimental components from the embryo to enhance embryo development or blastomeres for preimplantation genetic diagnosis and embryo splitting. Evaluating the impact(s) of different micromanipulation techniques on epigenetics of the embryo and considering quality control during these techniques are important issues in this regard. This review aims to discuss the micromanipulation of cleavage-stage embryos in clinical assisted reproductive technology (ART). Abbreviations: ART: assisted reproductive technology; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; PGD: preimplantation genetic diagnosis; PZD: partial zona dissection; ZP: zona pellucida; SUZI: subzonal insemination; PVS: perivitelline space; AH: assisted hatching; LAH: laserassisted hatching; ZT: zona thinning; UV: ultraviolet; IR: infrared; PCR: polymerase chain reaction; FISH: fluorescent in situ hybridization; NGS: next generation sequencing; QC: quality control; QA: quality assurance [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF), a neurotrophin traditionally associated with neural plasticity, has more recently been implicated in fluid balance and cardiovascular regulation. It is abundantly expressed in both the central nervous system (CNS) and peripheral tissue, and is also found in circulation. Studies suggest that circulating BDNF may influence the CNS through actions at the subfornical organ (SFO), a circumventricular organ (CVO) characterized by the lack of a normal blood-brain barrier (BBB). The SFO, well-known for its involvement in cardiovascular regulation, has been shown to express BDNF mRNA and mRNA for the TrkB receptor at which BDNF preferentially binds. This study was undertaken to determine if: (1) BDNF influences the excitability of SFO neurons in vitro; and (2) the cardiovascular consequences of direct administration of BDNF into the SFO of anesthetized rats. Electrophysiological studies revealed that bath application of BDNF (1 nmol/L) influenced the excitability of the majority of neurons (60%, n = 13/22), the majority of which exhibited a membrane depolarization (13.8 ± 2.5 mV, n = 9) with the remaining affected cells exhibiting hyperpolarizations (-11.1 ± 2.3 mV, n = 4). BDNF microinjections into the SFO of anesthetized rats caused a significant decrease in blood pressure (mean [area under the curve] AUC = -364.4 ± 89.0 mmHg 9 sec, n = 5) with no effects on heart rate (mean AUC = -12.2 ± 3.4, n = 5). Together these observations suggest the SFO to be a CNS site at which circulating BDNF could exert its effects on cardiovascular regulation. [ABSTRACT FROM AUTHOR]

Here we described an experimental protocol for in vivo imaging of macropinocytosis and subsequent intracellular events. By microinjection, we delivered fluorescence dextrans together with or without ATPγS into transparent Drosophila melanogaster embryos. Using a confocal microscope for live imaging, we monitored the generation of dextran-positive macropinosomes and subsequent intracellular events. Our protocol provides a continent and reliable way for investigating macropinocytosis and its underlying mechanisms, especially when combined with genetic strategies. [ABSTRACT FROM AUTHOR]

Objective: To study the regulation of orexin on the gastric afferent information in the septal nuclei of rats. Methods: 138 adult male Wistar rats weighed 250-300 g were selected, extracellular discharges of single unit neuron in the septal nuclei were used to identify the gastric distension(GD) sensitive neurons. Microinjection of orexin-A or orexin-A receptor antagonist SB334867 into septal nuclei was performed to explore the changes of GD sensitive neurons in the septal nuclei. The effects of different concentrations of orexin- A on the gastric motility of rats were observed. Results: The gastric motility experiments showed that administration of orexin-A in the septal nuclei could significantly increase the amplitude and frequency of gastric motility in a dose-dependent manner (P<0.05-0.01). Microinjection of SB-334867 could completely block the effect of orexin-A on the gastric motility. After the septal nuclei Microinjection of orexin-A, there were 36 GD excitatory (GD-E) neurons and 16 GD inhibitory (GD-I) neurons. The effects induced by orexin-A on GD sensitive neurons were completely abolished with administration of SB-334867. Conclusion: Injection of orexin into the septal nuclei could promote the gastric motility in rats and also change the discharge activity of gastric distention (GD) sensitive neurons. [ABSTRACT FROM AUTHOR]

