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Journal articles on the topic 'Retrograde viral tracing'
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1
Banfield, Bruce W., Jessica D. Kaufman, Jessica A. Randall, and Gary E. Pickard. "Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications." Journal of Virology 77, no. 18 (2003): 10106–12. http://dx.doi.org/10.1128/jvi.77.18.10106-10112.2003.
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ABSTRACT The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.
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Rouiller, Eric M., Mauricette Capt, Michel Dolivo, and Francois De Ribaupierre. "Tensor tympani reflex pathways studied with retrograde horseradish peroxidase and transneuronal viral tracing techniques." Neuroscience Letters 72, no. 3 (1986): 247–52. http://dx.doi.org/10.1016/0304-3940(86)90521-5.
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Streefland, Cerien, Frans W. Maes, and Béla Bohus. "Autonomic brainstem projections to the pancreas: a retrograde transneuronal viral tracing study in the rat." Journal of the Autonomic Nervous System 74, no. 2-3 (1998): 71–81. http://dx.doi.org/10.1016/s0165-1838(98)00047-2.
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Song, C. Kay, Gary J. Schwartz, and Timothy J. Bartness. "Anterograde transneuronal viral tract tracing reveals central sensory circuits from white adipose tissue." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 296, no. 3 (2009): R501—R511. http://dx.doi.org/10.1152/ajpregu.90786.2008.
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The origins of the sympathetic nervous system (SNS) innervation of white adipose tissue (WAT) have been defined using the transneuronal viral retrograde tract tracer, pseudorabies virus. Activation of this SNS innervation is acknowledged as the principal initiator of WAT lipolysis. The central control of WAT lipolysis may require neural feedback to a brain-SNS-WAT circuit via WAT afferents. Indeed, conventional tract tracing studies have demonstrated that peripheral pseudounipolar dorsal root ganglion (DRG) sensory cells innervate WAT. The central nervous system projections of WAT afferents remain uncharted, however, and form the focus of the present study. We used the H129 strain of the herpes simplex virus-1 (HSV-1), an anterograde transneuronal viral tract tracer, to define the afferent circuits projecting from WAT to the central nervous system. Siberian hamster inguinal (IWAT) or epididymal WAT was injected with H129 and the neuraxis processed for HSV-1 immunoreactivity. We found substantial overlap in the pattern of WAT sensory afferent projections with multiple SNS outflow sites along the neuraxis, suggesting the possibility of WAT sensory-SNS circuits that could regulate WAT SNS drive and thereby lipolysis. Previously, we demonstrated that systemic 2-deoxy-d-glucose (2DG) elicited increases in the SNS drive to IWAT. Here, we show that systemic 2DG administration also significantly increases multiunit spike activity arising from decentralized IWAT afferents. Collectively, these data provide structural and functional support for the existence of a sensory WAT pathway to the brain, important in the negative feedback control of lipid mobilization.
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Rouiller, Eric M., Mauricette Capt, Michel Dolivo, and Francois De Ribaupierre. "Neuronal organization of the stapedius reflex pathways in the rat: a retrograde HRP and viral transneuronal tracing study." Brain Research 476, no. 1 (1989): 21–28. http://dx.doi.org/10.1016/0006-8993(89)91532-1.
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Hanchate, Naresh K., Eun Jeong Lee, Andria Ellis, et al. "Connect-seq to superimpose molecular on anatomical neural circuit maps." Proceedings of the National Academy of Sciences 117, no. 8 (2020): 4375–84. http://dx.doi.org/10.1073/pnas.1912176117.
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The mouse brain contains about 75 million neurons interconnected in a vast array of neural circuits. The identities and functions of individual neuronal components of most circuits are undefined. Here we describe a method, termed “Connect-seq,” which combines retrograde viral tracing and single-cell transcriptomics to uncover the molecular identities of upstream neurons in a specific circuit and the signaling molecules they use to communicate. Connect-seq can generate a molecular map that can be superimposed on a neuroanatomical map to permit molecular and genetic interrogation of how the neuronal components of a circuit control its function. Application of this method to hypothalamic neurons controlling physiological responses to fear and stress reveals subsets of upstream neurons that express diverse constellations of signaling molecules and can be distinguished by their anatomical locations.
