Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 μL instillate volume can be introduced into mouse lung.
In mice that express SOD1 mutations found in human motor neuron disease, degeneration begins in the periphery for reasons that remain unknown. At the neuromuscular junction (NMJ), terminal Schwann cells (TSCs) have an intimate relationship with motor terminals and are believed to help maintain the integrity of the motor terminal. Recent evidence indicates that TSCs in some SOD1 mice exhibit abnormal functional properties, but other aspects of possible TSC involvement remain unknown. In this study, an analysis of TSC morphology and number was performed in relation to NMJ innervation status in mice which express the G93A SOD1 mutation. At P30, all NMJs of the fast medial gastrocnemius (MG) muscle were fully innervated by a single motor axon but 50% of NMJs lacked TSC cell bodies and were instead covered by the processes of Schwann cells with cell bodies located on the preterminal axons. NMJs in P30 slow soleus muscles were also fully innervated by single motor axons and only 5% of NMJs lacked a TSC cell body. At P60, about 25% of MG NMJs were denervated and lacked labeling for TSCs while about 60% of innervated NMJs lacked TSC cell bodies. In contrast, 96% of P60 soleus NMJs were innervated while 9% of innervated NMJs lacked TSC cell bodies. The pattern of TSC abnormalities found at P30 thus correlates with the pattern of denervation found at P60. Evidence from mice that express the G85R SOD1 mutation indicate that TSC abnormalities are not unique for mice that express G93A SOD1 mutations. These results add to an emerging understanding that TSCs may play a role in motor terminal degeneration and denervation in animal models of motor neuron disease.
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Suhasa B. Kodandaramaiah;
Gregory L. Holst;
Ian R. Wickersham;
Annabelle Singer;
Giovanni Talei Franzesi;
Michael L. McKinnon;
Craig R. Forest;
Edward S. Boyden
Whole-cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both suprathreshold spiking and subthreshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, which is a robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings in vivo. By following this protocol, a functional experimental rig for automated whole-cell patch clamping can be set up in 1 week. High-quality surgical preparation of mice takes ∼1 h, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it enables labs that are already doing in vivo patch clamping to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
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Megan F. Duffy;
Timothy J. Collier;
Joseph R. Patterson;
Christopher J. Kemp;
Kelvin C. Luk;
Malu Tansey;
Katrina L. Paumier;
Nicholas M. Kanaan;
Luke D. Fischer;
Nicole K. Polinski;
Olivia L. Barth;
Jacob W. Howe;
Nishant N. Vaikath;
Nour K. Majbour;
Omar M. A. El-Agnaf;
Caryl E. Sortwell
Background: Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson's disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS). Methods: Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis. Results: Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~35%) of substantia nigra pars compacta dopamine neurons within 5-6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum. Conclusions: Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD.
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Jeremy Epah;
Katalin Palfi;
Franziska Luise Dienst;
Pedro Felipe Malacarne;
Rolf Bremer;
Michael Salamon;
Sandeep Kumar;
Hanjoong Jo;
Christoph Schuermann;
Ralf Peter Brandes
Rationale: Classic histology is the gold standard for vascular network imaging and analysis. The method however is laborious and prone to artefacts. Here, the suitability of ultramicroscopy (UM) and micro-computed tomography (CT) was studied to establish potential alternatives to histology. Methods: The vasculature of murine organs (kidney, heart and atherosclerotic carotid arteries) was visualized using conventional 2D microscopy, 3D light sheet ultramicroscopy (UM) and micro-CT. Moreover, spheroid-based human endothelial cell vessel formation in mice was quantified. Fluorescently labeled Isolectin GS-IB4 A647 was used for in vivo labeling of vasculature for UM analysis, and analyses were performed ex vivo after sample preparation. For CT imaging, animals were perfused postmortem with radiopaque contrast agent. Results: Using UM imaging, 3D vascular network information could be obtained in samples of animals receiving in vivo injection of the fluorescently labeled Isolectin GS-IB4. Resolution was sufficient to measure single endothelial cell integration into capillaries in the spheroid-based matrigel plug assay. Because of the selective staining of the endothelium, imaging of larger vessels yielded less favorable results. Using micro-CT or even nano-CT, imaging of capillaries was impossible due to insufficient X-ray absorption and thus insufficient signal-to-noise ratio. Identification of lumen in murine arteries using micro-CT was in contrast superior to UM. Conclusion: UM and micro-CT are two complementary techniques. Whereas UM is ideal for imaging and especially quantifying capillary networks and arterioles, larger vascular structures are easier and faster to quantify and visualize using micro-CT. 3D information of both techniques is superior to 2D histology. UM and micro-CT together may open a new field of clinical pathology diagnosis.
The oxytocin receptor has been implicated in the regulation of reproductive physiology as well as social and emotional behaviors. The neurochemical mechanisms by which oxytocin receptor modulates social and emotional behavior remains elusive, in part because of a lack of sensitive and selective antibodies for cellular localization. To more precisely characterize oxytocin receptor-expressing neurons within the brain, we generated an oxytocin receptor-reporter mouse in which part of the oxytocin receptor gene was replaced with Venus cDNA (a variant of yellow fluorescent protein). Examination of the Venus expression revealed that, in the raphe nuclei, about one-half of tryptophan hydroxylase-immunoreactive neurons were positive for Venus, suggesting a potential role for oxytocin in the modulation of serotonin release. Oxytocin infusion facilitated serotonin release within the median raphe nucleus and reduced anxiety-related behavior. Infusion of a 5-HT 2A/2C receptor antagonist blocked the anxiolytic effect of oxytocin, suggesting that oxytocin receptor activation in serotonergic neurons mediates the anxiolytic effects of oxytocin. This is the first demonstration that oxytocin may regulate serotonin release and exert anxiolytic effects via direct activation of oxytocin receptor expressed in serotonergic neurons of the raphe nuclei. These results also have important implications for psychiatric disorders such as autism and depression in which both the oxytocin and serotonin systems have been implicated.
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Lihe Chen;
Jae Wook Lee;
Chung-Lin Chou;
Anil V. Nair;
Maria A. Battistone;
Teodor G. Paunescu;
Maria Merkulova;
Sylvie Breton;
Jill W. Verlander;
Susan M Wall;
Dennis Brown;
Maurice B. Burg;
Mark A. Knepper
Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
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Wilson C. Fok;
Yiqiang Zhang;
Adam B. Salmon;
Arunabh Bhattacharya;
Rakesh Gunda;
Dean Jones;
Walter Ward;
Kathleen Fisher;
Arlan Richardson;
Viviana I. Perez
Because rapamycin, an inhibitor of the nutrient sensor mammalian target of rapamycin, and dietary restriction both increase life span of mice, it has been hypothesized that they act through similar mechanisms. To test this hypothesis, we compared various biological parameters in dietary restriction mice (40% food restriction) and mice fed rapamycin (14 ppm). Both treatments led to a significant reduction in mammalian target of rapamycin signaling and a corresponding increase in autophagy. However, we observed striking differences in fat mass, insulin sensitivity, and expression of cell cycle and sirtuin genes in mice fed rapamycin compared with dietary restriction. Thus, although both treatments lead to significant downregulation of mammalian target of rapamycin signaling, these two manipulations have quite different effects on other physiological functions suggesting that they might increase life span through a common pathway as well as pathways that are altered differently by dietary restriction and rapamycin.