To establish an entirely cerebrospinal fluid (CSF)-contacting nucleus-deficient model animal, we used cholera toxin B subunit (CB)- saporin (SAP), which is an analog of CB-HRP that specifically labels the CSF-contacting nucleus, to exclusively damage the nucleus. The effectiveness and specificity of the ablation were evaluated upon days 1-10 after CB-SAP microinjection into the brain ventricular system. The vital status, survival, and common physiological parameters of the model animals were also assessed during the experimental period. The results demonstrated that CB-SAP damaged only the CSF-contacting nucleus, but not other functional structures, in the brain. The complete ablation occurred by day 7 after CB-SAP microinjection. A model animal that had no CSF-contacting nucleus was established after survival beyond that time point. No obvious effects were observed in the vital status of the model animals, and their survival was ensured. The common physiological parameters of model animals were stable. The present study provides a method to establish a CSF-contacting nucleus "knockout" model animal, which is similar to a gene knockoutmodel animal for studying this particular nucleus in vivo. [ABSTRACT FROM AUTHOR]

The inferior colliculus (IC) is an important midbrain relay station for the integration of descending and ascending auditory information. Additionally, the IC has been implicated in processing sensorimotor responses. Glutamatergic and GABAergic manipulations in the IC can improve motor deficits as demonstrated by the animal model of haloperidol-induced catalepsy. However, how the IC influences motor function remains unclear. We investigated the effects of either intracollicular deep brain stimulation (DBS) or microinjection of the glutamatergic antagonist MK-801 or the agonist NMDA in C57BL/6J mice chronically treated with saline or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). After DBS or microinjections, the mice were submitted to rotarod and open field tests, respectively. DBS in the IC was effective to increase the time spent on the rotarod in MPTP-treated mice. After unilateral microinjection of MK-801, but not NMDA, MPTP-treated mice increased the distance travelled in the open field (p < 0.05). In conclusion, intracollicular DBS or MK-801 microinjection can improve motor performance in parkinsonian mice suggesting the IC as a new and non-conventional therapeutic target in motor impairment. [ABSTRACT FROM AUTHOR]

Micro-injection moulding (μIM) represents the only technology currently capable of enabling the mass production of polymer micro-components. Although this process is mainly utilized to process rigid thermoplastics, the development of new fields of application asks for the extension of the technology potential to novel types of polymeric materials such as soft thermoplastic elastomers (TPEs). In this work, the authors studied the μIM technology of TPE micro suspension rings for sensor applications. An initial benchmark study, based on microscopy inspections and weld line depth measurements, allowed identifying suitable process parameters settings. Then, the effects of the process parameters on the dimensional variation of the outer and inner diameter of the produced micro rings were quantified. A focus variation microscope and an optical coordinate measuring machine were employed for the measurements of parts and mould cavities respectively. The results of this study showed that the outer diameter of the rings was mostly affected by mould temperature and holding pressure, while the inner one depended mainly on mould and melt temperature. It was also found that the investigated process parameters had an opposite effect on the outer and inner diameter variations, posing great challenges in the achievement of the part geometry specified in the design. [ABSTRACT FROM AUTHOR]