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Li, Dong, Hong Yang, Feng Xiong, et al. "Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives." International Journal of Molecular Sciences 21, no. 16 (2020): 5937. http://dx.doi.org/10.3390/ijms21165937.
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Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.
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Campbell, Rebecca E., and Allan E. Herbison. "Definition of Brainstem Afferents to Gonadotropin-Releasing Hormone Neurons in the Mouse Using Conditional Viral Tract Tracing." Endocrinology 148, no. 12 (2007): 5884–90. http://dx.doi.org/10.1210/en.2007-0854.
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Brainstem monoamines have long been considered to play a role in regulating the activity of GnRH neurons, although their neuroanatomical relationship with these cells has remained unclear. Using a Cre-dependent pseudorabies virus (Ba2001) technique that permits retrograde tracing selectively from GnRH neurons in the mouse, we have examined the organization of brainstem inputs to rostral preoptic area (rPOA) GnRH neurons. Two days after injection of Ba2001 into the rPOA of adult female GnRH-Cre transgenic mice, five to nine GnRH neurons located immediately adjacent to the injection site were found to express green fluorescent protein (GFP), the marker of virus infection, with no GFP expression anywhere else in the brain. In mice killed 24 h later (3 d after injection), GFP-expressing cells were identified (in order of density) in the raphe nuclei, periaqueductal grey, locus coeruleus, nucleus tractus solitarius, and area postrema. This time course is compatible with these neurons representing primary afferent inputs to the GnRH neurons. Four and 6 d after Ba2001 injection, GFP-expressing cells were found in additional brain regions. Dual-label immunofluorescence experiments in 3-d postinjection mice demonstrated that 100% of GFP-expressing neurons in the raphe were positive for tryptophan hydroxylase, whereas 100% and approximately 50% of GFP neurons in the locus coeruleus and nucleus tractus solitarius, respectively, expressed tyrosine hydroxylase. These observations demonstrate that rPOA GnRH neurons receive direct projections from brainstem A2 and A6 noradrenergic neurons and that, surprisingly, the largest afferent input from the brainstem originates from raphe serotonin neurons in the mouse.
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Krieger, Jean-Philippe, Ellen Paula Santos da Conceição, Graciela Sanchez-Watts, et al. "Glucagon-like peptide-1 regulates brown adipose tissue thermogenesis via the gut-brain axis in rats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 315, no. 4 (2018): R708—R720. http://dx.doi.org/10.1152/ajpregu.00068.2018.
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Endogenous intestinal glucagon-like peptide-1 (GLP-1) controls satiation and glucose metabolism via vagal afferent neurons (VANs). Recently, VANs have received increasing attention for their role in brown adipose tissue (BAT) thermogenesis. It is, however, unclear whether VAN GLP-1 receptor (GLP-1R) signaling affects BAT thermogenesis and energy expenditure (EE) and whether this VAN mechanism contributes to energy balance. First, we tested the effect of the GLP-1R agonist exendin-4 (Ex4, 0.3 μg/kg ip) on EE and BAT thermogenesis and whether these effects require VAN GLP-1R signaling using a rat model with a selective Glp1r knockdown (kd) in VANs. Second, we examined the role of VAN GLP-1R in energy balance during chronic high-fat diet (HFD) feeding in VAN Glp1r kd rats. Finally, we used viral transsynaptic tracers to identify the possible neuronal substrates of such a gut-BAT interaction. VAN Glp1r kd attenuated the acute suppressive effects of Ex4 on EE and BAT thermogenesis. Consistent with this finding, the VAN Glp1r kd increased EE and BAT activity, diminished body weight gain, and improved insulin sensitivity compared with HFD-fed controls. Anterograde transsynaptic viral tracing of VANs infected major hypothalamic and hindbrain areas involved in BAT sympathetic regulation. Moreover, retrograde tracing from BAT combined with laser capture microdissection revealed that a population of VANs expressing Glp1r is synaptically connected to the BAT. Our findings reveal a novel role of VAN GLP-1R signaling in the regulation of EE and BAT thermogenesis and imply that through this gut-brain-BAT connection, intestinal GLP-1 plays a role in HFD-induced metabolic syndrome.
