Jason Chen

Serotonin 1A Receptors, Serotonin Transporter Binding and Serotonin Transporter mRNA Expression in the Brainstem of Depressed Suicide Victims

Suicide and depression are associated with reduced serotonergic neurotransmission. In suicides, there is a reduction in serotonin transporter (SERT) sites and an increase in postsynaptic 5-HT1A receptors in localized regions of the prefrontal cortex. In depression, there is a diffuse decrease in SERT binding throughout the dorsoventral extent of the prefrontal cortex. Serotonergic innervation of the prefrontal cortex arises predominantly from neurons in the brainstem dorsal raphe nucleus (DRN). We, therefore, examined postmortem SERT binding and mRNA expression, as well as 5-HT1A autoreceptor binding in the DRN of 10 matched pairs of controls and depressed suicide victims. The concentration of SERT sites, SERT mRNA, and 5-HT1A binding was not different between controls and suicides (p > .05). In the DRN of suicides, the volume of tissue defined by 5-HT1A binding was 40% smaller than controls. An index of the total number of 5-HT1A receptors (receptor binding × volume of receptor distribution) was 43.3% lower in the DRN of suicides, compared with controls. The suicide group had 54% fewer DRN neurons expressing SERT mRNA compared with controls. In the serotonin neurons that expressed the SERT gene, expression per neuron was greater in suicides. Less total 5-HT1A and SERT binding is consistent with results of in vivo studies in depression. Less feedback inhibition of serotonin DRN firing via 5-HT1A autoreceptors and enhancement of serotonin action due to less uptake of serotonin, is consistent with compensatory changes in response to hypofunction in depressed suicides.

Enteric Dopaminergic Neurons: Definition, Developmental Lineage, and Effects of Extrinsic Denervation

The existence of enteric dopaminergic neurons has been suspected; however, the innervation of the gut by sympathetic nerves, in which dopamine (DA) is the norepinephrine precursor, complicates analyses of enteric DA. We now report that transcripts encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the murine bowel (small intestine > stomach or colon; proximal colon > distal colon). Because sympathetic neurons are extrinsic, transcripts encoding TH and DAT in the bowel are probably derived from intrinsic neurons. TH protein was demonstrated immunocytochemically in neuronal perikarya (submucosal >> myenteric plexus; small intestine > stomach or colon). TH, DA, and DAT immunoreactivities were coincident in subsets of neurons (submucosal > myenteric) in guinea pig and mouse intestines in situ and in cultured guinea pig enteric ganglia. Surgical ablation of sympathetic nerves by extrinsic denervation of loops of the bowel did not affect DAT immunoreactivity but actually increased numbers of TH-immunoreactive neurons, expression of mRNA encoding TH and DAT, and enteric DOPAC (the specific dopamine metabolite). The fetal gut contains transiently catecholaminergic (TC) cells. TC cells are the proliferating crest-derived precursors of mature neurons that are not catecholaminergic and, thus, disappear after embryonic day (E) 14 (mouse) or E15 (rat). TC cells appear early in ontogeny, and their development/survival is dependent on mash-1 gene expression. In contrast, the intrinsic TH-expressing neurons of the murine bowel appear late (perinatally) and are mash-1 independent. We conclude that the enteric nervous system contains intrinsic dopaminergic neurons that arise from a mash-1-independent lineage of noncatecholaminergic precursors.

Bone Morphogenetic Protein-2 and -4 Limit the Number of Enteric Neurons But Promote Development of a TrkC-Expressing Neurotrophin-3-Dependent Subset

The hypothesis that BMPs (bone morphogenetic proteins), which act early in gut morphogenesis, also regulate specification and differentiation in the developing enteric nervous system (ENS) was tested. Expression of BMP-2 and BMP-4, BMPR-IA (BMP receptor subunit), BMPR-IB, and BMPR-II, and the BMP antagonists, noggin, gremlin, chordin, and follistatin was found when neurons first appear in the primordial bowel at embryonic day 12 (E12). Agonists, receptors, and antagonists were detected in separated populations of neural crest- and noncrest-derived cells. When applied to immunopurified E12 ENS precursors, BMP-2 and BMP-4 induced nuclear translocation of phosphorylated Smad-1 (Sma and Mad-related protein). The number of neurons developing from these cells was increased by low concentrations and decreased by high concentrations of BMP-2 or BMP-4. BMPs induced the precocious appearance of TrkC-expressing neurons and their dependence on neurotrophin-3 for survival. BMP-4 interacted with glial cell line-derived neurotrophic factor (GDNF) to enhance neuronal development but limited GDNF-driven expansion of the precursor pool. BMPs also promoted development of smooth muscle from mesenchymal cells immunopurified at E12. To determine the physiological significance of these observations, the BMP antagonist noggin was overexpressed in the developing ENS of transgenic mice under the control of the neuron-specific enolase promoter. Neuronal numbers in both enteric plexuses and smooth muscle were increased throughout the postnatal small intestine. These increases were already apparent by E18. In contrast, TrkC-expressing neurons decreased in both plexuses of postnatal noggin-overexpressing animals, again an effect detectable at E18. BMP-2 and/or BMP-4 thus limit the size of the ENS but promote the development of specific subsets of enteric neurons, including those that express TrkC.