The dorsal raphe nucleus (DRN) is a key site for 5-hydroxytryptamine (5-HT) synthesis and release. DRN dysfunction has been implicated in several stress-related disorders, including depression and anxiety. The lateral habenular nucleus (LHb) has been shown to inhibit the activity of DRN 5-HT neurons, and thus the LHb-DRN pathway plays an important role in the pathogenesis of depression. Although it is known that the LHb also receives the projection from the 5-HT neuron in the DRN, whether 5-HT neurons in the DRN can influence activity of the LHb in vivo and whether this effect is related to the induced behavioral changes have not been investigated. In the current study, we determined how injecting γ-aminobutyric acid (GABA) into the DRN to inhibit 5-HT neurons affected behavior and the changes in the activity of LHb neurons in mice. We found that GABA injection into the DRN induced depression-like behavior in mice, as indicated by increased immobility time, and decreased climbing time in the forced swimming test and the tail suspension test, decreased time spent in the center and total distance moved in the open field test. Using extracellular single unit recording, we showed that the firing rate of LHb neurons decreased after GABA microinjection into the DRN. Further, c-Fos expression in LHb neurons was inhibited. Together our results indicate that inhibition of DRN 5-HT neurons can cause decreased LHb activity and depression-like behavior in mice, however this depression-like behavior could be independent of the LHb activity. The observed decrease in LHb activity is probably due to the presence of a negative feedback loop between the DRN and the LHb, which may play a role in maintaining emotional homeostasis. [ABSTRACT FROM AUTHOR]

Plants respond to insect attack by releasing blends of volatile chemicals that attract their herbivores' specific natural enemies, while insect herbivores may carry endosymbiotic microorganisms that directly improve herbivore survival after natural enemy attack. Here we demonstrate that the two phenomena can be linked. Plants fed upon by pea aphids release volatiles that attract parasitic wasps, and the pea aphid can carry facultative endosymbiotic bacteria that prevent the development of the parasitic wasp larva and thus markedly improve aphid survival after wasp attack. We show that these endosymbionts also attenuate the systemic release of volatiles by plants after aphid attack, reducing parasitic wasp recruitment and increasing aphid fitness. Our results reveal a novel mechanism through which symbionts can benefit their hosts and emphasise the importance of considering the microbiome in understanding insect ecological interactions. [ABSTRACT FROM AUTHOR]

Background: The Midas cichlid species complex (Amphilophus spp.) is widely known among evolutionary biologists as a model system for sympatric speciation and adaptive phenotypic divergence within extremely short periods of time (a few hundred generations). The repeated parallel evolution of adaptive phenotypes in this radiation, combined with their near genetic identity, makes them an excellent model for studying phenotypic diversification. While many ecological and evolutionary studies have been performed on Midas cichlids, the molecular basis of specific phenotypes, particularly adaptations and their underlying coding and cis-regulatory changes have not yet been studied thoroughly. Results: For the first time in any New World cichlid, we use Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus). By adapting existing microinjection protocols, we established an effective protocol for transgenesis in Midas cichlids. Embryos were injected with a Tol2 plasmid construct that drives enhanced green fluorescent protein (eGFP) expression under the control of the ubiquitin promoter. The transgene was successfully integrated into the germline, driving strong ubiquitous expression of eGFP in the first transgenic Midas cichlid line. Additionally, we show transient expression of two further transgenic constructs, ubiquitin::tdTomato and mitfa::eGFP. Transgenesis in Midas cichlids will facilitate further investigation of the genetic basis of species-specific traits, many of which are adaptations. Conclusion: Transgenesis is a versatile tool not only for studying regulatory elements such as promoters and enhancers, but also for testing gene function through overexpression of allelic gene variants. As such, it is an important first step in establishing the Midas cichlid as a powerful model for studying adaptive coding and non-coding changes in an ecological and evolutionary context. [ABSTRACT FROM AUTHOR]

There is still a large gap in our understanding between the functional complexity of cells and the reconstruction of partial cellular functions in vitro from purified or engineered parts. Here we have introduced artificial vesicles of defined composition into living cells to probe the capacity of the cellular cytoplasm in dealingwith foreignmaterial and to develop tools for the directed manipulation of cellular functions. Our data showthat protein-free liposomes, after variable delay times, are captured by the Golgi apparatus that is reached either by random diffusion or, in the case of large unilamellar vesicles, by microtubuledependent transport via a dynactin/dynein motor complex. However, insertion of early endosomal SNARE proteins suffices to convert liposomes into trafficking vesicles that dock and fuse with early endosomes, thus overriding the default pathway to the Golgi. Moreover, such liposomes can be directed tomitochondria expressing simple artificial affinity tags, which can also be employed to divert endogenous trafficking vesicles. In addition, fusion or subsequent acidification of liposomes can be monitored by incorporation of appropriate chemical sensors. This approach provides an opportunity for probing and manipulating cellular functions that cannot be addressed by conventional genetic approaches. We conclude that the cellular cytoplasm has a remarkable capacity for self-organization and that introduction of such macromolecular complexes may advance nanoengineering of eukaryotic cells. [ABSTRACT FROM AUTHOR]