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Tsetsenis, Theodoros, Julia K. Badyna, Julianne A. Wilson, et al. "Midbrain dopaminergic innervation of the hippocampus is sufficient to modulate formation of aversive memories." Proceedings of the National Academy of Sciences 118, no. 40 (2021): e2111069118. http://dx.doi.org/10.1073/pnas.2111069118.
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Aversive memories are important for survival, and dopaminergic signaling in the hippocampus has been implicated in aversive learning. However, the source and mode of action of hippocampal dopamine remain controversial. Here, we utilize anterograde and retrograde viral tracing methods to label midbrain dopaminergic projections to the dorsal hippocampus. We identify a population of midbrain dopaminergic neurons near the border of the substantia nigra pars compacta and the lateral ventral tegmental area that sends direct projections to the dorsal hippocampus. Using optogenetic manipulations and mutant mice to control dopamine transmission in the hippocampus, we show that midbrain dopamine potently modulates aversive memory formation during encoding of contextual fear. Moreover, we demonstrate that dopaminergic transmission in the dorsal CA1 is required for the acquisition of contextual fear memories, and that this acquisition is sustained in the absence of catecholamine release from noradrenergic terminals. Our findings identify a cluster of midbrain dopamine neurons that innervate the hippocampus and show that the midbrain dopamine neuromodulation in the dorsal hippocampus is sufficient to maintain aversive memory formation.
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Csáki, Ágnes, Katalin Köves, Zsolt Boldogkői, Dóra Tombácz, and Zsuzsanna E. Tóth. "The Same Magnocellular Neurons Send Axon Collaterals to the Posterior Pituitary and Retina or to the Posterior Pituitary and Autonomic Preganglionic Centers of the Eye in Rats." NeuroSci 2, no. 1 (2021): 27–44. http://dx.doi.org/10.3390/neurosci2010002.
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In rats, some parvocellular paraventricular neurons project to spinal autonomic centers. Using the virus tracing technique, we have demonstrated that some magnocellular paraventricular neurons, but not supraoptic neurons, also project to autonomic preganglionic centers of the mammary gland, gingiva, or lip. A part of these neurons has shown oxytocin immunoreactivity. In the present experiment, we have examined whether the same magnocellular neuron that sends fibers to the retina or autonomic preganglionic centers of the eye also projects to the posterior pituitary. Double neurotropic viral labeling and oxytocin immunohistochemistry were used. After inoculation of the posterior pituitary and the eye with viruses, spreading in a retrograde direction and expressing different fluorescence proteins, we looked for double-labeled neurons in paraventricular and supraoptic nuclei. Double-labeled neurons were observed in non-sympathectomized and cervical-sympathectomized animals. Some double-labeled neurons contained oxytocin. After the optic nerve was cut, the labeling did not appear in the supraoptic nucleus; however, it could still be observed in the paraventricular nucleus. In the paraventricular nucleus, the double-labeled cells may be the origin of centrifugal visual fibers or autonomic premotor neurons. In the supraoptic nucleus, all double-labeled neurons are cells of origin of centrifugal visual fibers.
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Li, Sa, Xinwen Dong, and Gilbert J. Kirouac. "Extensive divergence of projections to the forebrain from neurons in the paraventricular nucleus of the thalamus." Brain Structure and Function 226, no. 6 (2021): 1779–802. http://dx.doi.org/10.1007/s00429-021-02289-6.
