Michael D. Gershon, Md - US grants

The enteric nervous system (ENS), the largest and most phenotypically diverse region of the PNS, has unique characteristics, which are reminiscent of the brain, and are not seen in other ganglia. We have found that the neural crest-derived precursors of enteric neurons probably begin to differentiate as neurons before they reach the bowes; however, the microenvironment provided by the non-neuronal cells of the wall of the gut may be important in determining what type of neuron these precursors will form and also whether other precursors will express the phenotype of enteric glia or Schwann cells. The goal of the current proposal is to test the hypothesis that the development of the nuerons and gial of the ENS, as well as its unique pattern of organization, results from a determinative interaction between neural crest emigres and the enteric mesenchyme. In vitro experiments, in which the conditions of this interaction can be controlled, will be done to test this hypothesis and determine the nature of responsible factors. Quail neural crest, which can be recognized and traced, will be combined in co-culture with chick enteric mesenchyme. Intermediate filament content, neurotransmitter-related properties, and ultrastructure will be used as markers of the enteric pattern of ganglionic development. Developmental regulation, regional specificity and the role of soluble factors, extracellular matrix and/or cell to cell contact in the neural crest-enteric mesenchyme interaction will be explored. The development of the progeny of single clones of neural crest cells in the enteric mesenchyme will be followed to evaluate the extent to which single migrating neural crest cells are multipotential. Regions of the neuraxis that do or do not normally provide cells for the ENS will be compared. Finally, the regulation of specific transient changes in the enteric mesenchyme that may be associated with the development of particular types of enteric neuron will be analyzed. These changes include the specific uptake of serotonin by mesenchymal cells and the expression of aspects of a catecholaminergic phenotype by proliferating cells in the enteric mesenchyme that may be neuroblasts. Analysis of transient monoaminergic expression will use cell culture, in situ hybridization with a radiolabeled cDNA probe for tyrosin hydroxylase mRNA and the type-specific cellular localization of monamine oxidase. Selection of the serotonergic neuronal phenotype and the possibility that ingrowing sympathetic nerves regulate gene expression by enteric neuroblasts will be investigated.

[unreadable] DESCRIPTION (provided by applicant): Serotonin (5-HT) plays critical roles in enteric physiology and pathophysiology. Gastrointestinal (GI) motility is abnormal, in humans as well as in animals, when mucosal expression of the serotonin transporter (SERT), the molecule mainly responsible for 5-HT inactivation, is deficient. Enteric serotonergic signaling is inadequately understood despite its importance and clinical significance. Two isoforms of tryptophan hydroxylase (TpH-1 and TpH-2) have recently been demonstrated. Although both are expressed in the gut, neither the identities of the enteric cells that express TpH-1 and/or TpH-2, nor how either isoform is regulated in the bowel are known. We thus propose to identify cells in the gut that express TpH-1 and/or TpH-2 and to utilize transgenic mice lacking SERT to determine how increased 5-HT availability affects the expression of TpH-1 and TpH-2. Transcripts encoding 5-HT1B/1D, 5-ht1f, 5-ht5, 5-ht6, and 5-HT7 receptors have been detected in the gut but their enteric actions are unknown; moreover, important responses of enteric neurons to 5-HT have not been attributed to a classified 5-HT receptor. The gut thus contains 5-HT receptors that are "looking for a function" and functions of 5-HT that are "looking for a receptor." Some of these functions are mediated by a receptor activity called 5-HT1p, which has been characterized pharmacologically and with respect to transduction coupling, but has resisted cloning. Conceivably, the properties of 5-HT receptors expressed in enteric neurons may be different from those of the same receptors expressed in heterologous cells of the CNS, where most have been characterized. 5-HT receptors may also acquire novel properties in enteric neurons by forming homo- and/or hetero-oligomers. We now propose to identify cells of the bowel that express receptors that are "looking for a function" (5-HT1B/1D, 5-ht1f, 5-ht5, 5-ht6, and 5-HT7) and to identify enteric neuronal responses these receptors mediate. We will also determine whether 5-HT1B or other 5-HT receptors in enteric neurons form homo- or hetero-oligomers with 5-HT or a D2 dopamine receptor and, if they do, whether homo- or hetero-oligomers acquire 5-HT1p-like activity. Finally, intrinsic enteric dopaminergic neurons have recently been identified by not functionally characterized. We now propose to identify neurons that express DA receptors, to determine whether they receive a dopaminergic innervation, and how these neurons respond to DA and to DA receptor subtype-selective agonists and antagonists. [unreadable] [unreadable]