Ultrasonic micro injection molding was confirmed to be an efficient processing technique for the fabrication of a well-filled miniaturized dog-bone shaped specimen of ultra-high molecular weight polyethylene (UHMWPE). The influence of four process parameters on the filling phase of the reduced-size cavity was then analysed. It was established that it is possible to fabricate well-defined specimens when the highest ultrasonic amplitude is applied intermittently at specific intervals during the ultrasonic process to small compacted irregularly shaped UHMWPE samples and the mold temperature is set to 100 °C. GPC results showed a decrease in the molecular weight, which was the greatest when 100% of the ultrasonic amplitude was applied. The degree of crystallinity of the processed sample was increased because the reduction of the molecular weight. TGA showed that the thermal stability of UHMWPE fabricated by ultrasonic processing was not significantly influenced by the decrease in the molecular weight. FTIR spectra indicated oxidative degradation in three different regions of the processed UHMWPE specimen. Additionally, the band identified at the wavenumber 910 cm − 1 indicated a chain scission phenomenon the polymer experienced during the ultrasonic processing. [ABSTRACT FROM AUTHOR]

The delivery of exogenous biomolecules into teleost eggs is currently mostly relying on the manual microinjection methods, which, due to their high costs and low throughput, are not economically feasible for large-scale aquaculture applications. The main objective of this study was to develop a convenient, simple immersion delivery model for unfertilized Atlantic salmon ( Salmo salar L.) eggs using liposomes as a system for the delivery of selected molecules. We used a lipophilic marker, Rhodamine B isothiocyanate (RBITC) dye, to follow liposomal uptake, marker release and marker distribution within incubated egg. We investigated the influence of the incubation time, liposome surface charges and liposome sizes on the uptake into the eggs. After incubation with liposomes, the exposed eggs were fertilized and embryonic development was monitored until complete yolk sac absorption. The result showed that RBITC, was successfully delivered to the yolk of the incubated eggs. Moreover, during embryonic development, liposomal RBITC remained in the yolk sac until the yolk was completely absorbed. The findings demonstrated a novel approach for the delivery of exogenous molecules to unfertilized Atlantic salmon eggs, opening an avenue for large-scale aquaculture therapeutic applications. [ABSTRACT FROM AUTHOR]

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-β. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation. [ABSTRACT FROM AUTHOR]

Morphine, commonly used to relieve the acute or chronic pain, has a high potential for addiction and exerts rewarding effects via a critical role for mesolimbic dopamine system. Studies suggest that addiction-related behavior is highly associated with inflammatory immune response, but the mechanisms are poorly understood. The present study showed that intra-VTA microinjection of TLR4 antagonist LPS-RS prevented the acquisition and maintenance, but not the expression, of morphine-induced CPP in rats. In addition, chronic morphine treatment significantly activated STAT3 on day 6 and 11 in VTA, and bilateral microinjection of STAT3 inhibitor S3I-201 into the VTA suppressed the acquisition and maintenance of morphine-induced CPP in rats. Furthermore, local knockout of STAT3 by injection of the AAV-Cre-GFP into the VTA area of STAT3 flox/flox mice also significantly impaired the acquisition of morphine CPP. Importantly, the TLR4 expression is colocalized with p-STAT3-positive cell in VTA, and repeated injection of LPS-RS significantly attenuated the STAT3 activation in VTA induced by chronic morphine treatment. Collectively, these data suggest that TLR4/STAT3 signaling pathway in VTA might play a critical role in the acquisition and maintenance of morphine CPP, and provides new evidence that TLR4/STAT3 signaling pathway might be a potential target for treatment of morphine addiction. [ABSTRACT FROM AUTHOR]