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AbstractNeurons in the paraventricular nucleus of the thalamus (PVT) respond to emotionally salient events and project densely to subcortical regions known to mediate adaptive behavioral responses. The areas of the forebrain most densely innervated by the PVT include striatal-like subcortical regions that consist of the shell of the nucleus accumbens (NAcSh), the dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral-capsular division of the central nucleus of the amygdala (CeL). A recent tracing experiment demonstrated that the PVT is composed of two intermixed populations of neurons that primarily project to either the dorsomedial (dmNAcSh) or ventromedial region of the NAcSh (vmNAcSh) with many of the vmNAcSh projecting neurons providing collateral innervation of the BSTDL and CeL. The present study used triple injections of the retrograde tracer cholera toxin B to provide a detailed map of the location of PVT neurons that provide collaterals to the vmNAcSh, BSTDL and CeL. These neurons were intermixed throughout the PVT and did not form uniquely localized subpopulations. An intersectional viral anterograde tracing approach was used to demonstrate that regardless of its presumed target of innervation (dmNAcSh, vmNAcSh, BSTDL, or CeL), most neurons in the PVT provide collateral innervation to a common set of forebrain regions. The paper shows that PVT-dmNAcSh projecting neurons provide the most divergent projection system and that these neurons express the immediate early gene product cFos following an aversive incident. We propose that the PVT may regulate a broad range of responses to physiological and psychological challenges by simultaneously influencing functionally diverse regions of the forebrain that include the cortex, striatal-like regions in the basal forebrain and a number of hypothalamic nuclei.
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Antinone, Sarah Elizabeth, and Gregory Allan Smith. "Retrograde Axon Transport of Herpes Simplex Virus and Pseudorabies Virus: a Live-Cell Comparative Analysis." Journal of Virology 84, no. 3 (2009): 1504–12. http://dx.doi.org/10.1128/jvi.02029-09.
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ABSTRACT Upon entry, neuroinvasive herpesviruses traffic from axon terminals to the nuclei of neurons resident in peripheral ganglia, where the viral DNA is deposited. A detailed analysis of herpes simplex virus type 1 (HSV-1) transport dynamics in axons following entry is currently lacking. Here, time lapse fluorescence microscopy was used to compare the postentry viral transport of two neurotropic herpesviruses: HSV-1 and pseudorabies virus (PRV). HSV-1 capsid transport dynamics were indistinguishable from those of PRV and did not differ in neurons of human, mouse, or avian origin. Simultaneous tracking of capsids and tegument proteins demonstrated that the composition of actively transporting HSV-1 is remarkably similar to that of PRV. This quantitative assessment of HSV-1 axon transport following entry demonstrates that HSV-1 and PRV share a conserved mechanism for postentry retrograde transport in axons and provides the foundation for further studies of the retrograde transport process.
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Kulik, Paweł, Anna Zacharko-Siembida, and Marcin B. Arciszewski. "The localization of primary efferent sympathetic neurons innervating the porcine thymus – a retrograde tracing study." Acta Veterinaria Brno 86, no. 2 (2017): 117–22. http://dx.doi.org/10.2754/avb201786020117.
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The autonomic nervous system is a sophisticated and independent structure composed of two antagonistic (opposing) divisions (sympathetic and parasympathetic) that control many vital functions including: homeostasis maintenance, heart rate, blood circulation, secretion, etc. Thymus is one of the most important primary lymphoid organs playing a role in the developing of a juvenile’s immune system mainly by maturation, development, and migration of T-cells (T lymphocytes). In the last decades, several studies identifying sources of the thymic autonomic supply have been undertaken in humans and several laboratory rodents but not in higher mammals such as the pig. Therefore, in the present work, retrograde tracing technique of Fast Blue and DiI was used to investigate the sources of sympathetic efferent supply to the porcine thymus. After Fast Blue injection into the right lobe of the thymus, the presence of Fast Blue-positive neurons was found in the unilateral cranial cervical ganglion (82.8 ± 3.0% of total Fast Blue-positive neurons) as well as in the middle cervical ganglion (17.2 ± 3.0%). Injection of DiI resulted in the presence of retrograde tracer in neurons of the cranial cervical ganglion (80.4 ± 2.3% of total amount of DiI-labelled neurons), the middle cervical ganglion (18.4 ± 1.9%), and the cervicothoracic ganglion (1.2 ± 0.8%). The present report provides the first data describing in details the localization of primary efferent sympathetic neurons innervating the porcine thymus.
