Murine prenatal expression of cholecystokinin in neural crest, enteric neurons, and enteroendocrine cells

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5–9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development. Dev Dyn 1999;216:190–200 . © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35165/1/9_ftp.pd

A Dual Infection Pseudorabies Virus Conditional Reporter Approach to Identify Projections to Collateralized Neurons in Complex Neural Circuits

Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners

Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes

Whether all the infectious herpesvirus particles entering a cell are able to replicate and/or express their genomes is not known. Here, we developed a general method to determine the number of viral genomes expressed in an infected cell. We constructed and analysed fluorophore expression from a recombinant pseudorabies virus (PRV263) carrying a Brainbow cassette (Cre-conditional expression of different fluorophores). Using three isogenic strains derived from PRV263, each expressing a single fluorophore, we analysed the colour composition of cells infected with these three viruses at different multiplicities. We estimate that fewer than seven incoming genomes are expressed per cell. In addition, those templates that are expressed are the genomes selected for replication and packaging into virions. This finite limit on the number of viral genomes that can be expressed is an intrinsic property of the infected cell and may be influenced by viral and cellular factors

Pseudorabies Virus Infected Porcine Epithelial Cell Line Generates a Diverse Set of Host MicroRNAs and a Special Cluster of Viral MicroRNAs

Pseudorabies virus (PRV) belongs to Alphaherpesvirinae subfamily that causes huge economic loss in pig industry worldwide. It has been recently demonstrated that many herpesviruses encode microRNAs (miRNAs), which play crucial roles in viral life cycle. However, the knowledge about PRV-encoded miRNAs is still limited. Here, we report a comprehensive analysis of both viral and host miRNA expression profiles in PRV-infected porcine epithelial cell line (PK-15). Deep sequencing data showed that the ∼4.6 kb intron of the large latency transcript (LLT) functions as a primary microRNA precursor (pri-miRNA) that encodes a cluster of 11 distinct miRNAs in the PRV genome, and 209 known and 39 novel porcine miRNAs were detected. Viral miRNAs were further confirmed by stem-loop RT-PCR and northern blot analysis. Intriguingly, all of these viral miRNAs exhibited terminal heterogeneity both at the 5′ and 3′ ends. Seven miRNA genes produced mature miRNAs from both arms and two of the viral miRNA genes showed partially overlapped in their precursor regions. Unexpectedly, a terminal loop-derived small RNA with high abundance and one special miRNA offset RNA (moRNA) were processed from a same viral miRNA precursor. The polymorphisms of viral miRNAs shed light on the complexity of host miRNA-processing machinery and viral miRNA-regulatory mechanism. The swine genes and PRV genes were collected for target prediction of the viral miRNAs, revealing a complex network formed by both host and viral genes. GO enrichment analysis of host target genes suggests that PRV miRNAs are involved in complex cellular pathways including cell death, immune system process, metabolic pathway, indicating that these miRNAs play significant roles in virus-cells interaction of PRV and its hosts. Collectively, these data suggest that PRV infected epithelial cell line generates a diverse set of host miRNAs and a special cluster of viral miRNAs, which might facilitate PRV replication in cells

The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the Neonatal Spinal Cord of the Mouse

The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies virus (PRV) Bartha has been used to map neuronal connectivity in other parts of the nervous system, due to its ability to travel trans-neuronally. Its use in spinal circuits regulating locomotion has been limited and no study has defined the time course of labelling for neurons known to project monosynaptically to motoneurons.Here we investigated the ability of PRV Bartha, expressing green and/or red fluorescence, to label spinal neurons projecting monosynaptically to motoneurons of two principal hindlimb muscles, the tibialis anterior (TA) and gastrocnemius (GC). As revealed by combined immunocytochemistry and confocal microscopy, 24-32 h after the viral muscle injection the label was restricted to the motoneuron pool while at 32-40 h the fluorescence was seen in interneurons throughout the medial and lateral ventral grey matter. Two classes of ipsilateral interneurons known to project monosynaptically to motoneurons (Renshaw cells and cells of origin of C-terminals) were consistently labeled at 40 h post-injection but also a group in the ventral grey matter contralaterally. Our results suggest that the labeling of last order interneurons occurred 8-12 h after motoneuron labeling and we presume this is the time taken by the virus to cross one synapse, to travel retrogradely and to replicate in the labeled cells.The study establishes the time window for virally-labelling monosynaptic projections to lumbar motoneurons following viral injection into hindlimb muscles. Moreover, it provides a good foundation for intracellular targeting of the labeled neurons in future physiological studies and better understanding the functional organization of the lumbar neural networks

Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals

Entry of Herpes Simplex Virus Type 1 (HSV-1) into the Distal Axons of Trigeminal Neurons Favors the Onset of Nonproductive, Silent Infection

Following productive, lytic infection in epithelia, herpes simplex virus type 1 (HSV-1) establishes a lifelong latent infection in sensory neurons that is interrupted by episodes of reactivation. In order to better understand what triggers this lytic/latent decision in neurons, we set up an organotypic model based on chicken embryonic trigeminal ganglia explants (TGEs) in a double chamber system. Adding HSV-1 to the ganglion compartment (GC) resulted in a productive infection in the explants. By contrast, selective application of the virus to distal axons led to a largely nonproductive infection that was characterized by the poor expression of lytic genes and the presence of high levels of the 2.0-kb major latency-associated transcript (LAT) RNA. Treatment of the explants with the immediate-early (IE) gene transcriptional inducer hexamethylene bisacetamide, and simultaneous co-infection of the GC with HSV-1, herpes simplex virus type 2 (HSV-2) or pseudorabies virus (PrV) helper virus significantly enhanced the ability of HSV-1 to productively infect sensory neurons upon axonal entry. Helper-virus-induced transactivation of HSV-1 IE gene expression in axonally-infected TGEs in the absence of de novo protein synthesis was dependent on the presence of functional tegument protein VP16 in HSV-1 helper virus particles. After the establishment of a LAT-positive silent infection in TGEs, HSV-1 was refractory to transactivation by superinfection of the GC with HSV-1 but not with HSV-2 and PrV helper virus. In conclusion, the site of entry appears to be a critical determinant in the lytic/latent decision in sensory neurons. HSV-1 entry into distal axons results in an insufficient transactivation of IE gene expression and favors the establishment of a nonproductive, silent infection in trigeminal neurons