The transition from wakefulness to general anesthesia is widely attributed to suppressive actions of anesthetic molecules distributed by the systemic circulation to the cerebral cortex (for amnesia and loss of consciousness) and to the spinal cord (for atonia and antinociception). An alternative hypothesis proposes that anesthetics act on one or more brainstem or diencephalic nuclei, with suppression of cortex and spinal cord mediated by dedicated axonal pathways. Previously, we documented induction of an anesthesia-like state in rats by microinjection of small amounts of GABAA-receptor agonists into an upper brainstem region named the mesopontine tegmental anesthesia area (MPTA). Correspondingly, lesioning this area rendered animals resistant to systemically delivered anesthetics. Here, using rats of both sexes, we applied a modified microinjection method that permitted localization of the anesthetic-sensitive neurons with much improved spatial resolution. Microinjected at the MPTA hotspot identified, exposure of 1900 or fewer neurons to muscimol was sufficient to sustain whole-body general anesthesia; microinjection as little as 0.5 mm off-target did not. The GABAergic anesthetics pentobarbital and propofol were also effective. The GABA-sensitive cell cluster is centered on a tegmental (reticular) field traversed by fibers of the superior cerebellar peduncle. It has no specific nuclear designation and has not previously been implicated in brain-state transitions. [ABSTRACT FROM AUTHOR]

Cholinergic mechanisms in supramammillary nucleus (SuM), especially the lateral SuM (lSuM) modulates septo-hippocampal neural activity. The lSuM, as compared to the contiguous medial SuM (mSuM) has relatively dense projections to hippocampus and cingulate cortex (Cg). In the present study, we have investigated whether the effects of cholinergic activation of SuM on hippocampal and cortical neural activities involve a cooperative interaction with the medial septum (MS). Microinjection of the broadspectrum cholinergic agonist, carbachol, or the cholinergic-nicotinic receptor agonist, nicotine, into the lSuM and the mSuM in urethane anesthetized rat evoked a similar pattern of hippocampal theta rhythm. Despite that, only the lSuM microinjections resulted in an increase in expression of c-Fos-like immunoreactivity (c-Fos-ir) in neurons, including interneurons, of the ipsilateral hippocampus with a very dense expression in dentate gyrus. Likewise, a robust induction of c-Fos-ir was also observed in the ipsilateral Cg. Inhibition of the MS with muscimol pre-treatment attenuated both carbachol-evoked c-Fos-ir and theta activation. The findings indicate that cholinergic- nicotinic mechanisms in lSuM evoke not only neural activation via the ascending synchronizing pathway but also an MS-modulated expression of the plasticity-related molecule c-Fos in cortical regions that are strongly innervated by the lSuM. [ABSTRACT FROM AUTHOR]

The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. [ABSTRACT FROM AUTHOR]

Extinction of conditioned fear has been suggested to be a new form of learning instead of erasure of what was originally learned, and the process is NMDA ( N -methyl d -aspartate) receptor (NMDAR) dependent. Most of studies have so far revealed the important roles of NMDARs in the amygdala and medial prefrontal cortex (mPFC) in cued fear extinction. Although the ventral hippocampus has intimately reciprocal connections with the amygdala and mPFC, the role of its NMDARs in cued fear extinction remains unclear. The present experiment explored the issue by bilateral pre-extinction microinjection of the noncompetitive NMDAR antagonist MK-801 into the ventral hippocampus. Four groups of rats were given habituation, tone cued fear conditioning, fear extinction training and extinction test. Prior to extinction training, rats received bilateral infusions of either MK-801 (1.5, 3, or 6 μg/0.5 μl) or saline. Our results showed that MK-801 reduced freezing on the first trial of extinction training with no impact on within-session acquisition of extinction, and that the lower doses of MK-801 resulted in increased freezing on the extinction retrieval test. These findings suggest that ventral hippocampal NMDARs are necessary for the consolidation of tone cued fear extinction. [ABSTRACT FROM AUTHOR]