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Klingen, Yvonne, Karl-Klaus Conzelmann, and Stefan Finke. "Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport." Journal of Virology 82, no. 1 (2007): 237–45. http://dx.doi.org/10.1128/jvi.01342-07.
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ABSTRACT Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.
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Jovasevic, Vladimir, Mojgan H. Naghavi, and Derek Walsh. "Microtubule plus end–associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells." Journal of Cell Biology 211, no. 2 (2015): 323–37. http://dx.doi.org/10.1083/jcb.201505123.
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Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end–tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170–mediated capture to initiate transport in primary human cells.
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Scherer, Julian, Zachary A. Yaffe, Michael Vershinin, and Lynn W. Enquist. "Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics." Journal of Virology 90, no. 21 (2016): 9997–10006. http://dx.doi.org/10.1128/jvi.01122-16.
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ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
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Brown, Brandon L., Rachel M. Zalla, Courtney T. Shepard, et al. "Dual-Viral Transduction Utilizing Highly Efficient Retrograde Lentivirus Improves Labeling of Long Propriospinal Neurons." Frontiers in Neuroanatomy 15 (March 22, 2021). http://dx.doi.org/10.3389/fnana.2021.635921.
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The nervous system coordinates pathways and circuits to process sensory information and govern motor behaviors. Mapping these pathways is important to further understand the connectivity throughout the nervous system and is vital for developing treatments for neuronal diseases and disorders. We targeted long ascending propriospinal neurons (LAPNs) in the rat spinal cord utilizing Fluoro-Ruby (FR) [10kD rhodamine dextran amine (RDA)], and two dual-viral systems. Dual-viral tracing utilizing a retrograde adeno-associated virus (retroAAV), which confers robust labeling in the brain, resulted in a small number of LAPNs being labeled, but dual-viral tracing using a highly efficient retrograde (HiRet) lentivirus provided robust labeling similar to FR. Additionally, dual-viral tracing with HiRet lentivirus and tracing with FR may preferentially label different subpopulations of LAPNs. These data demonstrate that dual-viral tracing in the spinal cord employing a HiRet lentivirus provides robust and specific labeling of LAPNs and emphasizes the need to empirically optimize viral systems to target specific neuronal population(s).
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Poinsatte, Katherine, Denise M. Ramirez, Apoorva Ajay, et al. "Abstract 121: Visualization and Quantification of Post-Stroke Neural Connectivity in Mice Using Serial Two-Photon Tomography." Stroke 51, Suppl_1 (2020). http://dx.doi.org/10.1161/str.51.suppl_1.121.
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Background: It is a challenge to characterize post-stroke changes in neural connectivity at microscopic scale across the entire rodent brain. Serial two-photon tomography (STPT) is an advanced laser-scanning microscopy technique which collects serial fluorescence images across the brain and reconstructs 3D datasets. We examined changes in motor connectivity after cortical infarcts in mice, using retrograde viral tract tracing, STPT imaging, automated registration workflow, and machine learning algorithms. Methods: Young male C57/B6 mice received a photothrombotic motor cortex (M1) stroke (n=3) or sham surgery (n=3). 15 days later, a retrograde pseudorabies trans-synaptic virus encoding fluorescent protein was injected into the left forelimb flexor muscles to label motor system projections. Mice were sacrificed 3 weeks post-stroke. STPT images were analyzed using supervised machine learning (pixel-wise random forest via the “ilastik” software package) and datasets were mapped to the Allen Mouse Brain Atlas for region-specific visualization and quantification of fluorescent signals. Results: Machine learning algorithms successfully identified neuronal cell bodies, neuronal processes, and ischemic tissue throughout the brain. The fluorescent signal of cells and neuronal processes was higher in the right M1 and SS of uninjured mice than the left M1 and SS. After stroke, this signal was diminished in the right M1 and SS. Labeled neurons were also reduced in the left M1 suggesting the presence of secondary transcortical connections. Conclusions: STPT generates whole brain datasets that when analyzed with ML algorithms show early alterations in post-stroke neural connectivity in the corticospinal tract. Further studies utilizing monosynaptic and conditional viral tracers will better assess the full spectrum of connectivity changes during post-stroke functional recovery.