5-hydroxytryptamine (5-HT) is a neurotransmitter in the enteric nervous system (ENS). Two types of enteric neural 5-HT receptor have been identified, 5-HT1P and 5-HT3. The 5-HT1P receptor is labeled by 3H-5-HT; antagonists at 5-HT1P receptors include N- acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and BRL 24924. During developments 5-HT1P receptors arise after the appearance of serotonergic neurons. 5-HT3 receptors are not labeled by 3H-5-HT; antagonists at 5-HT3 receptors include ICS 205- 930, BRL 43694 (granisetron) and GR 65630. Neither the distribution in the wall of the bowel, nor the ontogeny of 5-HT3 receptors have previously been studied. It is now proposed to study enteric 5-HT3 receptors in the murine gut by radioligand binding techniques, using both rapid filtration of enteric membranes and radioautography with 3H-granisetron and 3H-GR 65630. Specific binding will be defined as that displaced by ICS 205-930. The location of 5-HT3 receptors will be determined in the adult and fetal bowel and the timing of their appearance in ontogeny will be related to that of 5-HT1P receptors. Selective destruction of serotonergic neurites with 5,7-dihydroxytryptamine (5,7-DHT) will be used to explore their influence on the development, maturation, and maintenance of 5-HT1P receptors. The ability of 5-HT itself to affect 5-HT1P receptor development will be separately explored. The role of 5-HT will be studied by examining 5-HT1P receptor development and maturation in the presence of specific antagonists and after depletion of 5-HT by inhibition of tryptophan hydroxylase. Experiments will be done with explants of fetal gut grown in organotypic tissue culture and in vivo with developing hindgut, in which 5-HT1P receptors develop during the first 3 weeks of life. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) induces a rapid upregulation of 5-HT1P receptors. The effect of 6-OHDA on 5-HT3 receptors will now be ascertained. In addition, studies will be done to determine whether the effect of 6-OHDA on enteric neural 5-HT receptors is due to loss of norepinephrine (NE), and is specifically related to destruction of the noradrenergic innervation of serotonergic neurons. These experiments will utilize tyrosine hydroxylase inhibition to deplete BE and the quantitative cytochemical demonstration of cytochrome oxidase activity to evaluate effect of sympathectomy on the tonic activity of serotonergic neurons. Finally, since both serotonergic neurons and 5-HT1P receptors are already present while the gut contains the proliferating precursors of neurons that contain vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) immunoreactivity the possibility exists that serotonergic neurons may influence the phenotypic expression of these late-developing neurons. This possibility will be tested by determining the effects of 5,7-DHT or chronic inhibition of 5-HT1P and/or 5-HT3 receptors on the birthdays and ultimate numbers of VIP- and CGRP-immunoreactive neurons. The effect of these treatments on the earlier-developing neuropeptide Y-immunoreactive neurons will also be examined as a control. Finally, if effects ar found, studies will be done to determine whether affected neurons actually receive serotonergic synapses. These experiments will enhance understanding of the pathogenesis of congenital defects of enteric neuromuscular development and will be important in evaluating the safety of psychoactive drugs that affect serotonergic neurons of 5-HT receptors in pregnancy.

5-hydroxytryptamine (5-HT) is a neurotransmitter in the enteric nervous system (ENS). Two types of enteric neural 5-HT receptor have been identified, 5-HT1P and 5-HT3. The 5-HT1P receptor is labeled by 3H-5-HT; antagonists at 5-HT1P receptors include N- acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and BRL 24924. During developments 5-HT1P receptors arise after the appearance of serotonergic neurons. 5-HT3 receptors are not labeled by 3H-5-HT; antagonists at 5-HT3 receptors include ICS 205- 930, BRL 43694 (granisetron) and GR 65630. Neither the distribution in the wall of the bowel, nor the ontogeny of 5-HT3 receptors have previously been studied. It is now proposed to study enteric 5-HT3 receptors in the murine gut by radioligand binding techniques, using both rapid filtration of enteric membranes and radioautography with 3H-granisetron and 3H-GR 65630. Specific binding will be defined as that displaced by ICS 205-930. The location of 5-HT3 receptors will be determined in the adult and fetal bowel and the timing of their appearance in ontogeny will be related to that of 5-HT1P receptors. Selective destruction of serotonergic neurites with 5,7-dihydroxytryptamine (5,7-DHT) will be used to explore their influence on the development, maturation, and maintenance of 5-HT1P receptors. The ability of 5-HT itself to affect 5-HT1P receptor development will be separately explored. The role of 5-HT will be studied by examining 5-HT1P receptor development and maturation in the presence of specific antagonists and after depletion of 5-HT by inhibition of tryptophan hydroxylase. Experiments will be done with explants of fetal gut grown in organotypic tissue culture and in vivo with developing hindgut, in which 5-HT1P receptors develop during the first 3 weeks of life. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) induces a rapid upregulation of 5-HT1P receptors. The effect of 6-OHDA on 5-HT3 receptors will now be ascertained. In addition, studies will be done to determine whether the effect of 6-OHDA on enteric neural 5-HT receptors is due to loss of norepinephrine (NE), and is specifically related to destruction of the noradrenergic innervation of serotonergic neurons. These experiments will utilize tyrosine hydroxylase inhibition to deplete BE and the quantitative cytochemical demonstration of cytochrome oxidase activity to evaluate effect of sympathectomy on the tonic activity of serotonergic neurons. Finally, since both serotonergic neurons and 5-HT1P receptors are already present while the gut contains the proliferating precursors of neurons that contain vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) immunoreactivity the possibility exists that serotonergic neurons may influence the phenotypic expression of these late-developing neurons. This possibility will be tested by determining the effects of 5,7-DHT or chronic inhibition of 5-HT1P and/or 5-HT3 receptors on the birthdays and ultimate numbers of VIP- and CGRP-immunoreactive neurons. The effect of these treatments on the earlier-developing neuropeptide Y-immunoreactive neurons will also be examined as a control. Finally, if effects ar found, studies will be done to determine whether affected neurons actually receive serotonergic synapses. These experiments will enhance understanding of the pathogenesis of congenital defects of enteric neuromuscular development and will be important in evaluating the safety of psychoactive drugs that affect serotonergic neurons of 5-HT receptors in pregnancy.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In the past 10 years, there have been many studies published on the development of the enteric nervous system in journals including Science, Nature, Neuron, Nature Genetics, Gastroenterology, Journal of Neuroscience and Development. These studies have included a broad diversity of approaches including genetic studies of humans with Hirschsprung's disease, zebrafish genetics, and cell and molecular biological studies using avians and a variety of mutant and transgenic mice and rats. Many of the studies were motivated by a desire to understand a number of developmental disorders of the enteric nervous system including Hirschsprung's disease, slow transit constipation and infantile hypertrophic pyloric stenosis. This research field is an excellent example of the power of animal models to understand a human disease (Hirschsprung's disease). Recent studies have shown that stem cells can be isolated from the developing and adult enteric nervous system, and there is enormous interest in the use of these enteric neural stem cells for therapeutic purposes. Importantly, the developing enteric nervous system has also provided many insights into neural development and cell migration in general. There has not yet been a conference devoted to the development of the enteric nervous system. The main goal of our meeting is to bring together, for the first time, the diversity of researchers interested in the development of the enteric nervous system. Our meeting will also be of interest to researchers interested in the control of adult motility as some of the molecules identified in developmental studies are likely to play key roles in the maintenance of function in adults, and are likely to be major targets in strategies to restore or repair enteric neural circuits. Topics to be covered include stem cells, migratory pathways, genetics of Hirschsprung's disease, genetic screens, development of gut motility, Sox10, endothelin-3, Ret signaling, interactions between pathways. The invited speakers include all of the leading researchers in the field, including Professor Nicole Le Douarin. We also anticipate that many post-graduate students and post-doctoral fellows will also attend. [unreadable] [unreadable]