Heretofore, transgenesis in the parasitic nematode genus Strongyloides has relied on microinjecting transgene constructs into gonadal syncytia of free-living females. We now report transgenesis in Strongyloides stercoralis by microinjecting constructs into the syncytial testes of free-living males. Crosses of individual males microinjected with a construct encoding GFP with cohorts of 12 non-injected females produced a mean of 7.28 ± 2.09 transgenic progeny. Progeny of males and females microinjected with distinct reporter constructs comprised 2.6% ± 0.7% of individuals expressing both paternal and maternal transgenes. Implications of this finding for deployment of CRISPR/Cas9 mutagenesis in Strongyloides spp. are discussed. [ABSTRACT FROM AUTHOR]

In order to elucidate whether cytokine mechanisms of the cingulate cortex (cctx) are important in the central regulation of homeostasis, in the present study, feeding-metabolic effects of direct bilateral microinjection of interleukin-1β (IL-1β) into the cctx of the rat have been investigated. Short- (2 h), medium (12 h) and long-term (24 h) food and water intakes and body temperature were measured after the intracerebral administration of this primary cytokine or vehicle solution, with or without paracetamol pretreatment. The effect of IL-1β on the blood glucose level of animals was examined in glucose tolerance test (GTT), and concentrations of relevant plasma metabolites (total cholesterol, HDL, LDH, triglycerides, uric acid) were additionally also determined following the above microinjections. In contrast to causing no major alteration in the food and water intakes, the cytokine treatment evoked significant increase in the body temperature of the rats. Prostaglandin-mediated mechanisms were shown to have important role in the mode of this action of IL-1β, since paracetamol pretreatment partially prevented the development of the above mentioned hyperthermia. In the GTT, no considerable difference was observed between the blood glucose levels of the cytokine treated and control animals. Following IL-1β microinjection, however, significant decrease of HDL and total cholesterol was found. Our present findings indicate that elucidating the IL-1β mediated homeostatic control mechanisms in the cingulate cortex may lead to the better understanding not only the regulatory entities of the healthy organism but also those found in obesity, diabetes mellitus and other worldwide rapidly spreading feeding-metabolic disorders. [ABSTRACT FROM AUTHOR]

This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7. Those 3 mMt pigs had a 4 bp deletion, 5 bp insertion, or 7 bp insertion at site I, and the bMt pig had 5 types of mutations at cleavage sites I and III. The expression of alpha-Gal on the bMt peripheral blood mononuclear cells (PBMCs) was reduced, and survival rate of bMt PBMCs was maintained as indicated by results of cultivation with sera of humans or Formosan Macaques. We concluded that mutant pigs could be generated by direct pronuclear microinjection of ppU6-GGTA1-gRNA CRISPR vectors with the pCX-Flag2-NLS1-Cas9-NLS2plasmid and that the 4-site strategy has a better mutant efficiency. Porcine U6 promoter was firstly used to express KO vectors and effectively generate mutant pigs, worthily to adopt for future KO studies. [ABSTRACT FROM AUTHOR]

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 h, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB. [ABSTRACT FROM AUTHOR]