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Shi, Mei-Yu, Lu-Feng Ding, Yu-Hong Guo, Yu-Xiao Cheng, Guo-Qiang Bi, and Pak-Ming Lau. "Long-range GABAergic projections from the nucleus of the solitary tract." Molecular Brain 14, no. 1 (2021). http://dx.doi.org/10.1186/s13041-021-00751-4.
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AbstractThe nucleus of the solitary tract (NTS) plays a crucial role in integrating peripheral information regarding visceral functions. Glutamate decarboxylase 2 (GAD2) inhibitory neurons are abundant in the NTS, and are known to form local and short-range projections within the NTS and nearby hindbrain areas. Here we performed whole-brain mapping of outputs from GAD2 neurons in the NTS using cell-type specific viral labeling together with ultrahigh-speed 3D imaging at 1-μm resolution. In addition to well-known targets of NTS GAD2 neurons including the principle sensory nucleus of the trigeminal (PSV), spinal nucleus of the trigeminal (SPV), and other short-range targets within the hindbrain, the high sensitivity of our system helps reveal previously unknown long-range projections that target forebrain regions, including the bed nuclei of the stria terminalis (BST) involved in stress and fear responses, and the paraventricular hypothalamic nucleus (PVH) involved in energy balance and stress-related neuroendocrine responses. The long-range projections were further verified by retrograde labeling of NTS GAD2 neurons with cholera toxin B (CTB) injections in the BST and PVH, and by Cre-dependent retrograde tracing with rAAV2-retro injections in the two regions of GAD2-Cre mice. Finally, we performed complete morphological reconstruction of several sparsely labeled neurons projecting to the forebrain and midbrain. These results provide new insights about how NTS might participate in physiological and emotional modulation.
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Viney, Tim James, Minas Salib, Abhilasha Joshi, Gunes Unal, Naomi Berry, and Peter Somogyi. "Shared rhythmic subcortical GABAergic input to the entorhinal cortex and presubiculum." eLife 7 (April 5, 2018). http://dx.doi.org/10.7554/elife.34395.
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Rhythmic theta frequency (~5–12 Hz) oscillations coordinate neuronal synchrony and higher frequency oscillations across the cortex. Spatial navigation and context-dependent episodic memories are represented in several interconnected regions including the hippocampal and entorhinal cortices, but the cellular mechanisms for their dynamic coupling remain to be defined. Using monosynaptically-restricted retrograde viral tracing in mice, we identified a subcortical GABAergic input from the medial septum that terminated in the entorhinal cortex, with collaterals innervating the dorsal presubiculum. Extracellularly recording and labeling GABAergic entorhinal-projecting neurons in awake behaving mice show that these subcortical neurons, named orchid cells, fire in long rhythmic bursts during immobility and locomotion. Orchid cells discharge near the peak of hippocampal and entorhinal theta oscillations, couple to entorhinal gamma oscillations, and target subpopulations of extra-hippocampal GABAergic interneurons. Thus, orchid cells are a specialized source of rhythmic subcortical GABAergic modulation of ‘upstream’ and ‘downstream’ cortico-cortical circuits involved in mnemonic functions.
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Bertero, Alice, Charles Garcia, and Alfonso junior Apicella. "Corticofugal VIP Gabaergic Projection Neurons in the Mouse Auditory and Motor Cortex." Frontiers in Neural Circuits 15 (July 23, 2021). http://dx.doi.org/10.3389/fncir.2021.714780.