Summary Varicella zoster virus (VZV) famously establishes latency in dorsal root (DRG) and cranial nerve (CNG) ganglia after its disseminated primary infection (varicella; chickenpox). VZV can reactivate from latency to cause a localized secondary infection (zoster; shingles). VZV latency, however, is not restricted to DRG/CNG; latent VZV is present in the enteric nervous system (ENS) in virtually everyone who has experienced varicella or received the live attenuated varicella vaccine. VZV reactivates in the ENS (enteric zoster) as it does in DRG/CNG but because enteric neurons lack cutaneous projections, enteric zoster occurs without rash and may be an unsuspected cause of GI disease. A major hindrance to research on VZV has been the absence of a suitable animal model. To overcome this difficulty, we demonstrated that VZV infects, establishes latency, and reactivates in isolated guinea pig enteric neurons; moreover, VZV infects guinea pigs in vivo, establishes latent infection in their DRG/CNG and ENS, and can be reactivated to produce a secondary infection resembling disseminated zoster. VZV can be transported to the ENS from infected epidermis in axons of DRG neurons that project both to the skin and gut but intravenous injection of VZV- infected T lymphocytes establishes latency in almost every ENS and DRG neuron of the animal. It had been thought that latent infection of enteric neurons could be established by cell-free VZV (VZVCF) but not by cell associated VZV (VZVCA). VZV-infected lymphocytes, however, do not secrete VZVCF but they are able transmit infection to neurons in vitro and in vivo that is exclusively latent. Aim 1 tests hypotheses that: (i) evanescent cell fusion is responsible for transmission of VZV from lymphocytes to neurons; (ii) exosomes derived from VZV-infected lymphocytes introduce stimulator of interferon genes (STING) to neurons; (iii) STING induces a type1 interferon response in neurons that inhibits VZV proliferation and facilitates establishment of latency. Aim 2 tests hypotheses that: (i) VZV-infected lymphocytes can induce a varicella- like primary infection in guinea pigs if immunosuppression and stress precedes infection; (ii) restriction of VZV latency allows localized reactivations to be confined to gut or skin; (iii) continuous activation of a receptor tyrosine kinase transduction pathway, similar to that in HSV1 reactivation in sympathetic neurons, regulates latent VZV genomes in enteric neurons. Aim 3 directly tests the hypothesis that salivary VZV DNA in patients with unexplained abdominal pain severe enough to warrant endoscopy and biopsy is a marker of enteric zoster. To validate this idea with a tissue diagnosis, we will analyze VZV DNA in saliva and GI mucosal expression of gE transcripts and protein which would indicate productive VZV infection (enteric zoster) in the bowel. This research makes the first use a novel animal model in which VZV reactivates in vivo and the first application of a non-invasive technique to identify patients that might have enteric zoster.