Role of serotonin in intestinal inflammation: knockout of serotonin reuptake transporter exacerbates 2,4,6-trinitrobenzene sulfonic acid colitis in mice

Serotonin (5-HT) regulates peristaltic and secretory reflexes in the gut. The serotonin reuptake transporter (SERT; SLC6A4), which inactivates 5-HT, is expressed in the intestinal mucosa and the enteric nervous system. Stool water content is increased and colonic motility is irregular in mice with a targeted deletion of SERT. We tested the hypotheses that 5-HT plays a role in regulating intestinal inflammation and that the potentiation of serotonergic signaling that results from SERT deletion is proinflammatory. Rectal installation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce an immune-mediated colitis, which was compared in SERT knockout mice and littermate controls. Intestinal myeloperoxidase and histamine levels were significantly increased, whereas the survival rate and state of health were significantly decreased in TNBS-treated mice that lacked SERT. Deletion of SERT thus increases the severity of TNBS colitis. These data suggest that 5-HT and its SERT-mediated termination play roles in intestinal immune/inflammatory responses in mice.

Mannose 6-Phosphate Receptor Dependence of Varicella Zoster Virus Infection In Vitro and in the Epidermis during Varicella and Zoster

Varicella zoster virus (VZV) is a highly infectious human pathogen; nevertheless, infectious virions are not released in vitro where infection is cell associated. Four VZV envelope glycoproteins contain mannose 6-phosphate (Man 6-P), and Man 6-P blocks infection of cells by cell-free VZV. Expression of antisense cDNA or siRNA-like transcripts were used to generate five stable human cell lines deficient in cation-independent mannose 6-phosphate receptors (MPRci). All 5 MPRci-deficient lines resisted infection by cell-free, but not cell-associated, VZV, secreted lysosomal enzymes, and released infectious virions when infected by cell-associated VZV. Intracellular MPRci thus appear to divert newly enveloped VZV to late endosomes, and plasmalemmal MPRci are necessary for entry by cell-free VZV. Biopsies from VZV-infected human skin supported the idea that because MPRci expression is naturally lost in maturing superficial epidermal cells, these cells do not divert VZV to endosomes and constitutively secrete infectious VZV.

Expression and developmental regulation of oxytocin (OT) and oxytocin receptors (OTR) in the enteric nervous system (ENS) and intestinal epithelium

Although oxytocin (OT) and oxytocin receptor (OTR) are known for roles in parturition and milk let‐down, they are not hypothalamus‐restricted. OT is important in nurturing and opposition to stress. Transcripts encoding OT and OTR have been reported in adult human gut, and OT affects intestinal motility. We tested the hypotheses that OT is endogenous to the enteric nervous system (ENS) and that OTR signaling may participate in enteric neurophysiology. Reverse transcriptase polymerase chain reaction confirmed OT and OTR transcripts in adult mouse and rat gut and in precursors of enteric neurons immunoselected from fetal rats. Enteric OT and OTR expression continued through adulthood but was developmentally regulated, peaking at postnatal day 7. Coincidence of the immunoreactivities of OTR and the neural marker Hu was 100% in the P3 and 71% in the adult myenteric plexus, when submucosal neurons were also OTR‐immunoreactive. Co‐localization with NeuN established that intrinsic primary afferent neurons are OTR‐expressing. Because OTR transcripts and protein were detected in the nodose ganglia, OT signaling might also affect extrinsic primary afferent neurons. Although OT immunoreactivity was found only in ∼1% of myenteric neurons, extensive OT‐immunoreactive varicosities surrounded many others. Villus enterocytes were OTR‐immunoreactive through postnatal day 17; however, by postnatal day 19, immunoreactivity waned to become restricted to crypts and concentrated at crypt‐villus junctions. Immunoelectron microscopy revealed plasmalemmal OTR at enterocyte adherens junctions. We suggest that OT and OTR signaling might be important in ENS development and function and might play roles in visceral sensory perception and neural modulation of epithelial biology. J. Comp. Neurol. 512:256–270, 2009.