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Anatomical and physiological studies have described the cortex as a six-layer structure that receives, elaborates, and sends out information exclusively as excitatory output to cortical and subcortical regions. This concept has increasingly been challenged by several anatomical and functional studies that showed that direct inhibitory cortical outputs are also a common feature of the sensory and motor cortices. Similar to their excitatory counterparts, subsets of Somatostatin- and Parvalbumin-expressing neurons have been shown to innervate distal targets like the sensory and motor striatum and the contralateral cortex. However, no evidence of long-range VIP-expressing neurons, the third major class of GABAergic cortical inhibitory neurons, has been shown in such cortical regions. Here, using anatomical anterograde and retrograde viral tracing, we tested the hypothesis that VIP-expressing neurons of the mouse auditory and motor cortices can also send long-range projections to cortical and subcortical areas. We were able to demonstrate, for the first time, that VIP-expressing neurons of the auditory cortex can reach not only the contralateral auditory cortex and the ipsilateral striatum and amygdala, as shown for Somatostatin- and Parvalbumin-expressing long-range neurons, but also the medial geniculate body and both superior and inferior colliculus. We also demonstrate that VIP-expressing neurons of the motor cortex send long-range GABAergic projections to the dorsal striatum and contralateral cortex. Because of its presence in two such disparate cortical areas, this would suggest that the long-range VIP projection is likely a general feature of the cortex’s network.
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Hanchate, Naresh, Eun Jeong Lee, Andria Ellis, et al. "SUN-LB55 Connect-seq to Superimpose Molecular on Anatomical Neural Circuit Maps." Journal of the Endocrine Society 4, Supplement_1 (2020). http://dx.doi.org/10.1210/jendso/bvaa046.2259.
Full textAbstract:
Abstract Animals exhibit instinctive behavioral and physiological responses to a variety of stressors to overcome danger and restore homeostasis. The physiological response to stress is governed by hypothalamic corticotropin-releasing hormone (CRH) neurons which regulate the hypothalamic-pituitary-adrenal axis to control blood levels of stress hormones. At present, the neural circuits and signaling mechanisms through which different stress signals are transmitted to CRH neurons are poorly understood. Here, we devised a new method, termed “Connect-Seq,” which couples single-cell transcriptomics and retrograde viral tracing to define the molecular identities of individual neurons in neural circuits. As a proof of concept, using Connect-Seq, we profiled single-cell transcriptomes of 124 brain neurons upstream of CRH neurons and identified subpopulations that are likely to communicate stress-related signals to CRH neurons. Analyses of single-cell transcriptomes for ‘fast-acting’ neurotransmitters revealed subsets of upstream neurons that expressed markers of inhibitory GABAergic neurons or excitatory glutamatergic neurons. Further analyses showed a number of other neuromodulators/neurotransmitters in upstream neurons, including acetylcholine, dopamine, histamine, and 43 different neuropeptides, each expressed in individual neurons or subsets of neurons. These findings reveal extreme molecular heterogeneity among upstream neurons and suggest the upstream neurons use diverse neurochemical messengers to transmit signals to CRH neurons. Many neurons coexpressed different neurotransmitters/neuromodulators, suggesting the co-release of neurochemical messengers. Dual labeling of brain sections verified expression of specific neuromodulators in virus-infected neurons upstream of CRH neurons in selected brain areas. Our results indicate that Connect-Seq can be applied to genetically dissect neural circuits and uncover molecular identities of neurons upstream of specific neuronal types of known function. Molecular markers identified in those neurons lay a foundation for the application of cell-specific genetic tools to investigate the functions and physiological significance of diverse neuronal subsets within complex neural circuits.
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Hogue, Ian B., Jolie Jean, Andrew D. Esteves, Nikhila S. Tanneti, Julian Scherer, and Lynn W. Enquist. "Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26." Journal of Virology 92, no. 1 (2017). http://dx.doi.org/10.1128/jvi.01193-17.
Full textAbstract:
ABSTRACTFluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCEAlphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.