The importance of neurons in the nucleus of the solitary tract (NTS) in the production of coughing was tested by microinjections of the nonspecific glutamate receptor antagonist kynurenic acid (kyn; 100 mM in artificial cerebrospinal fluid) in 15 adult spontaneously breathing anesthetized cats. Repetitive coughing was elicited by mechanical stimulation of the intrathoracic airway. Electromyograms (EMG) were recorded from inspiratory parasternal and expiratory transversus abdominis (ABD) muscles. Bilateral microinjections of kyn into the NTS rostral to obex [55 ± 4 nl total in 2 locations (n = 6) or 110 ± 4 nl total in 4 locations (n = 5)], primarily the ventrolateral subnucleus, reduced cough number and expiratory cough efforts (amplitudes of ABD EMG and maxima of esophageal pressure) compared with control. These microinjections also markedly prolonged the inspiratory phase, all cough-related EMG activation, and the total cough cycle duration as well as some other cough-related time intervals. In response to microinjections of kyn into the NTS rostral to the obex respiratory rate decreased, and there were increases in the durations of the inspiratory and postinspiratory phases and mean blood pressure. However, bilateral microinjections of kyn into the NTS caudal to obex as well as control vehicle microinjections in the NTS location rostral to obex had no effect on coughing or cardiorespiratory variables. These results are consistent with the existence of a critical component of the cough rhythmogenic circuit located in the rostral ventral and lateral NTS. Neuronal structures of the rostral NTS are significantly involved specifically in the regulation of cough magnitude and phase timing. NEW & NOTEWORTHY The nucleus of the solitary tract contains significant neuronal structures responsible for control of 1) cough excitability, 2) motor drive during cough, 3) cough phase timing, and 4) cough rhythmicity. Significant elimination of neurons in the solitary tract nucleus results in cough apraxia (incomplete and/or disordered cough pattern). The mechanism of the cough impairment is different from that for the concomitant changes in breathing. [ABSTRACT FROM AUTHOR]

The Betaproteobacteria - Euplotes association is an obligatory symbiotic system involving a monophyletic group of ciliate species and two betaproteobacteria species which can be alternatively present. Recent data showed that this relationship has been established more than once and that several symbiont-substitution events took place, revealing a complex and intriguing evolutionary path. Due to the different evolutionary pathways followed by the different symbionts, each bacterial strain could have differentially evolved and/or lost functional traits. Therefore, we performed re-infection experiments, both by phagocytosis and by microinjection, to test the possible functional role of the different bacteria towards the ciliates. Our results confirm that the growth capacity of the host is indissolubly linked to the presence of its original symbionts. Results of the attempts of re-infection by phagocytosis showed that none of the bacteria is able to successfully colonize the host cytoplasm in this way, even if regularly ingested. Re-infection by microinjection succeed only in one case. Such results point to a high degree of specificity in the interactions between bacteria and Euplotes even after the invasion step. Due to a co-evolutive pathway of reciprocal adaptation, different degree of re-colonization ability could have been conserved by the different species and strains of the symbionts. [ABSTRACT FROM AUTHOR]

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster is a well-established model organism to understand biological processes and study human diseases at the molecular-genetic level. The central nervous system (CNS) of Drosophila larvae is widely used as a model to study neuron development and network formation. This has been achieved by using various genetic manipulation tools such as microinjection to knock down certain genes or over-express proteins for visualizing the cellular activities. However, visualization of an intact-live neuronal response in larva's Central Nervous System (CNS) is challenging due to robust digging/burrowing behaviour that impedes neuroimaging. To address this problem, dissection is used to isolate and immobilize the CNS from the rest of the body. In order to obtain a true physiological response from the Drosophila CNS, it is important to avoid dissection, while the larva should be kept immobilized. In this paper, a series of microfluidic clamps were investigated for intact immobilization of the larva. As a result, an optimized structure for rapid mechanical immobilization of Drosophila larvae for CNS imaging was determined. The clamping and immobilization processes were characterized by imaging and movement measurement of the CNS through the expression of genetically encoded Calcium sensor GCaMP5 in all sensory and cholinergic interneurons. The optimal structure that included two 3D constrictions inside a narrowed channel considerably reduced the internal CNS capsule movements. It restricts the CNS movement to 10% of the motion from a glued larva and allows motion of only 10±30 lm over 350 s immobilization which was sufficient for CNS imaging. These larva-on-a-chip platforms can be useful for studying CNS responses to sensory cues such as sound, light, chemosensory, tactile, and electric/magnetic fields. [ABSTRACT FROM AUTHOR]