A Model of Lytic, Latent, and Reactivating Varicella-Zoster Virus Infections in Isolated Enteric Neurons

Because human primary afferent neurons are not readily obtained, we sought to develop a model in which the lytic, latent, and reactivating phases of varicella-zoster virus (VZV) infection were recapitulated in neurons from an animal source. Enteric neurons were obtained from the small intestine of adult guinea pigs and from the bowel of fetal mice. Latency was established when these neurons were infected by cell-free VZV in the absence of fibroblasts or other cells of mesodermal origin. In contrast, lytic infection ensued when fibroblasts were present or when the enteric neurons were infected by cell-associated VZV. Latency was associated with the expression of a limited subset of viral genes, the products of which were restricted to the cytoplasm. Lysis was associated with the expression of viral glycoproteins, nuclear translocation of latency-associated gene products, and rapid cell death. Reactivation was accomplished by expressing VZV open reading frame (ORF) 61p or herpes simplex virus ICP0 in latently infected neurons. Isolated enteric neurons from guinea pigs and mice recapitulate latent gene expression in human cranial nerve and dorsal root ganglia. Expression of latency-associated VZV gene products was detected in 88% of samples of adult human intestine, suggesting that VZV not only infects enteric neurons but also is latent in the human enteric nervous system. This in vitro model should facilitate further understanding of latency and reactivation of VZV.

Genetic analysis of the VP1 region of enterovirus 71 reveals the emergence of genotype A in central China in 2008

Enterovirus 71 (EV71) strains from children were characterized by full-length VP1 nucleotide sequencing. Out of 22 clinical specimens, five isolates identified as EV71 were recovered by virus isolation. The VP1 sequences of the five isolates had more than 97.4% sequence identity with prototype virus BrCr, clustering in the genotype A lineage. This represents the first record of genotype A EV71 in China since the BrCr prototype strain was discovered in the USA in 1969.

Netrin/DCC-Mediated Attraction of Vagal Sensory Axons to the Fetal Mouse Gut

Vagal sensory axons and migrating neural crest‐derived precursor cells follow similar pathways to reach the gut. The crest‐derived cells express the netrin receptor deleted in colorectal cancer (DCC) and migrate toward netrins expressed by the intestinal mucosa and pancreas; this attraction is required for the formation of submucosal and pancreatic ganglia. We tested the hypothesis that enteric netrins also attract vagal sensory fibers. These axons were located as a function of age in fetal mice by applying the lipophilic tracer 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) bilaterally to nodose ganglia. DiI‐labeled axons were found in the esophagus and proximal stomach by E12 and, more distally, in the small bowel at E14–E16. Transcripts encoding DCC were expressed in the nodose ganglia of mice from E12 to adulthood but were developmentally regulated. Paraesophageal anterior and posterior vagal trunks were DCC immunoreactive from E12 to E16. Transcripts encoding netrin‐1 were expressed in the developing foregut and midgut; netrin‐1 immunoreactivity was detected in the outer gut mesenchyme and mucosal epithelium. Neurites from explanted E14 nodose ganglia grew selectively toward cocultured E14 distal foregut explants (P < 0.01). Antibodies to DCC specifically abolished this preferential outgrowth (P < 0.05). Nodose axons also grew selectively toward cocultured netrin‐secreting 293‐EBNA cells (P < 0.005); antibodies to DCC again blocked this preferential outgrowth (P < 0.05). These data suggest that netrins, which are expressed in the bowel, attract DCC‐expressing vagal sensory axons. J. Comp. Neurol. 498:567–580, 2006.

Disseminated, Persistent, and Fatal Infection Due to the Vaccine Strain of Varicella-Zoster Virus in an Adult Following Stem Cell Transplantation

Live attenuated varicella vaccine is recommended for healthy individuals who are susceptible to varicella. Although the vaccine is safe, effective, and used worldwide, serious adverse events have been reported, mainly in immunocompromised patients who subsequently recovered. Here, we describe the fatality of an immunocompromised patient who received the varicella vaccine. His medical history provides a cautionary lens through which to view the decision of when vaccination is appropriate. A middle-aged man with non-Hodgkin lymphoma received chemotherapy and a stem cell transplant. He was vaccinated 4 years post-transplantation, despite diagnosis of a new low-grade lymphoma confined to the lymph nodes. Within 3 months of vaccination, he developed recurrent rashes with fever, malaise, weakness, hepatitis, weight loss, and renal failure. The syndrome was eventually determined to be associated with persistent disseminated zoster caused by the vaccine virus. This case illustrates a circumstance when a live viral vaccine should not be used.